22nd biennial evergreen international phage...

22nd Biennial Evergreen International

Phage Meeting

Aug. 6-11, 2017

The Evergreen State College, Olympia, WA

This abstract book is not a publication. Information can not be referenced without explicit

permission of the author(s).

22nd Biennial Evergreen International Phage Meeting – Aug. 6-11, 2017


Organizing committee: Elizabeth Kutter (chair), Jason Gill, Susan Lehman,

Krystyna Dabrowska, Peter Fineran, Debbie Hinton, Ramy Aziz, Paul Hyman, Dan Nelson, Joana

Azeredo, Bob Blasdel, Martha Clokie, Sandra Morales, and Martha Vives

Daniel Bryan, co-coordinator

Meeting goals: Building on the original Cold Spring Harbor phage meetings, the Evergreen

meeting’s primary goal is to provide an opportunity for members of the ever-broadening phage

community from various backgrounds, ages and places to build strong working relationships and

share skills and ideas, leading to new insights and collaborations and, in recent years, helping spur

on many kinds of applications. People are encouraged to present work that is still in progress.

Posters are strongly encouraged, both as independent presentations with their own abstracts

(grouped by general topic) and as a way of letting participants more closely examine the detailed

data of those giving talks. They remain up throughout the meeting, rather than having specific

presentation times for individual groups of posters.

Note that presenting at this meeting does not constitute a publication. Abstracts will not be

published online, and permission must be obtained from the respective authors before

quoting any of their abstracts or data, or applying it in a way that may be inappropriate.

Thanks to contributors: The Evergreen State College and the Phagebiotics Research Foundtion,

cosponsors and planners; Evergreen grants writer Dorothea Collins; Matthew Stidham, digital

support; the TESC Longhouse crew for taking over our traditional Native American salmon bake;

and the many of you who have helped with meeting planning. Special thanks to the following

organizations for helping support young scholars and participants from underdeveloped countries:

AmpliPhi Biosciences Inc (APHB): AmpliPhi is a biopharmaceutical company focused on the

development of an internally generated pipeline of bacteriophage-based products for human

therapeutic use. The company operates from laboratories in Australia, the USA and Slovenia and

has a production facility exclusively dedicated to manufacture of therapeutic phage products under

current Good Manufacturing Practices (cGMP) standards.

The Capitol Aeroporter has gone from simply being the means by which most participants

reached Evergreen to becoming an active supporter of our work toward phage applications. This

year, they have donated transportation for a number of the participants in our Africa product, as

well as making special efforts to make the trip to Evergreen as positive as possible in terms of

paying attention to the schedules of all of the meeting attendants in making van assignments.

EpiBiome, Inc., based in South San Francisco, CA, is a venture-backed precision microbiome

engineering company that employs a genomics approach to understand complex microbial

communities and deploys bacteriophages to specifically eliminate problematic bacteria while

leaving the rest of the community intact.

Intralytix, Inc., based in Baltimore, has been successfully exploring phage applications to food

safety and human health since the 1990s. Their product targeting Listeria in ready to eat meats and



cheeses was the first FDA approved phage product for human ingestion, and they are now

producing approved products for a number of other livestock and human pathogens.

Jafral is a small contract development and manufacturing organization (CDMO) which provides

support with process development, analytical development and manufacturing for bacteriophage

products. They are set up to manufacture phages for almost any purpose, including environmental,

agricultural, food safety, diagnostic and veterinary applications.

Synthetic Genomics: Synthetic Genomics Inc. (SGI) uses their pioneering and proprietary science

and technology to develop products to positively impact the world. From new vaccines and

therapeutics, food and nutritional products, humanized organs for transplant, biofuels, biobased-

chemicals, and agricultural solutions, they are advancing products through their own programs and

with industry leading partners. They are fully leveraging their synthetic biology, genomics,

bioinformatics and viral engineering toolbox to engineer bacteriophage with improved therapeutic

potential against medically important MDR pathogens such as P. aeruginosa.

The Bill and Melinda Gates Foundation, for providing travel support for scientists from various

African countries who are working toward using phage to increase food safety, and also for helping

support the recent 11-day training led by Martha Clokie and Janet Nole at the Makerere University

College of Veterinary Medicine, in which a number of the participants here took part.

Thanks also to the National Institutes of Health and the US Department of Agriculture for their

many years-long history of supporting this collaborative Evergreen meeting through grant funding.

This abstract book is not a publication. Information can not be referenced without explicit permission of the author(s).

EVERGREEN 2017 -- CONFERENCE SCHEDULE 5th – 11th August Breakfast (7:30 - 8:45 Sun, 7:30 - 8:15 M-F), lunch (12:00 - 12:45 M-Th), and dinner (5:00 - 5:45 M, T) are supplied on the lower floor of the CAB

SUNDAY MORNING

WORKSHOP (Purce Bldg, Lecture Hall 1) Affiliation

9:00 AM -1:00 PM Ramy Aziz Cairo University (EGY)

Jason Gill A helping hand through the annotation bottleneck Texas A&M (USA)

Alejandro Reyes Univ. de los Andes (COL)

2:00 - 4:30 PM

Welcoming Barbecue @ Betty’s House (4510 Green Cove Court)

SUNDAY EVENING

Welcome and Plenary Talks -- Betty Kutter Purce Lecture Hall 1

5:00 PM Betty Kutter Welcome TESC (USA)

5:10 PM Martha Clokie Adventures with our African Contingent Univ. Leicester (GBR)

5:20 - 6:00 PM Jean-Paul Pirnay Phage therapy research in the Queen Astrid military hospital in Brussels Queen Astrid Military Hosp. (BEL)

6:00 - 6:30 PM Naomi Hoyle Eliava Phage Therapy Center: Current practice of phage therapy in multiple fields of medicine Eliava Institute (GEO)

6:30 - 7:00 PM Alejandro Reyes Exploring gut associated phage diversity through metaviromics Univ. of the Andes (COL)

7:00 - 7:30 PM Scott Stibitz Phage Therapy and the FDA FDA (USA)

SUNDAY 7:30 PM - 9:30 PM POSTER SESSION & OPENING MIXER Seminar II E (4th floor)

MONDAY MORNING

I: Phage Ecology; Chair: Mya Breitbart Purce Lecture Hall 1

8:30 - 9:00 AM Jennifer Brum Global ecology and ecosystem effects of marine viruses Louisiana State Univ. (USA)

9:00 - 9:20 AM Debbie Lindell Dramatic differences in cyanophage distribution across environmental gradients in the oceans Technion (ISR)

9:20 - 9:40 AM Eric Keen Novel “super spreader” bacteriophages promote horizontal gene transfer by transformation Washington Univ. (USA)

9:40 - 9:55 AM Bas Dutilh Towards modelling phage-host interactions in natural ecosystems Utrecht Univ. (NLD)

9:55 - 10:10 AM Bonnie Hurwitz iVirus: a cyberinfrastructure for large-scale computing to unravel virus-host interactions Univ. Arizona (USA)

BREAK

10:25 - 11:40 AM Brittany Leigh Unraveling a role for prophages in shaping gut microbiomes Univ. South Florida (USA)

10:40 - 11:00 AM Bridget Watson CRISPR-Cas affects horizontal gene transfer by transduction Univ. Otago (NZL)

11:00 - 11:15 AM Olivier Zablocki The virome of a South African scalding spring: phages & archaeal viruses share a pool Univ. Western Cape (ZAF)

11:15 - 11:30 AM Maria Letarova Phage stratification in monoclonal Chlorobium phaeobacteroides in the meromictic lake Trekhtsvetnoe INMI RAS (RUS)

11:30 - 11:55 AM Poster Previews


MONDAY AFTERNOON

II: Agriculture & Food Safety; Chairs: Jason Gill & Tom Denes Purce Lecture Hall 1

1:20 - 1:40 PM Lone Brøndsted Phage-host interactions: what have we learned from studying Campylobacter phages Univ. Copenhagen (DNK)

1:40 - 2:00 PM Mayukh Das Application of phages to control Pierce’s Disease Texas A&M (USA)

2:00 - 2:15 PM Andrew Millard Bacteriophage diversity in cattle slurry Univ. Warwick (GBR)

2:15 - 2:30 PM Hedieh Attai Phage peptidoglycan hydrolase as an antagonist of Agrobacterium tumefaciens Univ. of Missouri (USA)

2:30 - 2:45 PM Devon Radford Antimicrobial properties of Felix O1 nd A511 embedded in xanthan coatings on PLA films Agric. & Agri-Food Canada (CAN)

BREAK

3:00 - 3:15 PM Viviana Clavijo Effect of SalmoFree® cocktail on Salmonella reduction in commercial broiler's farms Univ. de los Andes (COL)

3:15 - 3:30 PM Hany Anany Using free and immobilized lytic phages to tackle Salmonella contamination in food Agric. & Agri-Food Canada (CAN)

3:30 - 3:50 PM Joelle Woolston Bacteriophage biocontrol for improving the safety of human and pet foods Intralytix (USA)

3:50 - 4:05 PM Tobi Nagel Phages applications for developing countries Phages for Global Health (USA)

4:05 - 4:15 PM Images of the Phages for Global Health Ugandan Phage Training, July 2017

4:15 - 4:40 PM Poster Previews

MONDAY EVENING:

Music, dancing, and socializing (Housing Community Center)

TUESDAY MORNING

III: Phage-Based Biotech; Chairs: Dan Nelson & Magda Barbu Purce Lecture Hall 1

8:40 - 9:05 AM Magda Barbu Engineered phage as therapeutics against P. aeruginosa Synthetic Genomics (USA)

9:05 - 9:30 AM Sebastien Lemire Teaching phages to think ahead: a structure-inspired rapid host range modification system MIT (USA)

9:30 - 9:55 AM Stefan Miller Artilysin®: Targeted elimination of bacterial pathogens resulting in faster wound healing Lisando (GER)

9:55 - 10:15 AM Dan Nelson Using bacteriophage endolysins to create custom immunotherapeutics Univ. Maryland (USA)

BREAK

10:30 - 10:55 AM Todd Parsley Development of phage particles for gene therapy of the microbiome SynPhaGen (USA)

10:55 - 11:10 AM Kelly Williams Reconsidering temperate phages for therapy Sandia National Laboratory (USA)

11:10 - 11:25 AM Samuel Kilcher A synthetic platform for rapid and reporter-free engineering of bacteriophage genomes ETH Zurich (CHE)

11:25 - 11:50 AM Poster Previews

1:00 - 3:00 PM

POSTER SESSION

3:00 - 5:00 PM

Group Meetings & Free Time (contact Betty IN ADVANCE to schedule meeting spaces, if desired)

TUESDAY EVENING

IV: Phage-Host Interactions; Chair: Peter Fineran Purce Lecture Hall 1

6:15 - 6:45 PM Rob Lavigne Molecular hijacking of Pseudomonas K.U. Leuven (BEL)

6:45 - 7:05 PM Kimberley Seed Fighting with phages: how epidemic Vibrio cholerae defends against viral attack Univ. California Berkeley (USA)

7:05 - 7:25 PM Joana Azeredo Phages & biofilms a complex interaction: strategies to improve phage efficacy against infectious biofilms Univ. Minho (PRT)

7:25 - 7:45 PM Joe Bondy-Denomy When CRISPR and phage collide: What to do when foe becomes friend? Univ. California San Francisco (USA)

BREAK


TUESDAY EVENING cont’d

8:00 - 8:20 PM Stan Brouns Understanding inter-species gene exchange and compatibility in a bacteriophage model Delft Univ. (NLD)

8:20 - 8:40 PM Edze Westra Evolutionary ecology of CRISPR-Cas Exeter Univ. (GBR)

8:40 - 8:55 PM Geoffrey Hutinet A variety of deazapurine modifications that protect the phage DNA from the restriction system of hosts Univ. Florida (USA)

8:55 - 9:10 PM Denish Piya The multi-component antirestriction system of phage P1 Texas A&M (USA)

WEDNESDAY MORNING

V: Phage Therapy (Part I) Chairs: Susan Lehman & Ryszard Międzybrodzki Purce Lecture Hall 1

8:40 - 9:00 AM Jonas Van Belleghem Pro- and anti-inflammatory responses of PMN cells induced by S. aureus and P. aeruginosa phages Ghent Univ. (BEL)

9:00 - 9:20 AM Michael Rouse Assessing immunomodulation and serum neutralizing antibodies in an A. baumannii mouse model Naval Medical Res. Center (USA)

9:20 - 9:40 AM Lorraine Draper Faecal Virome Transplants (FVTs) reshape the murine microbiota after antibiotic perturbation Univ. College Cork (IRL)

9:40 - 10:00 AM David Speicher Clostridium difficile-associated bacteriophage profiles in FMT patients McMaster Univ. (CAN)

BREAK

10:20 - 10:40 AM Janet Nale Efficacy of an optimised phage cocktail to clear C. difficile in a gut fermentation model Univ. Leicester (GBR)

10:40 - 11:00 AM Jeremy Barr Bacteriophage penetrate the body via epithelial transcytosis Monash Univ. (AUS)

11:00 - 11:20 AM Alyxandra Schubert Murine model of bacteriophage therapy for the decolonization of vancomycin-resistant enterococci FDA (USA)

11:20 - 11:35 AM Vera Morozova Phage application in therapy of local infections: advances and failures Inst. Chem. Biol. Fund. Med. (RUS)

11:35 - 11:55 AM Mikael Skurnik Updates on Yersinia phages and on phage therapy initiative in Finland Univ. Helsinki (FIN)

WEDNESDAY AFTERNOON VI: Phage Therapy (Part II) Chair: Martha Clokie Purce Lecture Hall 1

1:15 - 1:45 PM Ryszard Międzybrodzki Phage therapy: The Polish experience Inst. Immunol. Exp. Ther. (POL)

1:40 - 2:00 PM Sandra Morales Phase 1 studies to evaluate safety, tolerability, and preliminary effectiveness of AB-SA01 AmpliPhi Biosceinces(AUS)

2:00 - 2:15 PM Nina Tikunova Phage treatment of diabetic foot ulcers in Novosibirsk Inst. Chem. Biol. Fund. Med. (RUS)

2:15 - 2:30 PM Randy Fish Working collaboratively to jump-start phage therapy

2:30 - 2:45 PM Alina Negut Clinical case studies in Romania of Eliava Pyophage and Intestiphage Natl. Inst. Infectious Diseases (ROU)

2:45 - 3:00 PM Biswajit Biswas IV application of phage therapy to treat a terminally ill patient infected with MDR A. baumannii. Naval Medical Research Center

(USA)

3:00 - 3:15 PM Randy Kincaid Phage therapy and strategies for treatment of bacterial infections NIAID (USA)

3:30 PM - 5:30 PM

POSTER SESSION, WINE & CHEESE PARTY

6:00 PM - 9:00 PM

Native American Salmon Bake @ The Longhouse (TESC Campus)


THURSDAY MORNING

VII: Molecular Biology; Chair: Debbie Hinton Purce Lecture Hall 1

8:40 - 9:10 AM Bob Blasdel The implications of the dynamic interchange between killing a cell and letting it hang itself for picking the

right phages for therapy K.U. Leuven (BEL)

9:10 - 9:25 AM Debbie Hinton T4 MotA contains a novel DNA binding motif that specifically recognizes modified DNA NIH (USA)

9:25 - 9:45 AM Jennifer West The bacteriophage T4 MotB protein, a DNA binding protein, boosts the level of T4 late gene expression NIH (USA)

9:45 - 10:05 AM Scott Nelson The regulation and mechanism of the bacteriophage T4 MR complex (gp46/47) Iowa State Univ. (USA)

10:05 - 10:20 AM Betty Kutter Hot tales of T4’s transition from host to phage metabolism TESC (USA)

BREAK

10:40 - 10:55 AM Angela Makumi Superinfection exclusion systems as molecular drivers of lactococcal 936 phage evolution Univ. College Cork (IRL)

10:55 - 11:15 AM Nikolai Prokhorov Host recognition by podoviruses G7C and Alt Univ. Texas Medical Branch (USA)

11:15 - 11:30 AM Ines Staes Phages that farm: insights into the phage-carrier state dynamics K.U. Leuven (BEL)

11:30 - 11:45 AM Lanying Zeng Virus interactions inside the cell: competition or cooperation? Texas A&M (USA)

11:45 - 12:00 AM Junjie Zhang Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and

the genomic ssRNA in situ Texas A&M (USA)

1:00 - 3:00 PM

FINAL POSTER SESSION

THURSDAY AFTERNOON

VIII: Open Section; Chair: Paul Hyman Purce Lecture Hall 1

3:00 - 3:20 PM Krystyna Dabrowska Review of factors determining phage penetration in animals and humans, 1940-2016 Inst. Immunol. Exp. Ther. (POL)

3:20 - 3:35 PM Rachel Chang Anti-pseudomonal activity of dry powder PEV20 phage in mouse lung infection Univ. Sydney, (AUS)

3:35 - 3:50 PM Cas Mosterd CRISPR-Cas and phage-host interactions in Streptococcus mutans Laval Univ. (CAN)

3:50 - 4:10 PM Sydney Hayes Mucosal vaccine delivery for peptide antigens via lambda display phage Univ. Saskatchewan (CAN)

4:10 - 4:25 PM Dwayne Roach Immunophage synergy Pasteur Inst. (FRA)

4:25 - 4:40 PM Marcin Los Phage for construction of biosensors Phage Consultants (POL)

4:40 - 4:55 PM Amelia McKitterick Elucidating the specificity determinants of anti-phage genomic island excision in Vibrio cholera Univ. California Berkeley (USA)

4:55 - 5:10 PM Paul Hyman Is more better? Use of multiple hosts in phage isolation Ashland Univ. (USA)

5:10 - 5:20 PM Betty Kutter Final words, looking forward TESC (USA)

THURSDAY EVENING

FREE EVENING -- DINNER IN TOWN

Dancing and Socializing in the Housing Community Center

FRIDAY

8:45 AM: 2 Buses: trip to Mt Rainier (meet in dorm loop)

Timing may be extended by taking private cars.

Ride direct to airport for night flights is possible.

EVENING: Housing Community Center available



ORAL PRESENTATION ABSTRACTS

Abstracts are arranged in the order they appear on the presentation schedule. Sections are

as follows:

Sunday Genomics & Annotation Workshop

Plenary Talks

Section I: Phage Ecology

Section II: Agriculture & Food Safety

Section III: Phage-Based Biotech

Section IV: Phage-Host Interactions

Section V: Phage Therapy (Part I)

Section VI: Phage Therapy (Part II)

Section VII: Molecular Mechanisms

Section VIII: Open Session

The presenting author’s name appears in bold text. The names of other authors who are

attending the meeting have been underlined.

E-mail addresses are provided for the corresponding author, which may or may not be the

same as the presenting author.



Sunday Workshop 9

From Sequence to Knowledge: Assembly, Annotation, and Analysis

of Phage Genomes from Genomic and Metagenomic Data Sets

Alejandro Reyes1, Jason Gill2, Ramy K. Aziz3

1 Department of Biological Sciences, Universidad de los Andes, Bogotá, Colombia.

2 Department of Animal Science, Texas A&M University, College Station, TX

3 Faculty of Pharmacy, Cairo University, Egypt and member of the SEED/PATRIC team

Bios:

● Alejandro Reyes is an Associate professor at the Department of Biological Sciences,

School of Sciences, Universidad de los Andes, Bogotá, Colombia. He is also Adjunct

Assistant Professor at the Department of Pathology and Immunology, Washington

University School of Medicine, St Louis, MO, and the Group leader of the Max Planck

tandem group in Computational Biology, Universidad de los Andes, Bogotá, Colombia.

● Jason Gill is an Assistant Professor in the Department of Animal Science and the Faculty

of Genetics at Texas A&M University, College Station, Texas USA. He is also Associate

Director of the Center for Phage Technology (CPT) at Texas A&M, a state-funded center

devoted to the study of phages for applications in human health and agriculture.

● Ramy Aziz is a Professor and Acting Chair at the Department of Microbiology and

Immunology, Faculty of Pharmacy, Cairo University, Egypt. He is also a member of the

SEED/PATRIC team at the Argonne National Lab (where RAST is hosted) and has

formerly been involved with the PhAnToMe project for phage genome annotation.

Unprecedented advances in genome sequencing improved sequence quality and accuracy,

increased throughput, while decreasing cost and time. This sequencing revolution resulted in tens

of thousands of bacterial genomes that are more or less fully sequenced. Surprisingly, although

phages are an order of magnitude more abundant than bacteria, and although their genomes are

smaller and easier to sequence, fewer phage genomes are in public databases, and consequently

bacteriophage biodiversity is undersampled.

Currently, hundreds of phages are being sequenced from genomes and metagenomes, but

more sequences are needed, and more phage-specific tools are being developed to fill the gap in

phage sequence assembly, interpretation, annotation, and comparative genomics.



10 Sunday Workshop

This year, the “Phage Genomics Workshop” will have a dual focus: (i) Annotation of assembled

phage genome sequences (from isolated phages); (ii) Analysis, Assembly, and Annotation of

phages from environmental or human-associated metagenomes/microbiomes.

Optional hands-on and problem-solving activities will be suggested.

Major toolkits that will be demonstrated in this workshop:

- PhAnToMe (Phage Annotation Tools and Methods) developed by the laboratory of

Robert Edwards at SDSU in collaboration with Mya Breitbart, Jeff Elhai, and Matt

Sullivan (http://www.phantome.org)

- The SEED and RAST toolkits, including the latest development: RAST toolkit (RAST-

tk), developed by an international team lead by the Argonne National Laboratory, IL, USA

(http://www.theseed.org)

- The CPT deployments of Galaxy and WebApollo, which are customized for the analysis

and annotation of whole phage genomes. This system is still under development and

supports customizable workflows to facilitate many kinds of DNA and protein sequence

analysis. (https://cpt.tamu.edu/galaxy-pub/)

- GenSeed-HMM, developed by the laboratory of Arthur Gruber at USP, Brazil, in

collaboration with Alejandro Reyes and Alan Durham. This package is designed for

targeted assembly from metagenomes using specific motifs (represented as HMMs) from

variable proteins.

Links:

Tool or Database URL

RAST http://rast.nmpdr.org

RASTtk To download: https://github.com/TheSEED/RASTtk-Distribution/releases/

For tutorials: http://tutorial.theseed.org

MyRAST http://blog.theseed.org/servers/installation/distribution-of-the-seed-server-

packages.html

Download: http://blog.theseed.org/downloads/myRAST-Intel.dmg

Prokka http://www.vicbioinformatics.com/software.prokka.shtml

phAST http://www.phantome.org/PhageSeed/Phage.cgi?page=phast

BASys http://basys.ca/

GenSAS v3.0 http://gensas.bioinfo.wsu.edu/

IGS Prokaryotic Annotation

Pipeline

http://www.igs.umaryland.edu/research/bioinformatics/analysis/whole_ge

nome.php

MAKER Web Annotation

Service (MWAS)

http://www.yandell-lab.org/software/mwas.html

GenSEED HMM https://sourceforge.net/projects/genseedhmm/

Copies of the workshop presentations and related tutorials can be found online:

http://bit.ly/phigenomics2017; http://bit.ly/phantome2017; http://egybio.net/tutorial/

http://www.theseed.org/

https://cpt.tamu.edu/galaxy-pub/

http://bit.ly/phigenomics2017

http://bit.ly/phantome2017

http://egybio.net/tutorial/



Plenary Talks 11

Phage therapy research in the Queen Astrid military hospital in Brussels

Jean-Paul Pirnay

Queen Astrid Military Hospital, Brussels, Belgium

The worldwide emergence of “superbugs” and a dry pipeline for new antibiotics threaten modern

medicine with a return to the pre antibiotic era. Phages - the viruses of bacteria - could help fight

antimicrobial resistance. In 2003, a first phage therapy related study proposal was submitted to the

R&D department of Belgian Defense. It was dismissed as mere “science fiction” with a score of

4/20. Today, phage therapy research has become commonplace in the Queen Astrid military

hospital and encompasses different aspects:

i) The isolation, selection and characterization of candidate therapeutic phages active against

clinically important pathogens such as Acinetobacter baumannii, which is often associated with

military operations in the Middle East (PMID: 25111143), Pseudomonas aeruginosa,

Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli, including the O104:H4 strain

from the 2011 foodborne EAHEC outbreak in Germany (PMID: 23285164).

ii) Clinical trials:

A small clinical safety study (PMID: 25356373): 10 applications of phage cocktail BFC 1

(PMID: 19300511), active against P. aeruginosa and S. aureus, in burn wound infections.

PhagoBurn (www.phagoburn.eu), funded by the European Commission: Evaluating phage

therapy for the treatment of burn wounds, infected with E. coli and P. aeruginosa, through

a randomized controlled trial (results pending).

iii) Study of the bacterium-phage (host-parasite) relationship, with an emphasis on bacterial phage

resistance evolution and the development of adequate treatment protocols (PMID: 22660719,

PMID: 26476097).

iv) Elaboration of a dedicated regulatory framework for phage therapy (www.P-H-A-G-E.org)

(PMID: 21063753).

v) Development of realistic production and QC/QA regimens for therapeutic phage products

(PMID: 25585954).

In the margin of these studies, and under the umbrella of article 37 (unproven interventions) of

“The Declaration of Helsinki,” eleven patients with multidrug resistant infections were treated with

phages in the Queen Astrid military hospital.

http://www.phagoburn.eu/

http://www.p-h-a-g-e.org/



12 Plenary Talks

Eliava Phage Therapy Center: Current Practice of Phage Therapy in Multiple

Fields of Medicine

N. Hoyle*, L. Nadareishvili, P. Zhvania, L. Pipia, G. Khvichia, I. Tedoradze, N. Odishelidze, N.

Pruidze, D. Nizharadze

Eliava Phage Therapy Center, Tbilisi, Republic of Georgia 0160

*E-mail: [email protected]

Since Felix d’Herelle and George Eliava co-founded it in the 1930’s, what is now the Eliava

Institute has developed and produced therapeutic phage cocktails, building on the broad

explorations the two began at the Pasteur Institute 100 years ago. The current Eliava

Biopreparations LLD makes commercial cocktails such as Pyophage and Intestiphage which are

used throughout Georgia as a first line of defense where appropriate. The Institute also prepares

individual phages from their vast collection when called for by clinical testing. They have always

worked very closely with many Georgian clinics, hospitals and individual physicians and have their

own pharmacy. However, until a few years ago, we did not have our own clinic, specifically

focused on conditions where the use of phage can be particularly beneficial and sometimes using

modern tools to measure key parameters. Indeed, this Phage Therapy Center is now able to

leverage the Eliava Institute’s significant collection of phage as well as its extensive scientific

experience working with phage, collecting crucial clinical data to inform future formal trials.

In our clinical practice, we have gained unique experience in using phage therapy to treat various

infectious complications of genetic diseases, as well as other antibiotic resistant and refractory

infections. As allopathic physicians, we understand the importance of objective examination and

practice according to evidence based medicine. While the need for large scale clinical trials

(RCT’s) is indisputable, phage therapy is complex and diverse enough that choosing the right

conditions for a double-blind approach has proven very tricky in major trials to date, while demand

for an alternative to complement current approaches is ever increasing, as can be seen through our

broad patient and physician contacts. Patients from countries all over the world travel to our clinic

for treatment with phage therapy. This treatment is not considered experimental by the Georgian

Ministry of Health, and phage preparations are registered drugs in Georgia.

Many patients have chronic infections which have been refractory to antibiotic therapy. Infection

control and prevention is a priority for patients with genetic conditions such as cystic fibrosis as

well as conditions such as diabetes that predispose to infections that are very difficult to treat.

Other frequent problems treated at our center include chronic bacterial prostatitis, chronic sinusitis,

lung infections, irritable bowel syndrome, and infected wounds. Pre-operative elimination of

antibiotic resistant bacteria in patient carriers is another useful application. We would like to share

our experiences via a few case reports which represent interesting and important outcomes in phage

therapy. Some involve straightforward use of the standard commercial Eliava cocktails, while

others have required preparation of appropriate phage from Institute collections or fresh isolation of

phage specifically targeting the patient’s bacteria, through the mediation of the Eliava’s other

research laboratories and subsidiary companies.



Plenary Talks 13

Exploring gut associated phage diversity through metaviromics

Alejandro Reyes

Department of Biological Sciences, Max Planck tandem group in Computational Biology,

Universidad de los Andes, Bogotá, Colombia;

Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St

Louis, MO, USA

E-mail: [email protected]

Recent advances studying the microbial ecology of different environments have shown that any

microbial environment is not fully characterized until we understand its viral component. Viruses

are the most abundant and diverse biological entity found in any microbial environment, however,

their function and role on any of those environments is far from being elucidated. Current advances

in DNA sequencing has allowed for the first time, the characterization of the viral component

(virome) associated to environments without requiring culturing the cellular component. However,

to date, our capacity to annotate and understand the functional potential of the virome is limited

due to the minute component of viral diversity fully characterized in public databases and our

restriction to computational tools based on sequence comparison. Recently we surveyed over 140

samples originated from 59 individuals of 22 families, sequencing effort reached an average of

46,000 reads per sample. However, only 30% had significant similarity to public databases queried.

De novo assembly of the reads allowed the usage of 92.3% of the reads in a total of 16,766 contigs,

some of those reaching over 150,000 nucleotides. Even though it is widely known that with longer

reads (or contigs) more chances of identifying a significant similarity in databases, still less than

50% of the contigs showed significant similarity with known sequences. In parallel, looking for

novel strategies for contig annotation that go beyond sequence similarity, we generated clusters of

orthologous phage and viral domains (ViPhOGs), that regardless of a known function associated,

can be used with high sensitivity and specificity as classifiers for different viral taxonomical levels.

Furthermore, coupling the ViPhOGs with the generation of pHMM constitute a tool capable of

identifying and annotating far more viral diversity than previously possible. This methodology,

together with machine learning algorithms such as Random Forest, opens a way to identify

signature viral domains from viral metagenomic studies increasing our capacity to characterize and

annotate that huge challenge of the viral dark matter.



14 Plenary Talks

Phage Therapy and the FDA

Scott Stibitz

US Food and Drug Administration

This presentation is intended to give an overview of the US FDA’s regulatory approach

towards bacteriophage therapy. It will provide a brief description of the FDA’s regulatory

organization, and describe which division is responsible for regulation of products for

Phage Therapy. The presentation will also provide an overview of standard FDA

biologics development pathways with regards to investigational new drugs (IND), and

discuss FDA’s expanded access (compassionate use) programs. Finally, chemistry,

manufacturing, and control (CMC) issues specific to bacteriophage will be covered, along

with FDA’s expectations for characterization of phage for use in clinical trials



Section I: Phage Ecology 15

Global Ecology and Ecosystem Effects of Marine Viruses

Jennifer Brum

Louisiana State University, Baton Rouge, LA


Marine viruses have important roles in microbial mortality, gene transfer, metabolic

reprogramming and biogeochemical cycling. However, methodological limitations have

previously prevented a quantitative assessment of their community structure and ecosystem

impacts. Recent transformative advances have enabled quantitative assessments of environmental

viral communities using metagenomic techniques, facilitating a rapid increase in knowledge of

environmental viral ecology and their impacts on ecosystem function. In this presentation, I will

focus on how we are using advanced metagenomic-based analyses from global-scale datasets to

connect viruses with ecosystem function, including an exploration of the roles of marine viruses in

the oceanic carbon cycle.

.



16 Section I: Phage Ecology

Dramatic differences in cyanophage distribution patterns

across environmental gradients in the oceans Debbie Lindell, Ilia Maidanik, Nava Baran, Michael Carlson, Shay Kirzner, Irena Pekarsky, Sveta

Goldin

Faculty of Biology, Technion – Israel Institute of Technology, Haifa, Israel.

*E-mail:[email protected]

Viruses are globally abundant and extremely diverse in their genetic make-up and in the hosts they

infect. They influence the abundance, diversity and evolution of their hosts as well as the

biogeochemical cycling of matter in the oceans. Yet current methods are inadequate for gaining a

quantitative understanding of their impact on these processes. Here, we employ a new culture-

independent, single phage, solid-phase PCR method, polonies, to gain the first quantitative view of

cyanophages at the phage family level. Dramatic differences in abundances and distribution

patterns were found for different cyanophage families along a transect traversing large

environmental gradients in the North Pacific Ocean as well as over the seasonal cycle in the Red

Sea. In addition, stark differences in the abundances of different lineages (clades) of T7-like

cyanophages were found in both oceanic regions: clade B T7-like cyanophages that encode host-

derived photosynthesis genes and infect either Synechococcus or Prochlorococcus hosts were

considerably more abundant than clade A T7-like cyanophages that lack these photosynthesis

genes and primarily infect Synechococcus hosts. Intriguingly, laboratory studies show that the more

abundant clade B phages have a longer lytic cycle, are less virulent and produce fewer phages per

burst than the clade A phages. These findings highlight that phylogenetic diversity within a single

phage family reflect biologically and ecologically meaningful differences.




Novel “super spreader” bacteriophages promote horizontal gene transfer by

transformation

Eric Keen*, Valery Bliskovsky, Francisco Malagon, James Baker, Jeffrey Prince, James

Klaus, and Sankar Adhya

*Center for Genome Sciences, Washington University in St. Louis, St. Louis, MO 63108


Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry

physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even

though phage-plasmid interactions occur on a massive scale and have potentially significant

evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis

has not been investigated to date. Here we show that a subset of the natural lytic phage population,

which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid

DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel

Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct

plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance

vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic

resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the

addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus

simplex resulted in roughly 1,000-fold more kanamycin-resistant Bacillus simplex than arose in

phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs

to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the

superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the

nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade

chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken

together, our results suggest that phage superspreaders may play key roles in microbial evolution

and ecology but should be avoided in phage therapy and other medical applications.




Towards modelling phage-host interactions in natural ecosystems

Bas Dutilh

Utrecht University, The Netherlands

Phages are diverse and abundant in all ecosystems and influence microbial community dynamics.

As metagenomics is unveiling the composition of microbial ecosystems at an unprecedented rate, a

major remaining question is whether we understand enough of phage-host interactions to model

microbial community dynamics using an eco-systems biology approach. To do this, we should first

accurately link phages to their hosts using metagenomic data, a question that implies several others:

(i) How many hosts can a phage infect, how stable are phage-host interactions in evolution, and at

what taxonomic level can phage-host interactions be predicted the best? (ii) What aspects of the

phage life cycle leave measurable signals in the respective genome sequences, and which genomic

signals are the strongest predictors of phage-host interaction? Next, given that phage-host

interactions can be predicted, what dynamics do we see, and how can they be explained? Based on

the methods we developed to address the questions above, I will discuss a recent example showing

initially counter-intuitive dynamics, that left us with more questions than answers in the quest for a

mechanistic model of phages in microbial ecosystems.




iVirus: a cyberinfrastructure for large-scale computing to unravel virus-host

interactions

Bonnie Hurwitz

University of Arizona, Tuscon, AZ


Currently, a number of tools exist for analyzing viromes and finding viruses in both bacterial

genomes and metagenomes. Yet, most of these tools are available through disparate website or

code repositories that can be challenging to install and/or are not optimized for high performance

computing to meet scale of modern virome and microbiome datasets. To enhance reproducibility

and methods development we are building iVirus, a data repository and optimized high-

performance computing infrastructure for elucidating viral-host interactions. iVirus is built on the

existing CyVerse cyberinfrastructure and provides tools, data and metadata resources specific to

virus ecology. Over the next year, the iVirus project is adding new metadata search capabilities for

users to discover data in the iVirus Data Commons, add these data (and/or their own private data)

to a shopping cart, and launch CyVerse tools (built by the iVirus team or others) directly from the

iVirus site. All analyses will be output to the user’s private CyVerse account, and accessible either

through CyVerse or in the iVirus “sandbox”. The iVirus website will become a central hub for

discovering massive viral ‘omics datasets stored in CyVerse, and running high-performance

computing analyses at CyVerse using XSEDE high-performance computing resources.




Unraveling a role for prophages in shaping gut microbiomes

Brittany A. Leigh1, Zachary Graham2, Assunta Liberti3, Mya Breitbart1* and Larry J. Dishaw3

1. University of South Florida, College of Marine Sciences, 2. St Petersburg College, 3.

University of South Florida, College of Medicine, Department of Pediatrics

*E-mail: mya@usf@edu

Phages play important roles in shaping microbial communities. Phages integrated into bacterial

genomes are predicted to play equally important roles, however, to date very little is known about

how either influence in vivo systems, primarily due to a lack of tractable model systems. Here, we

present evidence that the recently developed model system for gut host-microbial interactions,

Ciona intestinalis, can provide information on the role that phages play in shaping the outcome of

colonization and in influencing homeostasis in gut microbiomes. As a protochordate, Ciona

possesses only an innate immune system, and sequencing has shown that it maintains a core

community of both bacteria and viruses in the gut. In addition, metavirome and 16S rDNA

amplicon sequencing also reveals compartmentalization (stomach, midgut, hindgut) of distinct

communities. We have successfully cultured approximately 80 bacteria that encompass 9 of the 13

core families and tested each isolate for inducible prophages using mitomycin C. At least 1/3 of the

cultured bacteria contain inducible prophages, and the number of lysogens in vivo could be even

higher. The morphology of each of the induced phages was determined using TEM and their

complete genomes were sequenced and annotated. In vitro assays demonstrate an increase in the

formation of biofilms by some lysogens in the presence of a Ciona secretory immune protein, the

Variable region-containing Chitin Binding Protein (VCBP) known to directly interact with

bacteria; this outcome appears to be coupled to the induction of virus-like particles. Each of the

induced prophages was also assessed for lytic activity against all other cultured isolates. We found

that a number of the induced prophages are capable of infecting other members of the Ciona

microbiome, suggesting that induced prophages have the capacity to shape bacterial community

structure within an animal. Furthermore, preliminary in vivo experiments also suggest that

colonization of germ-free Ciona with a single bacterial lysogen results in increased immune

activity and subsequent prophage induction early in the colonization process. Thus, Ciona affords

the unique opportunity to dissect the complex tripartite dynamics involving host, bacterial, and

phage interactions during colonization of mucosal surfaces.




CRISPR-Cas affects horizontal gene transfer by transduction

Bridget N. J. Watson1, Raymond H. J. Staals1,† and Peter C. Fineran1,2*

1Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054,

New Zealand; 2Bio-Protection Research Centre, University of Otago, PO Box 56, Dunedin 9054,

New Zealand; †Present Address: Laboratory of Microbiology, Department of Agrotechnology and

Food Sciences, Wageningen University, 6708 WE Wageningen, The Netherlands.


A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through

transformation, conjugation and transduction. Gene gain can be advantageous, neutral or

detrimental to bacterial fitness. Bacteria and archaea control both HGT and phage infection through

CRISPR-Cas adaptive immunity. Although the beneficial effects from resisting phage infection are

evident, the net result of CRISPR-Cas on HGT is currently enigmatic, due to the lack of detectable

effects over evolutionary timescales. The established ability of CRISPR-Cas to limit HGT through

conjugation and transformation has even been considered an evolutionary downside. However, the

role of CRISPR-Cas in controlling transduction is largely overlooked. Transduction is the phage-

mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT.

Here we addressed the role of the Pectobacterium atrosepticum type I-F CRISPR-Cas system on

horizontal gene transfer via generalised transduction. By assaying the effects of spacers with

different targets, we reach an unexpected conclusion regarding the role of CRISPR-Cas on the

transduction of plasmids and chromosomal loci.




The virome of a South African scalding spring: bacteriophages and archaeal

viruses share the pool

Olivier Zablocki*, Lonnie van Zyl, Bronwyn Kirby, Marla Trindade

Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, South

Africa


Virus communities in terrestrial boiling/acidic hot springs are typically composed of a low

diversity of archaeal virus genera with distinctive morphologies. However, limited virome data are

available from scalding springs (≥50°C - ≤90°C), which may allow for a broader selection of

microbial consortia given intermediate thermal regimes. We hypothesized that scalding

environments may contain a more heterologous virus community, composed of both

bacteriophages and archaeal viruses. In this study, the viral assemblage of the Brandvlei hot spring

(South Africa) was examined through electron microscopy and metagenomics. The majority of

viral morphotypes were tailed. Siphoviridae-like virus particles, jumbo-sized myoviruses and

lemon-shaped virions (Fuselloviridae) were all observed. Using metavirome data, homologous

sequence to the polymerase family B gene (polB2) was used to provide additional support for the

presence of closely related salterprovirus-like sequences in the sample. Taxonomic classification of

predicted virus genes showed a dominance of tailed cyanophages in the spring. A large number of

predicted CRISPR loci with no database homologs hinted at a novel pool of archaeal viruses,

further supported by a large number of archaea-derived genes with archaeal virus-specific functions

but with no reported viruses. The second most abundant predicted phage genome had host

assignment to the Planctomycetes species, Gemmata, which had not previously isolated from

thermal environments, and with no isolated viruses identified to date. This study demonstrated that

a scalding spring environment contained a complex viral diversity, encompassing both archaeal

viruses and bacteriophages, whilst showing evidence for previously unknown viruses associated

with a member of the phylum Planctomycetes.




Phage stratification in monoclonal Chlorobium phaeobacteroides in the

meromictic lake Trekhtsvetnoe

Maria Letarova

INMI, Russian Academy of Sciences


The metabolic activity of prokaryotic life has been one of the dominant forces shaping the

landscapes and chemistry of the surface of our planet since the Biosphere emerged ca. 3 billion

years ago. The bacterial communities in free living ecosystems, such as in water and in soils, are

generally very complex and diverse, with every component comprising only for a small fraction of

total microbial population. Microbial populations that develop at the sites with steep transition of

physic-chemical conditions, specially of red-ox potential (Eh), may feature much higher density

and reduced complexity. The metabolic work of such populations contributes to the large scale

convertion of organic and inorganic substance at these biogeochemical barriers.

In our study we focused on the microbial communities of a sharp biogeochemical barriers

developed in liquid environment of the stratified water column in the meromictic lakes separating

from the White sea. We analysed in substantial detail the matter and energy flow in Trekhcvetnoe

Lake located in Kandalaksha bay near the White Sea Biological Station of MSU. Stratification of

the Trekhcvetnoe Lake as well as of the other lakes separating from the White Sea is imposed by a

sharp salinity gradient. The energy flow in this lake is mainly based on the sulfur cycle. Hydrogen

sulfide flow from the bottom part of the water column is intercepted by the microbial community

of 'bacterial plate' or biofilter layer that is about 30 cm wide and situated at approximately 2 m

depth. This layer is dominated by green sulfur bacteria that use energy of the light to oxidise H2S

and produce organic matter in anoxigenic photosynthesis process. The total microbial cell count in

the thin biofilter layer is 2-3 orders of magnitude higher than in surrounding water layers and

reaches 2x108 cells/ml. Metagenomic analysis revealed that 70-95% of this biomass is represented

by Chlorobium phaeovibrioides. Interestingly genetic diversity of the dominant strain population in

Trekhcvetnoe Lake is extremely low and close to that of the clonal bacterial populations.

Nevertherless the viruse like particles (VLP) observed in biofilter layer water samples taken with

the intervals as small as 2.5 cm are significantly different suggesting the ultra-fine stratification

within this narrow horizon. Taking into account high optical absorbance (OD700 > 0,8), one can

expect that steep light intensity decline over first top 2 cm should impose marked differences in

physiological state of the dominant microbial population. The H2S gradient within the biofilter is

also very steep with the concentration dropping from 6 mM to zero within 30 cm distance. This

ultra-fine stratification is reflected in the composition of associated viral community suggesting

substantial differences in virus effects such as mortality and lateral gene transfer over genetically

similar bacteria within cm distances in the liquid environment. Despite high VLP concentration

associated with high density homogenous host population the biofilter layer is very stable over

many years. This suggests that some mechanisms limiting lytic phage growth should exist in this

habitat.

The comparative study of neighboring meromictic lakes revealed that they also feature the

highly populated microbial layers that differ however by their location in the water column, by the

community composition and stability. So the comparative biogeography of the viruses and bacteria

between this closely located objects may reveal the mechanisms of the virus – bacteria coexistence

in the communities of the biogeochemical barriers.



24 Section II: Agriculture & Food Safety

Phage-host interactions: what have we learned from studying Campylobacter

phages

Martine Holst Sørensen, Y. Emre Gencay and Lone Brøndsted*

Department of Veterinary and Animal Sciences, University of Copenhagen, Stigbøjlen 4, 1870,

Frederiksberg C, Denmark

*E-mail:[email protected]

Campylobacter jejuni remains to be the leading cause of bacterial foodborne illness in the

western world and is seriously affecting child health and mortality in developing countries. C.

jejuni encodes a unique and highly variable surface and while some diversity is due to variation in

genetic content, multiple phase variable genes are found in genetic loci encoding CPS, lipo-

oligosaccharides and O-linked glycosylation of flagella. In C. jejuni phase variable genes encode a

polyG tract that causes slipped strand mispairing during replication, leading to frame shift

mutations, thus turning expression of the gene ON or OFF.

Campylobacter phages are members of the Myoviridae and belong to the two genera of the

Eucampyvirinae subfamily; Cp220virus and Cp8virus. Using our large phage collection, we

showed that Campylobacter phages are either dependent on CPS or motile flagella for infection.

Interestingly, the receptor type dependency correlated with the phage genus; Cp220virus are

dependent on motile flagella, whereas Cp8virus rely on CPS for infection. Analysis of

flagellotropic phage F341 showed a reversible, yet specific binding to the flagellum, and

demonstrated that C. jejuni becomes phage resistant when motility is lost. Overall, our data suggest

that the expression of surface structures influence phage sensitivity and provide the bacterium with

an efficient defense mechanism against phage infection.

While the actual receptor for the flagelloptropic phages is under investigation, further

analysis of the CPS-dependent phage F336 identified the MeOPN modification of GalfNAc present

in the CPS as a phage receptor in strain NCTC11168. This lead to the hypothesis that MeOPN is a

common receptor for all CPS-dependent phages. To test this, we deleted the only MeOPN

transferase gene in strain NCTC12662 sensitive to all our CPS-dependent phages. By HR-MAS

NMR we showed that the mutant is deficient of MeOPN of the CPS and seven phages did not form

any plaques on this strain. On the other hand, 33 phages infected the mutant, although at a lower

efficiency compared to the wild type. Interestingly, the different levels of infectivity of the CPS-

dependent phages neither correlate with the time of isolation nor the origin of phages. To

investigate the phage diversity further and identify receptor-binding proteins all our CPS phages

are currently being sequenced using PacBio. Nevertheless, all our CPS-dependent phages were

affected by the lack of MeOPN, indicating the vast importance of this unique surface modification

for phage infection of C. jejuni.

Resistance to CPS phage F336 develops due to loss of the receptor by phase variation of a

polyG tract in the MeOPN-GalfNAc transferase gene cj1421. However, phage resistance is also

associated with phase variable expression of other CPS genes in vitro and in a chicken model; ON

expression states of cj1422 (attaching MeOPN to heptose) and cj1426 (attaching 6-O-Me to

heptose). Population analyses investigating all phase variable genes in C. jejuni NCTC11168

demonstrated a highly specific phase variable response after phage F336 exposure, only selecting

for specific phase variants of cj1421, cj1422 and cj1426. We hypothesize that the dynamic changes

of these modifications influence the conformation of CPS, hence impacting phage binding

depending on the interaction with the specific receptor binding protein. Finally, multiple phase

variation events led to phage resistance, thus increasing the chance of phage resistant sub-

populations present in a growing culture.



Section II: Agriculture & Food Safety 25

Application of Phages to Control Pierce’s Disease

Mayukh Das, Tushar Suvra Bhowmick and Carlos F. Gonzalez

Department of Plant Pathology & Microbiology,

Center for Phage Technology, Texas A&M University, College Station, Texas

Pierce's Disease (PD), caused by a xylem-blocking, insect-transmitting bacterium Xylella fastidiosa

subsp. fastidiosa, is a major threat to the wine industry in the USA. PD is prevalent from Florida to

California and causes major damage in wine-producing regions. Current approaches for control are

only partially successful and in most cases include the use of chemical pesticides. Phage therapy is

an alternative that could provide a treatment for PD. Phages are the most abundant and ubiquitous

genetic entity on earth. Besides ubiquity, two major properties of phages, specificity and

exponential propagation, make them attractive as antibacterial agents. A phage therapy system

could offer a novel control strategy that also provides high specificity and non-toxicity to animals,

plants or non-target bacteria that may be beneficial to plants against the PD pathogen. Specifically,

a cocktail of phages exhibiting broad host range activity that reflects a diversity of receptors would

maximize the potency of the treatment and minimize the possibility for development of resistance.

A cocktail of four virulent phages (Sano, Salvo, Prado, Paz) were both tested for the therapeutic

and prophylactic efficacy in Vitis vinifera (variety Cabernet Sauvignon) in greenhouse

experiments. Vines were inoculated with bacteria and/or phage(s) and were evaluated for symptom

development for 12 weeks. During the 12-week period, triplicate vines (two cordons each) were

harvested. Plants were segmented and individual segments were assayed using Real Time PCR

(qRTPCR) to quantify Xylella and/or phages. qRTPCR results showed movement of pathogen

and/or phage(s) in vines over a 12-week period. Typical PD symptoms were visible by week 8 in

pathogen inoculated control vines. PD symptoms ceased to progress one week post-therapeutic

treatment and symptoms were not observed in prophylactically treated grapevines. In therapeutic

and prophylactic studies, the PD pathogen declined to almost non-detectable levels as compared to

vines not challenged or pre-treated with phage(s), respectively. Phage(s) were able to replicate in

vines in the presence of the host. Field trials are being conducted in Texas and California to

determine field efficacy. Successful application of phages as biocontrol agents for X. fastidiosa will

offer a novel alternative method to wine industry for the treatment and/or prevention of PD that is

effective, sustainable and environmentally safe.




Bacteriophage diversity in cattle slurry

Pavelas Sazinas1, Tamsin Redgwell2, Branko Rihtman2, Aurelija Grigonyte2 & Andrew Millard1*

1 Warwick Medical School, University of Warwick, Coventry, UK; 2 School of Life Sciences,

University of Warwick, Coventry, UK


There are ~1.8 million dairy cows in the UK, with the dairy farming industry contributing £3.8

billion to the economy. The UK dairy herd produces ~15 billion litres of milk per year and ~30

million tonnes of cattle slurry. This slurry contains a mixture of bacteria, including potential human

pathogens, antibiotics, and co-selective anti-microbials, such as ionic copper and zinc. Current

legalisation requires that farms have the capacity to store five months worth of slurry, meaning that

millions of litres are stored in tanks before being used as fertiliser. The diversity of bacteriophage

within these slurry tanks is poorly characterised. In this study we have begun to characterise the

bacteriophage community, combining genomics of individual isolates along with metagenomics on

the total viral community over a three-year period. To date, greater than 100 bacteriophages

infecting Escherichia coli have been isolated and had their genome sequenced. The most

commonly isolated bacteriophages fall within the genus T4virus. However, discrete populations

can be identified based on the year of isolation. Furthermore, a large number of bacteriophages

have been isolated that fall into poorly represented genera. Exemplified by the isolation of 14

bacteriophages that fall within the genus Seuratvirus, that previously contained only two

representatives.

Analysis of the viral metagenome has allowed a many of what are thought to be complete

bacteriophage genomes to be assembled. These genomes share little similarity with known

bacteriophage isolates, yet are in high abundance within cattle slurry. In common with most viral

metagenomes, the vast majority of the metagenome has limited similarity to known phage isolates.

However, the simultaneous isolation of bacteriophage from the same sample that is used for the

construction of a viral metagenome, has proved fruitful in increasing the fraction of a viral

metagenome that can be assigned to known viruses.

mailto:[email protected]




Phage Peptidoglycan Hydrolase as an Antagonist of Agrobacterium

tumefaciens

Hedieh Attai, Kenya Phillips, Jeanette Rimbey, George Smith, Pamela Brown

Division of Biological Sciences, University of Missouri, Columbia, MO


Bacteriophages can be used as biocontrol agents to protect plants from phytopathogens such as

Agrobacterium tumefaciens. We have thus isolated five lytic bacteriophages from environmental

sources (AP 2, 3, 4, 7, 8) with narrow-host ranges. Preliminary results indicate that coinoculation

of A. tumefaciens with phage limits the effects of Crown Gall disease. In order to better understand

the mechanism of phage-mediated killing, we have sequenced the bacteriophage genomes and have

begun to characterize the phage endolysins, or proteins that cleave the bacterial peptidoglycan cell

wall. The genomes of AP2 and AP3 contain a putative endolysin, Phage Peptidoglycan Hydrolase

(PPH) with an atypical domain structure. PPH contains a predicted peptidoglycan-binding region, a

transmembrane domain and a positively charged C-terminal tail. Thus, PPH is predicted to be a

transmembrane protein with its N-terminus in the periplasm, suggesting that PPH may function

independent of accessory proteins to mediate host cell lysis. Expression of PPH from an inducible

promoter inhibits cell growth in A. tumefaciens. Time-lapse microscopy shows that PPH-

expressing A. tumefaciens exhibit a branching morphology, normally only observed when the

divisome is perturbed. In addition, expression of PPH also inhibits E. coli growth and triggers cell

filamentation. Zymography, an SDS-PAGE with peptidoglycan as a substrate, confirms that PPH

cleaves peptidoglycan. Together, these observations suggest that PPH is capable of gaining access

to the periplasm where it may interact with cell division machinery, cleave the peptidoglycan, and

ultimately lyse the host cell. Further characterization of PPH includes site-directed mutagenesis of

functional residues and purification of His-tagged PPH.




Characterization of Antimicrobial Properties of Salmonella Phage Felix O1

and Listeria phage A511 Embedded in Xanthan Coatings on Poly(lactic acid)

Films

Devon Radford a*, Brandon Guild b, Philip Strange a, Rafath Ahmed a, Loong-Tak Lim b,

Sampathkumar Balamurugan a

aGuelph Research and Development Centre, Agriculture and Agri-Food Canada, 93 Stone Road

West, Guelph, Ontario, N1G 5C9, Canada bDepartment of Food Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada


Beyond simply providing a barrier between food and external contaminants, active packaging

technologies aim to inhibit pathogen survival and growth within the packaged environment.

Bacteriophages have a proven track record as targeted antimicrobials but have yet to be

successfully integrated in active packaging without serious loss of activity. We have developed two

bacteriophage based xanthan coatings on poly(lactic acid) (PLA) film which significantly inhibits

Salmonella Typhimurium and Listeria monocytogenes growth in culture (P < 0.01), and

significantly reduces survival and growth of diverse cocktails of Salmonella sp. and L.

monocytogenes respectively on precooked sliced turkey breast over 30 days of anaerobic packaging

at 4 or 10°C (P < 0.05). Specifically, reductions of 0.832 log at 4°C and 1.30 log at 10°C for

Salmonella sp., and 6.31 log at 4°C and 1.52 log at 10°C for L. monocytogenes were observed. The

coating containing Listeria phage A511 also significantly inhibited growth of L. monocytogenes

over 14 days in aerobic packaging (3.79 log at 4 oC, 2.17 log at 10°C, P < 0.05). These coatings

showed 99.99% phage release within 30 minutes for both phages. Similar approaches could be

used to develop packaging inhibitory to other significant foodborne pathogens such as

Campylobacter, and Escherichia coli, as well as spoilage bacteria.




Effect of the phage cocktail SalmoFree(R) on Salmonella reduction in

commercial broiler's farms

Viviana Clavijo, Alejandro Reyes, Diana Baquero, Alejandra Arevalo, Pilar Donado, Martha

Josefina Vives*

Universidad de los Andes.Bogotá, Colombia.


The World Health Organization considers Salmonella one of the most important zoonotic

foodborne pathogens. Bacteriophages, acting as host-specific parasites of bacterial cells, are today

one of the possible alternatives to antibiotics in animal therapy which can also contribute to food

safety and security. Researchers at Universidad de los Andes developed a phage cocktail against

Salmonella strains, focusing on those of veterinarian and human importance. The product was

named SalmoFree® and its phages have been completely characterized by host range, infection

assays, stability in chlorine, transmission electron microscopy, genome sequencing and a safety

trial in broilers kept in cage batteries. Due to the lack of knowledge in Colombia of phage therapy

and the consequent distrust among poultry farmers, the technology needs to be tested at the

production scale to be considered as viable, safe and effective, especially when there are no prior

reports on use by commercial broiler farms. The present study aimed to evaluate SalmoFree® in a

commercial broiler farm which belongs to an integrated Colombian poultry company. The

objective of the assay was to test the effectiveness of SalmoFree® in controlling Salmonella and

also to assess the relationship between the use of phages and such productivity parameters as feed

conversion, weight gain and homogeneity. We selected four production houses in a farm which had

a record of the presence of Salmonella during two previous production cycles. The selected

production-houses had between 6100 and 13400 broilers each (Ross). A two-phase feeding

program was used in this experiment: a standard commercial starter diet from day 0 to day

21, and a grower diet from day 22 to day 42. Broilers in two of the production houses were

supplemented with SalmoFree® in drinking water at days 18, 27 and 34 and those in the other two

houses were supplemented with a suspension without phages for the same days. Weight and feed

intake were recorded for the four houses. The presence of Salmonella was determined in cloacal

swabs, feces and sampling-shoes samples, following standard protocols. Data of Salmonella

incidence, feed conversion and homogeneity were calculated for all production houses and

statistical analysis was performed to compare these parameters between the houses treated with

SalmoFree® and the control houses. All together, the results represent important information for

the development of phage therapy in Columbia.




Using Free and Immobilized lytic Bacteriophages to Tackle Salmonella

Contamination in Food

Hany Anany1,3, Noha Eldougdoug2, Hajar Hawsawi3, Stevan Cucic3, Vince Leung4, Monsur Ali4,

Carlos Filip4, Mansel Griffiths3

1Agriculture and Agri-Food Canada, Guelph, ON, Canada; 2Microbiology Department, Benha

University, Benha, Egypt; 3Canadian Research Institute for Food Safety, Food Science Department,

University of Guelph, Guelph, ON, Canada; 4Chemical Engineering Department, McMaster

University, Hamilton, ON, Canada

Bacteriophages have been envisioned as a novel and safe tool to control different types of

foodborne pathogenic bacteria. Salmonella is one of the most important foodborne pathogens that

are associated with various outbreaks around the world. Contaminated tomatoes and raw chicken

were linked to some of these outbreaks. Hence, the objective of this work was to isolate and

characterize different lytic bacteriophages against Salmonella Newport and apply these phages to

control the growth of Salmonella Newport and enhance the safety of cherry tomato and raw

chicken breast. Local sewage samples were used for the isolation of Salmonella Newport lytic

phages. The morphology of the isolated phages was determined by TEM and their restriction

digestion pattern was characterized using different restriction enzymes. Their stability, host range,

and ability to control the growth of Salmonella in broth were investigated.

For food application, phages were applied by dipping contaminated cherry tomato in a phage

cocktail solution or coated on the raw chicken breast packaging material (immobilized).

Polysaccharides were used to stabilize immobilized phages on the packaging material. Four lytic

phages, belonging to family Myoviridae (CGG4-1, CGG4-2) and Siphoviridae (CGG3-1, CGG3-

2), were selected from 15 isolated phages based on their broad host range patterns against 26

tested Salmonella serovars. The four phages behaved differently when stored at various

environmental conditions. One phage, CGG 4-1, was sequenced and showed no lysogenic or

virulent coding sequences in its genome. The isolated phages have a latent period of around 50 min

and burst size of around 100. When a cocktail of the isolated phages was used to control the growth

of Salmonella Newport in broth medium, complete inhibition of bacterial growth was observed at

25 and 12°C for 24 h. On the other hand, a 4.5 log CFU/g reduction in the bacterial count was

observed when applying the phage cocktail at MOI of 105 on contaminated tomato fruits stored at

25°C for 3 days. Pullulan and trehalose were able to stabilize dried phage cocktail and maintain its

infectivity on coated food packaging material. When phage cocktail coated on butcher’s paper and

applied on contaminated raw chicken breast samples, 2.1 log CFU/g reduction in Salmonella count

was achieved after 4 days storage at 12°C. These finding support the notion of considering lytic

phages as a biocontrol option for Salmonella spp contamination in different food matrices.

Furthermore, developing stable and infective phage-based bioactive packaging materials could

broaden phage applications as biocontrol agent and might alleviate some concerns of using

spraying/dipping approach to apply phages during food production.




Bacteriophage biocontrol for improving the safety of human and pet foods

Joelle Woolston

Intralytix, Inc., Baltimore, MD


Bacteriophages are naturally part of the normal microflora of many foods, and the 'phage

biocontrol' approach is based on the concept of using the right phage, in the right place, in the right

concentration to eliminate or significantly reduce pathogenic bacteria. Interest in using

bacteriophages to improve food safety has been driven by both the continued occurrence of

foodborne outbreaks worldwide and the desire of consumers for natural foods. The bacteriophage

biocontrol approach has been applied to three main areas of food safety: (i) pre-harvest treatment of

livestock, (ii) decontamination of inanimate surfaces in the processing environment, and (iii) post-

harvest treatment (i.e. direct food applications), the area which has received the most attention.

Bacteriophages can reduce levels of the targeted bacterial pathogen on a variety of foods,

including, but not limited to, dairy products, fruits and vegetables, and poultry. Because of the

specificity of bacteriophages, their application only affects the target bacteria (the pathogen) but

will not affect the other naturally present and potentially beneficial microflora. This presentation

will review the use of bacteriophage biocontrol as a food safety measure, in both human and pet

foods, as well as discuss regulatory and safety issues concerning their use.




Phage Applications for Developing Countries

Tobi Nagel1, Doudou Batumbo2, Didier Bompangue2,3,4, Nicholas Carrigy5, Benjamin Chan6,

Martha Clokie7, Ian Connerton8, Daniel De Vos9, Fitriya Dewi10, Ayman El-Shibiny11, Lasha

Gogokhia12, Diah Iskandriati10, Adamu Ahmad Kaikabo13, Guyguy Kamwiziku2, Erastus

Kang'ethe14, Samuel Kariuki15, Rudovick Kazwala16, Alice Maestri17, Alice Nyambura Maina14,15,

Angela Makumi15, Jesca Nakavuma18, Janet Nale7, George Nasinyama18,19, Julien Ntaongo2, Joko

Pamungkas10, Jean-Paul Pirnay9, Paul Turner6, and Reinhard Vehring5

1Phages for Global Health (USA), 2University of Kinshasa (DR Congo), 3Ministry of Public Health

(DR Congo), 4University of Franche-Comte (France), 5University of Alberta (Canada), 6Yale

University (USA), 7University of Leicester (UK), 8University of Nottingham (UK), 9Queen Astrid

Military Hospital (Belgium), 10Primate Research Center at Bogor Agricultural University

(Indonesia), 11University of Science and Technology (Egypt), 12University of Utah (USA), 13National Veterinary Research Institute (Nigeria), 14University of Nairobi (Kenya), 15Kenya

Medical Research Institute (Kenya), 16Sokoine University of Agriculture (Tanzania), 17University

of Turin (Italy), 18Makerere University (Uganda), 19Kampala International University (Uganda)

Phages for Global Health is a nonprofit organization that facilitates the development of phages for

applications in developing countries. We accomplish this by bringing together global phage experts

and developing world infectious disease specialists, including food safety scientists, veterinarians,

clinicians, regulatory advisors, and stakeholder engagement professionals. With this mix of

specialists, we create international, multidisciplinary teams that have both the technical expertise

necessary to develop phage products and the local scientific and cultural knowledge to deliver

products that will be socially accepted. Our growing consortium of partners includes leaders from

nonprofit organizations, universities and governmental institutions across Africa, Asia, Europe and

North America. We have also created a series of short-term laboratory training programs to teach

the key essentials of phage biology to scientists in developing countries. Current projects include:

(1) Training program (East Africa, West Africa, Indonesia)

We have developed a 2-week, hands-on laboratory workshop through which we can teach scientists

in developing countries how to isolate and characterize phages in their own regions. The first of

these workshops will be held in East Africa during July 2017 (hosted at Makerere University in

Uganda), and plans are underway for subsequent workshops in West Africa (at the National

Veterinary Research Institute in Nigeria) and Indonesia (at the Primate Research Center at Bogor

Agricultural University).

(2) Campylobacter phages for decontamination of retail poultry meat (Kenya, Egypt)

Campylobacter is the leading bacterial cause of gastroenteritis worldwide, and recent data

demonstrate that it is particularly detrimental for children under age 2 in developing countries.

Globally, Kenya has the highest case fatality rate of Campylobacteriosis, with 8.8% of infected

people dying, mostly children. In terms of overall incidence of Campylobacter-associated diarrhea,

Egypt has the highest reported rate of any developing country. To address this public health issue,

we have undertaken to develop phage products to decrease Campylobacter levels on retail poultry.

(3) Cholera phages for water decontamination and prophylactic treatment (DR Congo)

According to the World Health Organization, there are an estimated 1.4 - 4.3 million cholera cases

and up to 142,000 associated deaths worldwide each year. Over the past decade >60% of the

reported cholera cases have occurred in Africa, with the largest percentage of those in the

Democratic Republic of Congo. We are developing phage products to test for both water

decontamination and prophylactic treatment of people during cholera outbreaks.



Section III: Phage-Based Biotech 33

Engineered phage as therapeutics against P. aeruginosa

Yvette Del Rosario, Matt LaFave, Srividya Akella, Toby H. Richardson, Todd C. Peterson, Magda

Barbu*

Synthetic Genomics Inc., La Jolla, California, USA.


The emergence of bacterial clinical isolates resistant to nearly all-current use antibiotics has

renewed interest in the development of lytic bacteriophage (or phage) therapeutic and prophylactic

agents. The advantages of using phage to combat infectious diseases include minimal disruption of

resident flora, lack of cross-resistance with antibiotics, low toxicity and self-limiting dosing.

Despite these attractive features, translational development of natural phage has been hindered

mainly by the difficulty of accessing bacterial hosts within biofilms, the rapid emergence of

resistant bacteria to a single phage, and above all, by the narrow host specificity of phage compared

to antibiotics. The need for customized and complex combinations of natural phage to achieve

adequate host range activity has made their development as licensed therapeutics very difficult.

Genetic engineering of phage genomes can overcome these hurdles; however, broadly applicable

methods for efficient construction of defined mutations in virulent phage genomes are still in their

infancy. Using multidrug-resistant Pseudomonas aeruginosa (MDR PA) as proof-of-principle, we

developed a completely cell-free phage engineering platform that allows rapid and iterative editing

of viral genomic DNA. In parallel, we sequenced and annotated the genomes of several hundred

MDR P. aeruginosa clinical isolates, and subsequently determined the susceptibility of each isolate

to specific phages. Through bioinformatics analysis in combination with our engineering platform,

we have successfully collapsed the host range of a family into a representative phage. We further

engineered wide host range phage to express secondary payloads, such as biofilm degrading

enzymes and antimicrobial moieties, and demonstrated that these phages have improved activity

compared to natural phage in P. aeruginosa models of infection. We predict that our engineering

platform may be adapted for more refined applications, such as construction of viruses with

multiple receptor specificity, which may accelerate both development of phage therapeutics and

functional genomics.



34 Section III: Phage-Based Biotech

Teaching phages to think ahead: a structure-inspired rapid host range

modification system

Sébastien Lemire*, Kevin Yehl, Andrew Yang, Hiroki Ando and Timothy Lu

Research Laboratory of Electronics, Center for Synthetic Biology, Massachusetts Institute of

Technology


Bacterial antibiotic resistance development will be one of the most serious public health threat of

the 21st century. Still, few solutions have been put forth and antibiotic development is largely at a

standstill. Bacteriophages have a tremendous potential in the fight against antibiotic resistant

bacteria; however, naturally sourced bacteriophages often display a limited host range and bacteria

readily evolve resistance against them. While assembling cocktails of bacteriophages that

collectively provide the desired pathogen strain coverage is a convenient ad hoc solution to the

problem, current drug approval schemes make it hard for such cocktails to pass testing and become

routinely available for the treatment of patients.

Inspired by antibody specificity engineering principles, we have created vast libraries of phage

mutants (up to ~107 different mutants) by targeting the regions of the phage T3 tail fiber protein

most likely to direct receptor recognition. This approach maximizes the functional diversity while

minimizing the risk of crippling the protein. We call these mutants phagebodies and screened the

libraries for host-range alteration with a focus on isolating phagebodies capable of infecting

mutants E. coli BL21, the normal T3 host, that are resistant to T3. Despite sampling a very small

fraction of the total phagebody diversity we have created, we isolated phagebodies that were

capable of curbing resistance development in its host on plates and in liquid cultures for an

extended period and some even appeared to completely prevent resistance development, a feat we

could not reproduce through natural evolution of T3. We also demonstrate that the genetic diversity

we generated translates into an equally wide array of different host range phenotypes including

phagebodies that have not simply expanded their host range beyond binding to wild-type E. coli

BL21 but have actually lost the ability to grow on the wild-type bacterium and instead prefer the

T3-resistant mutants.

Our work shows that therapeutic phages can be programmed to brace for resistance and proposes a

pre-emptive design as opposed to deploy reactive coping strategies after resistance becomes an

issue. We also show that expanding host range to target receptor mutants does not necessarily come

at the expense of species selectivity, an advantage of phages over chemical antibiotics.

This is an emerging concept that we hope will attract attention to the possibilities of genetic

engineering and synthetic biology in promoting phage therapy as a viable therapeutic option for

bacterial pathogen control. We anticipate that our phagebody library strategy will help, not only to

create better therapeutic phages, but also explore the link between structure and function in phage-

host interactions.




Artilysin®: Targeted elimination of bacterial pathogens resulting in a faster

wound healing

Stefan Miller*

Lisando GmbH, Am BioPark 13, D-93053 Regensburg, Germany


Chronic wounds are becoming a growing problem for patients, healthcare professionals, and the

health care system. For example, the US health care system this means affecting about 5.7 million

patients and costing an estimated 20 billion dollars annually. Decubitus wounds e.g. are often

infected by multi-resistant strains of Pseudomonas aeruginosa, Staphylococcus aureus or

Acinetobacter baumanii. To accelerate wound healing and protecting the skin microbiome, there is

a clear need for effective antimicrobials, which show a targeted mode of action.

Artilysins display a new, targeted mode of action. Artilysins are recombinant fusion proteins

consisting of an endolysin combined with a targeting peptide that transfers the endolysin through

the outer membrane of Gram-negatives. Art-175 kills P. aeruginosa, including multidrug-resistant

strains, by puncturing the peptidoglycan layer within a minute. Artilysin® Art-175 is highly

effective against highly antibiotic resistant strains of P. aeruginosa and A. baumanii, without

observable resistance development. As Artilysins activity does not require an active bacterial

metabolism, they show a superior bactericidal effect against persisters of P. aeruginosa and A.

baumanii.

Preclinical data underline the broad applicability of Artilysins against bacterial infections being

non-cytotoxic and non-haemolytic. Clinical observation studies with several patients using standard

(homecare) or compassionate use (hospital) application show that on wounds, were conservative

treatments fail, Artilysins application results in removing the persisting bacterial pathogens and a

clearly accelerated wound healing. By removing pathogens, Artilysin facilitates the commensal

bacteria re-colonising skin and wound, thereby preventing pathogens from re-infecting. In

summary, Artilysins are novel antibacterial proteins for targeted elimination of bacterial infections

that favour the natural microbiome and thereby accelerate wound healing.




Using Bacteriophage Endolysins to Create Custom Immunotherapeutics

Daniel C. Nelson*

Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD


In the era of growing antibiotic resistance, life-threatening infections by several bacterial pathogens

are a cause for major concern. While the lytic activity of bacteriophage endolysins offers an

untapped reservoir of potential therapeutics, bacteriolytic therapies are not without concern,

including release of bacterial toxins, lipopolysaccharide, or other inflammatory mediators.

Additionally, as challenges with vaccines continue to rise, non-traditional concepts that engage the

innate and/or adaptive immune responses will be needed to meet these challenges. Toward this end,

we are developing two independent platform technologies that exploit the species-specific affinity

of endolysin cell wall binding domains (CBDs) for the bacterial surface of target organisms. In one

approach, CBDs are fused to specific antitoxin neutralizing monoclonal antibodies to generate

Infection Site Targeted Antitoxin antibodies, or ISTAbs. ISTAbs accumulate at the site of

infection where they are needed most and sequester the toxins at the bacterial surface, thus

immediately neutralizing the effects of the toxins and preventing their release into circulation.

More importantly, since the antitoxin antibodies are humanized, anchoring them to the bacterial

surface via the CBDs functionally opsonizes the bacterium targeting it for opsonophagocytic

killing. In a second approach, CBDs are fused to an antigen against which most people have

existing antibodies (i.e. childhood vaccines such as tetanus or diphtheria toxoid). The CBD acts as

an “ImmunoBridge”, anchoring the antigen to the bacterial surface thereby redirecting pre-existing

immunity against the new invading pathogen resulting in opsonophagocytic clearance of infection.

In this respect, the ImmunoBridge acts as a post-exposure vaccine. Both ISTAbs and

ImmunoBridges possess the potential to have broad application across multiple pathogens based on

an array of available endolysin CBDs.




Development of Phage-derived Particles for Gene Therapy of the Microbiome

Todd B. Parsley*

SynPhaGen, LLC. Gaithersburg, MD USA.


Objective. At SynPhaGen we are developing a platform technology for in vivo transduction of the

microbiome. The platform uses non-lytic, non-bactericidal and replication-deficient bacteriophage-

derived particles (BP) that are engineered to target and deliver their therapeutic genetic cargo to

specific bacteria inhabiting the commensal microflora, transforming the bacterial cell into a local

protein factory. We hypothesize that the close association of the transduced bacteria with host cells

will deliver the therapy to the site of greatest medical need, thus increasing the efficacy of the

therapeutic and decreasing the potential adverse systemic side-effects. The objective of the

presented studies is to demonstrate proof-of-concept using BPs for the in vitro and in vivo

transduction of bacteria and expression of therapeutic sequences.

Methods. BPs were synthesized in an E. coli packaging cell line using a two-plasmid system: one

plasmid encoding an engineered filamentous phage genome and the other a therapeutic plasmid

composed of the gene(s) of interest and a modified receptor binding protein engineered to

selectively bind to and transduce specific strains of E. coli or Pseudomonas aeruginosa. BPs

programmed with a nanoluciferase reporter gene were harvested from packaging cell line culture

supernatants and characterized for selective packaging of the therapeutic plasmid sequences,

examined for transduction specificity, titered in susceptible bacteria in vitro, and subsequently used

in vivo in murine models. BPs programmed to deliver genes encoding functional sRNA gene

sequences, an immunogenic peptide (TARP), or soluble therapeutic protein (rhIL-10), were also

engineered and evaluated in vitro.

Results. Characterization of BPs by PCR revealed selective packaging of the therapeutic plasmid

sequences. In vitro analysis of BPs encoding nanoluciferase demonstrated that the transduction

specificity could be tuned for transduction of specific strains of E. coli or P. aeruginosa by

modification of the N-terminus of the receptor binding protein. This was shown both in vitro as

well as in vivo where BPs could successfully transduce E. coli cells in the gastrointestinal tract of

CD-1 mice. Data on the in vivo transduction of P. aeruginosa in the lungs of Balb/c mice is in

progress and will be presented. Expression of TARP peptide and soluble rhIL-10 was achieved in

E. coli transduced with BPs programmed to deliver the respective genes. Transduction of E. coli

with BPs engineered to deliver genes encoding functional sRNA targeting lacZ resulted

suppression of endogenous ß-galactosidase expression.

Conclusions. As proof of concept, we have engineered BPs that selectively transduced specific

strains of E. coli or Pseudomonas aeruginosa. Selectivity was achieved by modification of the N-

terminus of the BP receptor binding protein. BPs can be programmed to deliver genes encoding

functional sRNA gene sequences, immunogenic peptides (TARP), or soluble therapeutic proteins

(rhIL-10).




Reconsidering temperate phages for therapy

Britney Y. Lau, Raga Krishnakumar, Julian M. Wagner, Corey M. Hudson, Joseph S. Schoeniger,

Kelly P. Williams*

Sandia National Laboratories, Livermore CA 94551


Temperate phages grow lytically in most of the bacterial cells they infect, yet produce a small

fraction of lysogen cells by repressing lytic growth and establishing stable inheritance, usually

through integration into the chromosome. Temperate phages have rightly been shunned for therapy

because the lysogens i) are not killed, ii) are resistant to reinfection by the original phage, iii) may

have gained virulence/resistance/toxin genes enhancing the pathogenicity phenotype (lysogenic

conversion), and iv) can occasionally transduce chromosomal markers flanking the integration site

[1]. However, this guideline had set in before the development of a general PCR-based approach

for multilocus engineering of bacteriophage genomes [2]. Engineering knockout mutations in

integrase or repressor genes essential for lysogeny (and in virulence/resistance/toxin genes if any)

would remove the above objections to temperate phages.

This lysogeny-knockout approach would allow exploitation of the vast number of temperate phages

that are integrated, in prophage form, within sequenced bacterial genomes. Prophages are the main

class (ICEs being the other major class) within the larger category of genomic islands, which can

be defined as mobile DNAs that integrate site-specifically into bacterial (or archaeal) genomes due

to an integrase (of the tyrosine recombinase family, or less frequently, of the serine recombinase

family). We have developed tools that identify and precisely map such genomic islands. Our

experimental method Juxtaposer [3] can detect mobility events due to genomic islands (and

insertion sequences) in subpopulations of a culture, through the circularization and deletion

junctions they produce among next-generation sequencing reads. For higher throughput, we have

developed two bioinformatic methods, and applied them to a large set of sequenced genomes.

Islander is based on the preference of genomic islands for inserting into tRNA genes [4].

Comparator is based on two principles: the cohesion of the integration module, which practically

means that the island’s integrase gene will be found near one end of the integrated island, and a

negative comparative genomic approach, meaning a search for a reference genome in which the

integration site of the candidate island is uninterrupted. These complementary bioinformatic tools

find large numbers of genomic islands, and Comparator can also be configured to find insertion

sequences. In a test set of 2168 genomes we found 7949 islands. We are scaling up the application

of these algorithms to the >80000 prokaryotic genomes at GenBank. This database will allow

selection/production of custom phage cocktails from very close relatives of nearly any target

bacterium, which are more likely to be efficacious at killing the target than phages “fished” from

environmental or clinical samples.

1. Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM. 2011. Bacteriophage 1:66-85.

2. Ando H, Lemire S, Pires DP, Lu TK. 2015. Cell Syst. 2015 1(3):187-196.

3. Schoeniger JS, Hudson CM, Bent ZW, Sinha A, Williams KP. 2016. Nucleic Acids Res 44:6830-6839.

4. Hudson CM, Lau BY, Williams KP. 2015. Nucleic Acids Res 43:D48-D53.

Sandia National Laboratories is a multimission laboratory managed and operated by National Technology

and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for

the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.




A Synthetic Platform for Rapid and Reporter-Free Engineering of

Bacteriophage Genomes

Samuel Kilcher*, Patrick Studer, Christina Muessner, Jochen Klumpp, and Martin J. Loessner

ETH Zurich; Institute of Food, Nutrition and Health


Targeted modification of virulent bacteriophage genomes is frequently achieved using homology-

directed allele replacement. This approach is difficult, time-consuming, and often requires the

incorporation of phage-selectable markers such as fluorescence or luminescence. Therefore, the use

of engineered phages in biotechnology and fundamental research has been limited to phages for

which efficient cloning systems are available. Our objective was to develop a fast, accurate, and

reporter-free method for targeted engineering of a wide range of phage genomes that is broadly

applicable to viruses of Gram-positive bacteria. To this end, we used L-form bacteria as recipients

of synthetic phage genomes: L-forms are cell wall-deficient variants of otherwise walled bacteria

that indefinitely proliferate in osmotically stabilized media. They lack the multi-layered

peptidoglycan envelope, which restricts transformation of Gram-positive bacteria with large DNA

molecules. We show that the Listeria monocytogenes L-form strain Rev2L can be transformed with

full-length bacteriophage DNA, resulting in genome rebooting and production of infectious viruses

from naked DNA. Listeria L-forms did not only reboot Listeria phage genomes, but also supported

reactivation of Bacillus and Staphylococcus phage DNA, effectively bypassing the evolutionary

conserved, strict genus barriers controlling virus infection. Based on these findings, we developed a

unique platform technology for phage engineering that uses Rev2L cells as rebooting

compartments for fully synthetic, in vitro-assembled genomes, allowing rapid, accurate and

reporter-free construction of tailored phage infecting Gram-positive pathogens. We used this

approach to produce strictly lytic bacteriophages by targeted modification of temperate phage

genomes, and demonstrated strongly enhanced killing efficacy of the synthetic viruses. Lytic

activity was further expanded by incorporation of heterogeneous pathogen-specific cell wall-

hydrolases into recombinant phages as genetic payload. Altogether, L-form bacteria as rebooting

tools in synthetic phage biology open unprecedented avenues not only for fundamental research,

but also for therapeutic and biotechnological phage development.



40 Section IV: Phage-Host Interactions

Molecular hijacking of Pseudomonas

Rob Lavigne

Laboratory of Gene Technology - KU Leuven, Leuven, Belgium.

Bacteriophages infecting Pseudomonas aeruginosa mirror the widespread and diverse nature of

their hosts. Tailed Pseudomonas phages are represented in different taxonomic clades and share

little or no sequence similarity between them, with dsDNA genomes ranging between 40kb to over

300kb. Most of this genomic diversity is found within a high number of functionally unknown

open reading frames (ORFans) that are present in hypervariable regions, which are often involved

in adaptation to host-specific conditions. Therefore, these phages are an ideal system to study the

molecular interactions that govern the virus-host interplay.

Our research aims to understand this virus-host interplay, elucidating the role of ORFans which

mediate the metabolic take-over of the bacterial cell. This subversion of host functions is incredibly

diverse, impacting replication, transcription, RNA turnover, (post)-translational modifications and

metabolic pathways. This lecture will focus on different examples which illustrate these hijacking

mechanisms and links them to potential applications in antibacterial design strategies and

biotechnological applications.



Section IV: Page-Host Interactions 41

Fighting with phages: how epidemic Vibrio cholerae defends against viral

attack

Kim Seed

Department of Plant & Microbial Biology, University of California, Berkeley


Vibrio cholerae is a globally important water-borne pathogen that is the causative agent of the

severe acute diarrheal disease cholera. Successful epidemic strains of V. cholerae must defend

against predatory phages in the aquatic environment and within the human intestinal tract. Our

work suggests that a key weapon for phage defense in epidemic V. cholerae are mobile genetic

elements called PLEs (for phage-inducible chromosomal island-like elements). We have identified

and characterized five unique PLEs in V. cholerae. PLE+ isolates have been recovered from

patients between 1949-2011 (spanning the entire collection period for which strains were

available), from different locations including Egypt, Mozambique and Bangladesh. All PLEs,

regardless of temporal or geographic origin, show specific activity towards a prominent phage

called ICP1. Our efforts to understand the molecular mechanisms underpinning PLE activity,

including how specificity is imparted onto PLE-phage interactions and how PLE blocks phage

production, will be discussed.




Phages and biofilms a complex interaction: strategies to improve phage

efficacy against infectious biofilms

Joana Azeredo, Priscila Pires, Luis Melo and Sanna Sillankorva

CEB – Centre of Biological Engineering, LIBRO - Laboratory of Research in Biofilms Rosário

Oliveira, University of Minho, 4710-057 Braga, Portugal

The intricate heterogeneous structure of biofilms confers to bacteria an important survival strategy

in times of adversities even against their natural predators – the phages. In these structures, phages

and cells establish a complex relationship to guarantee the long term survival of the progeny of

both entities. Theoretically, the close proximity of cells within the biofilm structure could enhance

phage-host interaction and facilitate phage infection. Conversely, the biofilm structure and

composition as well as the physiological state of the biofilm cells may be an obstacle to phage

infection. Nonetheless, phages have developed mechanisms to overcome biofilm barriers in a

natural evolutionary prey-predator model. A thorough characterisation of biofilm/phage interaction

and the identification of the weak aspects of biofilms and the strong features of phages are thus

important to develop efficient phage-based biofilm control strategies. In this presentation studies

involving the use of phages for the treatment or prevention of bacterial biofilms will be presented

highlighting the biofilm features that difficult phage infection and the phage characteristics that

enhances biofilm control. Also, some strategies based on combined therapies that can be used to

enhance phage therapy against infectious biofilms will be presented (Figure 1).

Figure 1. Schematic representation of strategies to overcome biofilm barriers using phages and

combined therapies. The biofilm is represented by cells under different metabolic conditions and

susceptibility to phages




When CRISPR and phage collide: What to do when foe becomes friend?

Benjamin J. Rauch, Adair L. Borges, Joseph Bondy-Denomy*

Department of Microbiology & Immunology and Quantitative Biosciences Institute, University of

California San Francisco


Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against

bacteriophage infection. As a counter-measure, numerous phages produce proteins to block the

function of the Class 1 CRISPR-Cas systems, which utilize multi-subunit protein complexes to

enact immunity. To find inhibitors of Class 2 systems (e.g. Cas9), we took a bioinformatics

approach to identify candidate microbes for downstream investigation. We searched bacterial

genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR

inhibition. This “self-targeting” is lethal if the CRISPR-Cas system is functional, as the bacterial

genome is cleaved. This analysis led to the discovery of four unique and pervasive type II-A

CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. Despite the utility

of genomic self-targeting as a discovery tool, in the Pseudomonas aeruginosa Class 1 CRISPR-Cas

system, we find that self-targeting (i.e. the acquisition of a prophage possessing a CRISPR target

and encoding an anti-CRISPR) is not a stable situation. Self-targeting P. aeruginosa strains have

growth defects, which can be suppressed by the deletion of cas genes. Therefore, our understanding

of self-targeting is currently incomplete and we are now investigating how bacteria cope with the

concomitant presence of a CRISPR guide RNA and its target in the same cell.




Understanding inter-species gene exchange and compatibility in a

bacteriophage model

F.L. Nobrega 1, Y. Liu2, N.M. Edner3, S.J.J. Brouns1-2

1Bionanoscience, TU Delft, Delft 2Wageningen UR, Wageningen, The Netherlands 3Division of

Infection and Immunity, University College London, London, United Kingdom

Bacteriophages are highly diverse viral entities that strongly influence the evolution of bacterial

communities. While the study of phages has provided significant knowledge in other fields, phages

themselves remain relatively obscure entities. Many important questions linger regarding phage

modular genome organization, rapid phage evolution, and host-specific interactions. Here we show

a tool to study gene function and essentiality, host specificity and evolution. This technique is

based on bacteriophage genome shuffling for the creation of chimeric phages. By applying this,

from all 3 major families of tailed phages (E. coli phage Myoviridae S2-36s, Siphoviridae S2-55s

and Podoviridae S1-55L) we were able to obtain many different recombinant phages with distinct

features from the parental phages. Most interestingly, we have sequenced and annotated the

genome of one recombinant phage (chimera phiChi3) using Nanopore sequencing, and this

revealed rearrangement of genes from the three parental phages. Sixty-five percent of the

recombinant phage genes derived from S2-36s, and 29 % and 7 % from phages S2-55s and S1-55l

respectively. Although most derived from S2-36s, the recombinant phage surprisingly does not

display the same morphology. Instead, it has a siphovirus morphology like phage S2-55s. This

morphology is mostly derived from the contribution of phage S2-55s to the tail structure of the

recombinant phage, while S2-55s provided capsid genes, and S2-36s donated the host recognition

structures. However, the host range of the recombinant phage is limited when compared to that of

S2-36s, which we discuss to be related to the absence of important receptor binding proteins, as tail

fibers with enzymatic activity. This confirm that phages have high levels of flexibility and

adaptability to accommodate and re-arrange genetic information from other phages, even from

distinct families, posing the hypothesis of exchangeable protein modules complementarity among

microbial populations.




Evolutionary ecology of CRISPR-Cas

Edze R. Westra

Environment and Sustainability Institute, Centre for Ecology and Conservation, University of

Exeter, Biosciences, Penryn, Cornwall, UK.


Bacteria have a range of sophisticated immune mechanisms to protect against virus infections, but

it is unclear why all these different mechanisms evolved in the first place. Under laboratory

conditions, bacteria typically evolve de novo virus resistance using either surface modification or

CRISPR-Cas adaptive immune systems. In this talk I will discuss ecological factors that can tip the

balance in the evolution of these two immune mechanisms and examine their distinct co-

evolutionary implications.

Recent publications: van Houte S, Buckling A, Westra ER. Evolutionary Ecology of Prokaryotic Immune

Mechanisms. 2016 Microbiol Mol Biol Rev. 80(3):745-63.

van Houte S, Ekroth AK, Broniewski JM, Chabas H, Ashby B, Bondy-Denomy J, Gandon S, Boots

M, Paterson S, Buckling A, Westra ER. 2016 The diversity-generating benefits of a prokaryotic

adaptive immune system. Nature 532(7599):385-8.

Westra ER, van Houte S, Oyesiku-Blakemore S, Makin B, Broniewski JM, Best A, Bondy-

Denomy J, Davidson A, Boots M, Buckling A. 2015 Parasite Exposure Drives Selective Evolution

of Constitutive versus Inducible Defense. Curr Biol. 25(8):1043-9.




A variety of deazapurine modifications that protect the phage DNA from the

restriction system of the hosts.

Geoffrey Hutinet1, Witold Kot, Roman Hillebrand, Alexander B. Carstens, Sylvain Moineau,

Graham F. Hatfull, Peter C. Dedon, Lars H. Hansen, Valérie de Crécy-Lagard.

1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA.


Escherichia coli phage 9g is resistant to many restriction enzymes and we recently discovered its

DNA was heavily modified with 2’-deoxyarchaeosine (dG+), a novel complex modification. This

7-deazaguanine-derived base (G+) is normally found in tRNA of Archaea. Both G+ and queuosine

(Q), another 7-deazaguanine derivative found in bacterial and eukaryotic tRNA, are synthesized

from GTP through the 7-cyanodeazaguanine (preQ0) intermediate. Phage 9g genome encodes

homologs of the preQ0 synthesis proteins folE, queD and queE. These are functional when

expressed in trans on multicopy plasmids and complement the Q- phenotype of the respective E.

coli mutants. A homolog of the archaeal Gat-QueC protein, previously shown to be involved in G+

synthesis, is also encoded in phage 9g genome and we propose it is involved in converting preQ0 to

the archaeosine base before or after its insertion in phage DNA. In Archaea, preQ0 is inserted into

tRNA by the tRNA-guanine transglycosylase (aTGT). A TGT-like enzyme is encoded in 9g

genome, and we renamed it DpdA for DeoxyPurine in DNA. Detailed multiple alignment analysis

revealed that all the DpdA proteins identified in phages contain aTGT catalytic and preQ0 binding

residues but lack the predicted Zinc binding residues. In addition, a histidine residue is highly

conserved in all these proteins, at position 196 of 9g DpdA, but is absent from the homologs that

modify tRNAs. Expression of the phage 9g dpdA gene did not complement the Q- of an E. coli tgt

strain, confirming that it has functionally diverged. Phage genomes encoding G+ biosynthesis

pathway proteins can be classified as follow: 1) those encoding both DpdA and other proteins of

the G+ or Q biosynthetic pathways; 2) those who only harbor DpdA; 3) those encoding no DpdA

nor TGT but some G+ biosynthetic pathway proteins. A Mass Spectrometry (MS) analysis of

genomic DNA from a subset of these phages showed that phages from the first group harbored dG+

or other 2’-deoxyribose-7-deazaguanine derivatives. For example, dpreQ0, the deoxynucleotide

form of the preQ0 base, was detected in the Mycobacterium Rosebush phage, whereas the

Streptococcus Dp-1 phage harbored dpreQ1 (the deoxynucleotide form of 7-aminomethyl-7-

deazaguanine or preQ1 base). Interestingly, these phages are also protected from various restriction

enzymes. Heterologous expression in E. coli of the two 9g genes thought to be required for

insertion of G+ in DNA, gat-queC and dpdA, conferred partial resistance to cleavage by EcoRI to

the expression plasmids. MS analysis of the corresponding plasmid DNA showed that it was

modified with 2’-deoxyribose-7-amido-7-deazaguanine (dADG) and not dG+, as the parent 9g

phage. This work extends the discovery of G+ in DNA to other 7-deazaguanine derivatives that

protect phages from a wide variety of restriction enzymes encoded by the hosts.




The multi-component antirestriction system of phage P1

Denish Piya, Leonardo Vara, William K. Russell, Ry Young, Jason J. Gill

Texas A&M University, College Station, TX

*E-mail: [email protected] / [email protected]

Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA

entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that

protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to

reconstitute the previously described restriction phenotypes associated with darA and darB. While

P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and

ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype,

implicating several new proteins of previously unknown function in the P1 dar antirestriction

system. Interestingly, disruption of ddrB decreased P1’s sensitivity to EcoB and EcoK restriction.

Proteomic analysis of purified virions suggests that packaging of antirestriction components into

P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and

concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants

also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf

and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than

previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in

addition to DarA and DarB.

mailto:[email protected]%20/

mailto:[email protected]



48 Section V: Phage Therapy (Part I)

Pro- and anti-inflammatory responses of peripheral blood mononuclear cells

induced by Staphylococcus aureus and Pseudomonas aeruginosa phages

Jonas D Van Belleghem1*, Maia Merabishvili2,1, Frédéric Clement3, Rob Lavigne4 and Mario

Vaneechoutte1.

1Laboratory Bacteriology Research, Department of Chemistry, Microbiology and Immunology,

University Ghent, 9000, Ghent, Belgium, 2Laboratory for Molecular and Cellular Technology

(LabMCT), Queen Astrid Military Hospital, 1120 Brussels, Belgium, 3Centre for Vaccinology,

Ghent University and Hospital, 9000, Ghent, Belgium, 4Division of Gene Technology, Katholieke

Universiteit Leuven, 3001, Leuven, Belgium


The ability of bacteriophages to kill bacteria is well known as well as their potential use to combat

bacterial infections, as an alternative to antibiotics. Bacteria that inhabit the intestine and skin are

generally regarded as stable residents that may confer metabolic and/or immune benefits to their

hosts. The host immune system has evolved mechanisms to tolerate these commensal organisms

while at the same time providing protection for the host from pathogens. Similarly, metagenomic

studies have revealed that a vast variety of bacteriophages are associated with healthy humans. It

has been demonstrated that oral uptake of phages by animals results in the translocation of phages

to systemic tissues. This suggests that mammals have mechanisms for the uptake and delivery of

phages and may allow intestinal phages to elicit innate and adaptive immune responses. We show

that individual phages can induce an immune response.

In order to assess a possible immune response against phages, we conducted a whole transcriptome

analysis of human peripheral blood mononuclear cells (PBMCs) stimulated with either

Pseudomonas aeruginosa or a P. aeruginosa phage PNM lysate. The phage lysate most closely

reflects the immunological state obtained during phage therapy, when the phage titer is the highest

and predominantly bacterial fragments, from the lysed bacterial cells, are present.

Next, to understand to what extent the phage particles interact with the human immune system, the

immune response induced by two different phage purification strategies (i.e. either a phage lysate

or a highly-purified phage) was compared. Using twelve immunity related genes, the immune

response induced by five different phages, representing the three major phage morphologies, were

compared, four infecting P. aeruginosa and one infecting S. aureus. This might give us an idea

whether the immune response might be phage host specific or phage morphology dependent.

We found that for all five phages, the overall immune response, as determined by RT-qPCR is very

comparable and largely in correspondence with the transcriptome analysis: down-regulation of LYZ

and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA.

Moreover, the immune response induced by a large number of phages (i.e. 1011 pfu/ml) was found

to be endotoxin independent, since addition of 106 EU/ml (or 1 EU/PBMC) to the highly-purified

S. aureus phage ISP or P. aeruginosa phage PNM did not subvert the immune response to the one

induced by the corresponding phage lysate.

The influence of phages on e.g. cytokine production, as observed in our study, may not be all that

unexpected, since there are several indications for other interactions of phages with human cells.

For example, it has been shown that phages are able to interact with human mucosal surfaces and

form a non-host derived immune barrier, as well as that they may play a significant role in clinical

transplantation as it was shown in mice that they reduce cellular infiltration of allogenic skin

allografts.



Section V: Phage Therapy (Part I) 49

Assessment of immunomodulation and serum neutralizing antibodies during

bacteriophage therapy in an A. baumannii mouse wound infection model

Michael D. Rouse1*, Anna Jacobs2, Biswajit Biswas3, LT James Regeimbal4, Matthew Henry3,

LCDR Theron Hamilton3, Jessica A. Roman1, Josh Stanbro1, Yonas Alemnah2, MAJ Jonathan

Shearer2, MAJ Samandra Demons2, LTC Stuart D. Tyner2, LT Chaselynn Watters1, CDR Michael

G. Stockelman1, LCDR Mark P. Simons1

1Naval Medical Research Command Silver Spring, MD, 2 Walter Reed Army Institute of Research

Silver Spring, MD, 3 Biological Defense Research Detachment FT Detrick, MD, 4 Naval Medical

Research Unit – 6 Lima, Peru


Objectives: The spread of multidrug antibiotic resistance (MDR) is a widely-recognized crisis in

the treatment of bacterial infections, and ranks in top tier of high operational risk and infectious

disease threats. Recently, the World Health Organization published its first ever list of antibiotic-

resistant "global priority pathogens": a catalogue of 12 genera of bacteria that pose the greatest

threat to human health, with Acinetobacter baumannii listed in the “Priority 1: Critical” category of

pathogens. Lytic bacteriophage cocktails specifically targeted against each of the high priority

bacterial pathogens are promising antimicrobial candidates; however, the possibility of host

immune responses interfering with phage therapy is a concern that must be investigated.

Methods: BALB/c mice were pre-immunized with control (PBS) or bacteriophage cocktail of 5

diverse phages previously shown to lyse A. baumannii strain AB5075. After 3 weeks, mice were

immunosuppressed with cyclophosphamide before wounding (dorsal full thickness injury) and

challenged topically with AB5075-lux. Following infection, mice were treated with PBS or the

phage at 4h post-infection and every 24h for 2 days. Mice were then evaluated for 3 more weeks to

assess the safety and efficacy of the bacteriophage treatment relative to the placebo. We assessed

mortality, bacterial burden, wound closure, systemic and local cytokine profiles, and alterations in

host cellular immunity, including serum neutralizing antibodies.

Results: While pre-immunization with bacteriophage cocktail did not modulate circulating

cytokines, it did lead to a significant increase in total immunoglobulins, specifically Ig2A and

IgG2b compared to control. Furthermore, we found that these immunoglobulins were capable of

specifically binding and neutralizing our phage cocktail from lysing the targeted bacteria. Our data

demonstrate that bacteriophage administration also decreased circulating neutrophils, while

elevating monocytes within 24h. Such effects were observed to last for 2 weeks following the first

phase of pre-immunization; however, no significant changes were observed in immune cell

populations of secondary lymphoid organs, including spleen and lymph nodes. Administration of

bacteriophage treatment effectively prevented wound expansion as assessed by bioluminescence.

Importantly, despite moderate immunological changes associated with pre-immunization, there was

no statistical difference observed in therapeutic efficacy of phage cocktail between phage-

immunized or control-immunized groups.

Conclusion: Our data demonstrate that wound infection outcome does not appear to be impaired

by prior exposure to the therapeutic phage. These findings are an important step in development

of personalized phage therapy that will continue to be studied in other animal infection models.




Faecal Virome Transplants (FVTs) reshape the murine microbiota after

antibiotic perturbation

Lorraine A. Draper1,2, Feargal J. Ryan1,2, Marion Dalmasso1,2,†, Pat G. Casey1,2, Angela

McCann1,2, Vimalkumar Velayudhan 1,2, R. Paul Ross1,3, Colin Hill1,2*

1 APC Microbiome Institute, University College Cork, Cork, Ireland. 2 School of Microbiology, University College Cork, Cork, Ireland

3 Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

†Present address: Normandie Univ, UNICAEN, ABTE, 14000 Caen, France


Background: It has become increasingly apparent that creating and maintaining a complex and

diverse gut microbiome is fundamental to human health. Thus, there are increasing efforts to

identify methods that can modulate and influence the microbiome, especially in those who, due to

disease or circumstance, have had their native microbiome disrupted. Faecal microbial

transplantation (FMT) is one such method and it has been shown to replenish the microbiome of an

individual with that of a healthy donor’s. In which case, the microbiome as a whole is reintroduced

into the recipient i.e. bacterial, archaeal, fungal and viral species. Within this study we determined

if the faecal virome of which bacteriophages predominate could alone transform an individual’s

microbiome via a Faecal Virome Transplant (FVT).

Methods: The current study describes how the administration of virus-like particles (VLPs) in the

form of an FVT influenced and reshaped the murine microbiome of test mice following microbial

perturbation with antibiotics. Subsequent to administration of penicillin and streptomycin, test mice

(n=8) received a bacteria free-VLP enriched faecal transplants, while control mice (n=8) received a

heat-treated nuclease treated version of the same.

Results: Mice that received FVTs, in which bacteriophages predominate, had a bacteriome (as

determined via 16S rRNA sequencing) that separated into distinct groups via principle co-ordinate

analysis (PCoA) and contained differentially abundant taxa when compared to the control group.

Additionally through metagenomic sequencing of Virome DNA, we were able to obtain a snapshot

of the putative viruses present in the gut of test and control mice at various time points. It was

observed that viral content appeared to differ in both abundance and diversity.

Conclusions: This study has validated the role of FVTs in gut microbiome population dynamics.

There is potential to take advantage of such, especially with the increasing trend toward performing

FMTs. Up until now these have focussed on transferring living bacteria and spores; as

bacteriophage are non-living proteinaceous entities they could form a robust, inexpensive

alternative that is susceptible to standardisation and could be delivered as a freeze dried

formulation.




Clostridium difficile-associated Bacteriophage profiles in Faecal Microbiota

Transplant Patients

David Speicher

McMaster University, Hamilton, Ontario, Canada

Clostridium difficile infection (CDI) is a serious hospital-associated infection, and relapsing or

refractory CDI may respond only to faecal microbiota transplantation (FMT). At St. Joseph’s

Healthcare Hamilton, we have performed over 1000 FMTs since 2010. In a recent study

comparing fresh vs frozen faecal samples delivered rectally, FMT was successful in 80% of

patients with 2 to 6 treatments, but 20% of cases never resolve, even after multiple FMTs.

Comparable results have been obtained using lyophilised faecal samples. The rapid clinical

response, usually in 2-5 days but in as little as 24 hours, and the rapid clearance of C. difficile

following FMT suggests that something other than bacterial recolonization is at work, presumably

phages. To elucidate the role of phages in recovery, we are currently comparing whole genome

sequences from C. difficile isolates from diagnostically confirmed CDI and asymptomatic carriers,

and scrutinizing the sequences for prophage DNA. We have also identified several myoviruses

sequences in samples from diagnostically confirmed CDI, albeit at much lower concentrations than

C. difficile. Understanding the role and profiles of phages may be critical to further improving

FMT efficacy and for making it available more widely in future.




Efficacy of an optimised phage cocktail to clear Clostridium difficile in a gut

fermentation model

Janet Y. Nale1, Tamsin T. Redgwell2, Andrew Millard2 and Martha R. J. Clokie1*

1Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, LE1

9HN, UK, 2Division of Biomedical Sciences, Unit of Microbiology and Infection, University of

Warwick, Coventry, CV4 7AL, UK


Clostridium difficile infection (CDI) is a significant cause of infectious diarrhoea and is responsible

for death in 10% of cases, and treatment is limited. Conventional antibiotics are ineffective for all

ribotypes or could trigger dysbiosis and resistance leading to recurrent infection. There is also

significant cost associated with the newest available antibiotic that has limited its use. Thus, there

has been a shift of interest towards exploring novel antimicrobials to either replace or supplement

antibiotics.

We have previously isolated a large bank of bacteriophages and described the activity of an

optimised 4-phage cocktail on C. difficile in vitro using cultures and biofilms, and in vivo using

hamster and wax moth larvae CDI models. Although the data obtained from these models provides

novel insights into the therapeutic applications of the phages, more information is needed to

determine their specificity and efficacy in the presence of human gut microbiome.

Here, we describe the activity of the cocktail using an in vitro human gut fermentation CDI model

with freshly voided fecal samples from 4 healthy volunteers covering diverse ethnicity and age

groups. The bacterial communities present in each sample were assayed on selective media prior to

mixing in equal proportions. The combined fecal slurry was added to a minimal medium and

treated prophylactically or remedially with phages and bacteria over a time course anaerobically.

We observed 6 and 1-log reductions in C. difficile counts in the prophylaxis and remedial regimens

respectively within the first 5h post-infection and complete eradication of bacteria at 24h in both

regimens. C. difficile remained undetected from this time till the experiment was terminated at 72

hour. Commensal Enterococci, Bifidobacteria, Lactobacilli, total Anearobes and

Enterobacteriaceae were not affected by both regimens. However, the phage control showed ~2

logs increase of the total Anaerobes and Enterobactericeae counts compared to the regimens and

the bacterial/untreated controls. Comparable impact of the phage treatments on culturable and

unculturable components of the microbiota was ascertained using metagenomics analysis.

This data supports the prophylactic application of the phages to treat CDI. The elevated levels of

specific commensals in the phage-treated control could be used to prevent colonisation of C.

difficile and provide protection from the infection.




Bacteriophage penetrate the body via epithelial transcytosis

Sophie Nguyen1, Kristi Baker2, Benjamin S. Padman3, Thomas A. Weston1, Kyle Schlosser1,

Barbara Bailey4,5, Michael Lazarou3, Antoni Luque4,5,6, Forest Rohwer1,5, Richard Blumberg2,

Jeremy J. Barr1,5,7*

1 San Diego State University, Department of Biology, San Diego, CA 92182, USA 2 Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital,

Harvard Medical School, Boston, MA 02115, USA 3 Monash University, Department of Biochemistry and Molecular Biology, Biomedicine Discovery

Institute, Melbourne, VIC 3800, Australia 4 San Diego State University, Department of Mathematics & Statistics, San Diego, CA 92182,

USA; 5 Viral Information Institute, San Diego State University, San Diego, CA 92182, USA 6 Computational Science Research Center, San Diego State University, San Diego, CA 92182,

USA; 7 Monash University, School of Biological Sciences, Melbourne, VIC 3800, Australia


Bacterial viruses are amongst the most numerous biological entities within the human body. These

viruses are found within regions of the body that have conventionally been considered sterile,

including the blood, lymph and organs. However, the primary mechanism that bacterial viruses use

to access the body remains unknown. Here we use in vitro studies to demonstrate the rapid and

directional transcytosis of diverse bacteriophages across confluent cell layers originating from the

gut, lung, liver, kidney and brain. Bacteriophage transcytosis across cell layers had a significant

preferential directionality for apical-to-basolateral transport, with approximately 0.1% of total

bacteriophage applied being transcytosed over a two hour period. Microscopy and subcellular

fractionation revealed bacteriophages accessed microsomal and cytosolic compartments of the

eukaryotic cell, passaging via the Golgi apparatus before being exocytosed. We posit that the

human body is continually absorbing phages from the gut and transporting them throughout the

epithelial cell structure and subsequently the body. From our experimental results we estimate that

thirty-one billion bacteriophage particles are transcytosed from the gut into the average human

body each day. The transcytosis of bacteriophage is a natural and ubiquitous process that enables

direct interactions between bacteriophages and the cells, organs, and immune systems of the human

body.




Murine Model of Bacteriophage Therapy for the Decolonization of

Vancomycin-Resistant Enterococci

Alyxandria M. Schubert, and Paul E. Carlson Jr.

Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review,

Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring,

MD


Since the advent of antibiotics in the 1920s, these drugs have saved millions of people from

diseases such as pneumonias, healthcare associated infections, and foodborne illnesses. However,

the continued use of antibiotics has led to several unintended consequences, including disruption of

the indigenous beneficial gut bacteria and a rise in antibiotic-resistant bacteria. The Centers for

Disease Control and Prevention (CDC) estimates that 23,000 deaths are caused by antibiotic-

resistant bacteria each year in the United States, and these organisms constitute a growing problem

worldwide. Vancomycin-resistant enterococci (VRE), which have been classified as a serious

threat by the CDC and World Health Organization, are responsible for 20,000 U.S. infections

annually. The inability to treat these infections with common antibiotics necessitates the

development of alternative methods of intervention.

The objective of this investigation was to develop and characterize an effective bacteriophage

therapy against VRE, including both vancomycin-resistant E. faecalis and E. faecium. To that end,

several naturally occurring phage isolates with activity against a range of VRE strains were isolated

from sewage. In a mice model, various antibiotics were used to disrupt the normal gut microbiota

and allow VRE to colonize and persist. A cocktail of lytic phages with high activity against the E.

faecalis strain used to infect mice. This cocktail and the constituent individual phages were

administered to VRE colonized mice to study their efficacy and the dynamics of phage resistance.

Preliminary data suggests that a phage cocktail could be successfully utilized to decolonize VRE-

infected mice. This treatment will be compared with a standard antibiotic intervention, ampicillin,

for differences in efficacy as well as their effects on the microbiota. We hypothesize that

bacteriophage therapy will have minimal effects on the microbiota compared with antibiotic

treatment. These bacteriophage therapy investigations will have a significant impact on a largely

understudied field and contribute to solving the antibiotic-resistant bacteria problem.

This work was funded by CBER/FDA and an Interagency Agreement with DMID/NIAID

(AAI15021-001-02000).




Phage application in therapy of different local infections: advances and

failures

V.V. Morozova, Yu.N. Kozlova, E.A. Yakovets2, N.V. Tikunova

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia


The traditional delivery method for phage therapy is direct application to the local site of infection.

Here, we describe four successful cases of localized phage therapy and two cases of ineffective

local phage application.

Two cases of local phage application to treat a nasopharyngeal S. aureus infection led to clinical

cure of patients. One more case of successful phage therapy was the treatment of a newborn girl

suffering from bronchititis associated with P. aeruginosa infection. Piobacteriophage and

Sextaphage preparations were applied together by inhalation. Pseudomonas aeruginosa was

eliminated and now the newborn recovered.

The treatment of polymicrobial urinary tract infection was carried out in cooperation with the

Centre of New Medical Technologies (Novosibirsk). A patient (51 years old woman) suffered from

a chronic urinary tract infection after surgical operation to eradicate a cancer. Multiple courses of

antibiotics were applied, and as a result, multidrug resistance strains were acquired. Five clinical

strains were cultured from the urine sample and phages were obtained to kill all the strains. Phages

were applied by urinary catheter and per rectal for seven days. Two strains (E. coli and K.

pneumoniae) were revealed a week after treatment: the K. pneumoniae strain was highly sensitive

to Piobacteriophage and the E. coli was sensitive to amoxiclav. A complex treatment with

piobacteriophage (per os + per rectum) in combination with amoxiclav was carried out for the next

10 days. A week after treatment no microbes were cultured from the urinary samples.

A patient (75 years old man) suffering from otitis showed only partial microbiological cure. Two

strains of P. aeruginosa were cultured from his ear, both sensitive to P. aeruginosa phage.

However, the treatment eliminated only one of the strains. The application of phage treatment after

an infection developed post orthopedic surgery was also unsuccessful. The negative outcome

suggests this direct method of application was inadequate.




Updates on Yersinia phages and on phage therapy initiative in Finland

Mikael Skurnik

Department of Bacteriology and Immunology, Medicum, and Research Programs Unit,

Immunobiology, University of Helsinki, and Helsinki University Central Hospital Laboratory

Diagnostics, Helsinki, Finland


Genus Yersinia belongs to Enterobacteriaeae and includes 17 species of which 4 cause diseases to

animals. Most famous and notorious pathogen is Y. pestis the causative agent of bubonic and

pneumonic plague, while Y. pseudotuberculosis and Y. enterocolitica cause mainly enteric

infections. Y. ruckeri is a fish pathogen causing the red mouth disease. We have isolated Yersinia –

specific phages from various sources and sequenced altogether 34 phages.

I will present new data on a couple of the phages and on application of phages in food biocontrol. I

will also summarize our efforts towards first phage therapy trials in Finland



Section VI: Phage Therapy (Part II) 57

Phage Therapy: the Polish Experience

Ryszard Międzybrodzki1,2,3, Beata Weber-Dąbrowska1,2, Sławomir Letkiewicz1, Wojciech

Fortuna1,2, Paweł Rogóż1, Andrzej Górski1,2,,3*

1Bacteriophage Laboratory, Hirszfeld Institute of Immunology and Experimental Therapy,

Polish Academy of Sciences, Wrocław, Poland 2Phage Therapy Unit, Hirszfeld Institute of Immunology and Experimental Therapy,

Polish Academy of Sciences, Wroclaw, Poland 3Department of Clinical Immunology, Medical University of Warsaw, Warsaw, Poland


Tradition of the therapeutic use of bacteriophages in Poland is almost as long as their discovery by

Twort and d’Hereulle. The first report on their therapeutic use in the treatment of diarrhea dates to

1923. Since that time phage therapy was undergoing continuous evolution from standard through

nonstandard to therapeutic treatment due to increasing standards of current pharmaceutical law and

ethical regulations. Since 1970s Hirszfeld Institute became a main center supplying phages and

supervising the phage therapy in Poland. Reports of its phage group, published between 1980 and

2000, on the clinical effects of the phage therapy of hundreds of patients were the most cited papers

on the clinical phage therapy in the English literature.

At the end of 2005 Hirszfeld Institute opened its own Phage Therapy Unit, so far the only such

center among the European Union countries. Since that time almost 600 patients with different

chronic bacterial infections resistant to antibiotic treatment were qualified for the phage therapy

and treated according to a detailed protocol approved by an independent bioethical committee. This

protocol was subjected to periodical changes including the most recent one applying phage therapy

in children older than 6 years, and the possibility of application of phage preparations by

intravesical instillations in patients with urinary tract infections. Monitoring of the production of

anti-phage antibodies was also introduced. We have possibility to apply in our patients not only

monovalent phage lysate but also cocktails of staphylococcal phages as well as their purified

preparations. Our general results show that a good response to the treatment could be achieved in

approximately 40% of the patients which we consider as very encouraging result for future clinical

trials, since it was achieved in a group of patients with complex clinical problems. We follow the

principle that our primary aim is to treat the patient and not only try to eradicate infection at all

cost. Moreover, our recent studies open new perspectives for phage therapy in immunomodulation.



58 Section VI: Phage Therapy (Part II)

Phase 1 studies to evaluate the safety, tolerability and preliminary

effectiveness of a novel bacteriophage-based treatment, AB-SA01

Sandra Morales

On behalf of the team members at AmpliPhi Biosciences, Queen Elizabeth Hospital, Adelaide, SA,

Australia, Walter Reed Army Institute of Research, Silver Spring, MD, USA


In 2016, AmpliPhi completed two clinical studies assessing the safety, tolerability and preliminary

effectiveness of a novel bacteriophage-based treatment, AB-SA01, when applied to the skin of

healthy volunteer subjects and to the nasal cavities and sinuses of subjects with chronic

rhinosinusitis (CRS) associated with Staphylococcus aureus. AB-SA01 is a therapeutic

bacteriophage mixture of three well-characterised individual S. aureus phages produced under

current good manufacturing practice (cGMP) in AmpliPhi’s own GMP certified facility.

The study on the skin of healthy volunteers was conducted in the USA, in collaboration with

colleagues at the Walter Reed Army Institute of Research, under an Investigational New

Drug Application (IND#16820). The study on patients suffering from CRS associated with S.

aureus, (PJPhage_SA01), was conducted at the Queen Elizabeth Hospital in Adelaide, South

Australia. Overall, AB-SA01 has been shown to be safe and well tolerated in the populations and

dose levels tested in the studies conducted to date. In the CRS study of patients with S. aureus,

reductions in S. aureus counts were observed over the course of treatment. A summary of the

results and practical lessons learnt from these studies will be presented.




Our experience in personalized phage therapy of patients with diabetic

ulcers

Tikunova, N.V.,* Morozova, V.V., Kozlova, Yu.N., Ganichev, D.A., Vlassov, V.V.

Institute of chemical biology and fundamental medicine SB RAS, Novosibirsk, Russia;

630090, Lavrent’ev av. 8, Novosibirsk,


Antibiotic-resistant bacteria become a global threat now. A promising approach to solving the

problem is the use of bacteriophages - viruses that can destruct bacteria, but not able to infect

humans. Bacteriophages do not cause side effects and are not toxic. Unlike antibiotics,

bacteriophages are mostly species- and strain-specific and therefore do not disrupt the normal

human microbiome. In Russia, there are approved phage preparations and cocktails, but most of

them were developed in the last century and their characteristics do not meet recent requirements.

In addition, the main criterion for the application of therapeutic bacteriophage should be sensitivity

of bacterial agent to the bacteriophage. This requires a fast and qualitative microbiological analysis,

rapid selection of the appropriate therapeutic phage or a cocktail, and microbiological control of

treatment. In ICBFM SB RAS, the approach to personalized implementation of phage therapy was

developed and tested. A number of original and unique bacteriophages that are effective against a

broad spectrum of bacteria were isolated and characterized. Several new phage cocktails, including

unique polyspecific cocktail containing bacteriophages against 11 species of pathogenic and

nosocomial bacteria were elaborated. Clinical data on personalized application of phage

preparations for the treatment of diabetic foot ulcers were analyzed. The use of phage therapy in

the clinic provided the healing of most patients, whose diseases were caused by resistant bacteria

and previous antibiotic therapy was unsuccessful.




Working Collaboratively to Jump-start Phage Therapy

E. M. Kutter1,2*, R. Fish2, S. Langevin3 G. Wheat2, B. Blasdel2, D. Bryan1,2, M. Kutateladze4, R.

Adamia4, Sarah Kuhl5

Evergreen State College1 and Phagebiotics Research Foundation2, Olympia, WA; University of

Washington3; Eliava Institute, Tbilisi, Republic of Georgia4;


Phage are increasingly being considered as part of a desperately needed solution to the

growing crisis of antibiotic resistance around the world. Progress toward successful clinical trials

and governmental approval is still frustratingly slow for a variety of reasons. A major public-health

problem lending itself to physician-initiated double-blind Staphylococcus phage trials is the

treatment of diabetic foot infections, where vascular and immune problems limit antibiotic

effectiveness and treatment frequently fails even with non-MRSA Staphylococcus. Infection often

extends into the bone and leads to amputation, even when treatment with antibiotics begins early.

Here we present our data on a set of compassionate-use cases in which the sequenced,

highly purified commercial Eliava Institute Staphylococcus phage Sb1 was added to standard

treatment after the failure of appropriate antibiotics for diabetic toe ulcers. As antibiotic treatment

and good surgical debridement had failed, amputation was the only standard alternative. The fact

that all cases of previously intransigent wounds, most with bone involvement, went on to heal and

none required amputation is a remarkable result for clinicians who know the unfortunate natural

history of such infections. The treatment consists of infusing packing gauze with the

Staphylococcus phage product after routine debridement. Localized injections were also

incorporated for those cases involving osteomyelitis or joint infection. It is remarkably easy to

apply phage treatment in routine practice and such treatment appears to be safe and leads to healing

of intransigent chronic wounds, reducing the rate of amputation. Phage treatment appears to have

the potential to improve the quality of care and decrease the costs of amputation, rehabilitation and

chronic wound management. We are now beginning to use metagenomics to analyze the microbial

composition of such diabetic toe wounds and try to understand why using phage targeting only

Staphylococcus has been so successful.

We propose that S. aureus phage targeting diabetic foot ulcers be the subject of a public-

private-academic initiative, moving quickly toward controlled clinical trials. Much data is already

available on Staphylococcus phage wound treatment, from the 1921 first phage therapy paper, to

Ward MacNeal’s US work in the 1940’s, to the extensive French, Polish and Georgian clinical

work.

Diabetic ulcer treatment occurs largely in civilian and military wound-care centers where

standardized monitoring and well-defined protocols facilitate cross-institutional data collection for

double-blind trials. No other variation from standard of care would be needed. Particular features

of the large, purely lytic Staphylococcus phage K genus are ideal for phage therapy. Many hit over

90% of clinical Staphylococcus isolates and all tested MRSA strains, provoke very little resistance,

and have been widely used both as monophages and in French, Georgian, Russian and Belgian

therapeutic cocktails. Two such phages were included in the first US FDA-approved phase 1

phage trial, in Lubbock, Texas. Broad US safety data comes from the decades-long human use of

Staphylococcus Phage Lysate (SPL), still marketed as a veterinary immune stimulant against

Staphylococcus. We have found SPL to contain as high a phage concentration as commercial phage

cocktails.




Clinical case studies in Romania of Eliava Pyophage and Intestiphage

Alina Cristina Neguț, Oana Săndulescu, Maria Magdalena Motoi, Anca Streinu-Cercel, Adrian

Streinu-Cercel

National Institute for Infectious Diseases Prof. Dr. Matei Balş, Romania


Background: Chronic infections have become of worldwide interest not only due to antibiotic

resistance and biofilm formation but also due to the presence of persister cells that are tolerant to

antimicrobials. New antimicrobials for combating this phenotype are needed. Can bacteriophages

be a solution?

Methods: We have performed an experimental study in the National Institute for Infectious

Diseases. Our group, in collaboration with Prof. Dr. Matei Balș have treated 7 patients with chronic

infections using antibiotics and bacteriophages in combination. The patients included in this study

suffered hard-to-treat infections due to antibiotic resistant bacteria, biofilm formation or hard to

sterilize infection sites and received antibiotics and commercial Georgian phage cocktails:

PyoPhage or IntestiPhage (Eliava BioPreparations, Tbilisi, Georgia). Before initiating therapy, we

evaluated the isolated microorganism for both antibiotic and phage susceptibility.

Results: We have included into the study the following cases: recurrent endocarditis with

Staphylococcus aureus, chronic hip osteomyelitis with S. aureus and P. aeruginosa, periprosthetic

dorsolumbar soft-tissue infection with S. aureus, chronic cutaneous infection with S. simulans, E.

coli and P. aeruginosa, axillary hidradenitis with Proteus mirabilis and S. epidermidis, chronic

osteomyelitis of the thigh-bone with S. aureus and chronic osteomyelitis of the humerus with S.

aureus. The combined therapy proved to be safe and the patients had no adverse reactions or

changes in laboratory safety parameters. A total of 6 patients had negative cultures during therapy

but only one case achieved sustained bacteriological response with clinical cure, the others relapsed

after stopping the therapy. For the patient with recurrent endocarditis the timespan between

relapses was longer when receiving combined therapy compared with antimicrobial treatment

alone. The sustained bacteriological response was achieved for the patient with periprosthetic

dorsolumbar soft-tissue infection with methicillin susceptible S. aureus. The case was considered

hard-to-treat due to a history of multiple drug allergy syndrome, presence of multiple foreign

bodies (metallic rods and screws), with biofilm formation and unsuccessful long-term antibiotic

therapy.

Conclusions: This is the first Romanian pilot study, since 1975, treating patients with combined

therapy antibiotics and bacteriophages. The results confirmed the safety and the importance of

future research in this domain.




Intravenous Application of Phage Therapy to Treat a Terminally Ill Patient

Who Was Infected with Multi Drug Resistant A. baumannii.

Biswajit Biswas1,2*, Robert Schooley4, Matthew Henry1,2, Javier Quinones1,2, Luis Estrella1, David

Wolfe1, Robert K. Pope5, Matthew Doan1, James Regeimbal3, Eric Hall3, Alfred Joseph Mateczun1

and Theron Hamilton1

1 Biological Defense Research Directorate, Naval Medical Research Center – Frederick, 84000

Research Plaza, Fort Detrick, MD; 2 Henry M. Jackson Foundation for the Advancement of

Military Medicine, 6720-A Rockledge Dr, Suite 100, Bethesda, MD; 3 Naval Medical Research

Center, Infectious Diseases Directorate 503 Robert Grant Blvd, Silver Spring, MD 20910; 4

University Of California, San Diego, CA; 5 National Bioforensic Analysis & Countermeasures

Center, Fredrick, MD.


Current global surveillance indicates that multi drug resistant (MDR) bacteria are emerging at an

alarming rate. There is also a significant concern about the potential of generating highly virulent

microorganisms using genetic engineering and synthetic biology. With the growing problem of

rapidly occurring and spreading antimicrobial resistance, alternative treatment must be sought and

developed. Phages or bacterial viruses are the most abundant biomolecules on the surface of the

earth and known to be bacterial killers. Furthermore without any partiality phage will kill both

MDR and non-MDR bacterial pathogens. In this regard a properly formulated phage cocktail

would be highly effective for overcoming antimicrobial resistance (AMR) of any bacteria.

Currently we are developing phage therapy to treat MDR bacterial infections in human. In this

project we invented a rapid process to select combination of broad spectrum phages which can

overcome the emergence of phage resistance bacteria and ultimately enhance the therapeutic

efficacy of the phages. Recently we received a request from University of California, San Diego

(UCSD) to provide cocktail phages which are enabled to kill an MDR A. baumannii clinical isolate

called the TP strain. We immediately engaged to evaluate the killing efficacy of 98 A. baumannii

phages from our library on the TP strain using our proprietary liquid assay system. Our rapid assay

results indicated that the phage library contained 10 very virulent phages which very effectively

killed the TP strain. After further analysis using our proprietary system we selected 4 phages to

prepare cocktail which can overcome the emergence of phage resistance when used of TP strain.

Selected phages were amplified on TP strain and purified using cesium chloride density gradient

technique to prepare phage cocktail. The cocktail was sent to UCSD where physician used the

phage cocktail to treat a terminally ill patient who was infected with MDR A. baumannii. This

phage therapy was done as an FDA-approved, off-use intravenous infusion to treat patient’s

systemic infection. Within 48 hours of intravenous administration of phage cocktail the patient

came out of his coma and within 7 days patient’s temperature and white blood cell count became

normal and his creatinine level improved significantly. Additionally phage therapy also re-

sensitizes the TP strain to several antibiotics to which it was previously acquired resistant. The

detail of this phage therapy study will be presented here.




Phage Therapy and Strategies for Treatment of Bacterial Infections

Randall L. Kincaid

National Institute of Allergy and Infectious Diseases, NIH Rockville, MD 20892


Clinical interventions using bacteriophages have been employed in some parts of the world for

more than 80 years, although such use has not been widely adopted by Western medicine.

Nonetheless, phage therapy is being viewed increasingly as a viable alternative strategy for

treatment of antibiotic-resistant infections, owing to the fundamentally different mechanisms by

which phage kill bacteria. Phages have co-evolved with our commensal bacteria and may also

provide protection from infection through their localization on mucosal surfaces; thus, they can be

viewed as a part of our natural microbiome and are unlikely to pose substantial safety risks to the

patient. Phages represent the most abundant form of “life” and constitute an almost inexhaustible

source of anti-infective options – a virtual pharmacopeia of medically-relevant solutions.

There are several challenges to broad adoption of phage therapy: 1) well-controlled clinical studies

are needed to establish efficacy of phage products, 2) the processes for the manufacture, assay and

formulation of phage preparations require characterization and standardization to ensure products

that are reliable and 3) medical indications for phage therapy that represent clear unmet needs must

be identified. These are not insurmountable issues but they require longer-term strategies to create

products that will meet regulatory expectations and provided clear medical benefit over existing

treatments. These challenges are very similar to those see for all new therapeutic and prophylactic

agents, and require product-specific planning and the design of appropriate clinical studies.

An overview of these planning needs will be presented at the meeting, with emphasis on the role

that the government may play. This includes a discussion of fundamentally different models for

phage therapy (treatment centers vs products), strategies for optimizing the effectiveness of phage

therapy formulations, product standards that might be needed to create repositories of phage and

the specific indications that would be of most value to the infectious disease community. We will

also consider paradigms for prevention of infectious disease, both in medical and community

settings. Lastly, we will consider the prospects for future investment in phage-based solutions,

both from the commercial and from government sectors.



64 Section VII: Molecular Biology

The implications of the dynamic interchange between killing a cell and letting

it hang itself for picking the right phages for therapy.

Bob Blasdel1,Hanne Hendrix1, Jeroen De Smet1, Rob Lavigne1*

1Laboratory of Gene Technology - KU Leuven, Leuven, Belgium


By performing RNA-Seq on the non-ribosomal RNA of synchronously infected Pseudomonas

aeruginosa cells we are able to compare host transcript data for infected cells to an uninfected

control with eight fundamentally different lytic phages. With data for so many distantly related

phages, we were able to highlight the host-mediated transcriptional stress responses to phage

infection by defining the genes that are significantly differentially expressed in common between

all of the different infections. Within this stress response we have discovered a number of

fundamentally new defenses used by P. aeruginosa PAO1 to inhibit phage infection. These include

a quorum sensing based mechanism using the Pseudomonas Quinolone Signal (PQS), has been

previously demonstrated to control an oxidative stress response encouraging metabolic dormancy,

to reduce infection vigor in sister bacteria by daughter phage.

At the same time, having defined the host-mediated transcriptional stress response to phage

infection, we can also define the phage-specific differential expression resulting from phage-

mediated stresses or molecular mechanisms. Indeed, we have found that by leaving the host

transcriptional apparatus intact, each of these phages appears to be able to direct the expression of

the host genome towards phage ends, using the host genome as if it were an auxiliary phage

genome. For example, we have found that PAK_P3 appears to rely on host machinery that it

expresses to manipulate the RNA metabolism of the phage-infected cell, while both YuA and

PAK_P4 impose an iron stress transcriptional response on their hosts. Allowing transcription of the

host genome to proceed during infection likely allows phage-infected cells to adapt to new

metabolic conditions, reduces latent periods, and increases burst sizes as evidenced by the impact

of rifampicin on infections by giant phage that do not require the host RNA polymerase to

transcribe their own genomes but appear to be aided by the transcription of the hosts’.

However, they may also have reduced host ranges due to the host defenses they allow to function,

and could also lead to less predictable results in patients, leading to interesting questions about

whether they may be the best fit for phage therapy. Indeed, like some antibiotics, we have observed

that each of the eight phages we have studied activates the transcription of at least one prophage in

P. aeruginosa PAO1 or PAK. Similarly, we have observed that infection by LUZ19 upregulates the

expression of the hcnABC operon that is responsible for the production of hydrogen cyanide, one

of the primary virulence factors of P. aeruginosa. By choosing phages that inhibit the expression of

the host, as well as other competing genetic elements within the cell, or by manipulating phages to

do so we can hope to gain more control over what happens within the infected cell – reducing the

chance of encouraging horizontal gene transfer or the activation of virulence factors with phage

therapy.



Section VII: Molecular Biology 65

The phage T4 MotA transcription factor contains a novel DNA binding motif

that specifically recognizes modified DNA

Maxime G. Cuypers1, Rosanna M. Robertson1, Leslie Knipling2, M. Brett Waddell1, Deborah M.

Hinton2* and Stephen W. White1

1Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105,

USA 2Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology,

NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA


Bacteriophage T4 temporally controls expression of its genome, giving rise to early,

middle, and late transcripts. T4 uses E. coli RNA polymerase throughout this process, but encodes

factors that change the specificity of the polymerase, switching recognition from early to middle to

late promoters. Like all bacterial polymerases, E. coli RNA polymerase is composed of a core that

synthesizes RNA (beta, beta’, 2 alphas, and omega) plus a sigma specificity subunit that recognizes

the promoter sequence. While recognition of T4 early promoters does not require additional

factors, activation of middle promoters requires the T4 co-activator AsiA and the T4 activator

MotA in a process called sigma appropriation.

AsiA remodels the C-terminal region of sigma, MotANTD interacts with sigma C-terminal

residues, and MotACTD binds to the middle promoter motif, the MotA box,

5’(a/t)(a/t)(a/t)TGCTTtA3’ centered at -30. Using structures (RNA polymerase, MotACTD,

MotANTD, and AsiA/sigma) and cleavage analyses obtained with FeBABE–conjugated MotA, we

generated a structure-based model for sigma appropriation, which indicates the positions of the

proteins relative to each other and to the DNA within the competent transcription complex (James

et al. NAR 2016 44, 7974). We have now determined the crystal structure of the MotAlinker-CTD with

MotA box DNA. The structure reveals a new type of protein/DNA interaction, with the saddle-

like, double-wing motif of MotACTD lying within the major groove and the arginine-rich MotAlinker

lying within the upstream minor groove. MotACTD binds DNA primarily through non-specific

interactions with the DNA backbone although there are two specific interactions with the MotA

box in the major groove. The MotAlinker plays a key role in stabilizing the complex via minor

groove interactions. The structure is remarkably similar to that of our transcription model,

justifying the use of FeBABE analyses to map protein/DNA interactions at a high resolution.

Furthermore, the structure predicted that the hydroxymethyl, glucosylation modification present on

cytosines within T4 DNA might provide an important MotA/DNA interaction. Using EMSAs and

SPR, we find that this modification on the nontemplate strand substantially improves binding,

primarily by significantly decreasing the off rate.

Although AsiA and MotA analogs have been identified in dozens of T4-like phages, a

process similar to sigma appropriation has not been observed outside phage. However, the novel

double-wing structure has been reported for two proteins of unknown function, the conserved E.

coli YjbR and the Pseudomonas syringae protein Pspto_3016. Thus, understanding how MotA

interacts with its partner DNA may yield insights into the functions of these proteins and other

unidentified members of the family. Another protein that contains the double wing motif is the T4

SF2 helicase UvsW, which has a key role in T4 recombination and is functionally and structurally

related to the eukaryotic Rad54 protein. The MotAlinker/CTD/DNA complex suggests a role for the

UvsW double wing structure. We speculate that the largely non-specific interactions allow both

MotA and UvsW to search for their motifs (the MotA box motif and 3- and 4-way junction DNA,

respectively) by ‘riding along’ the DNA via the saddle-like surface of the double-wing domain.




The bacteriophage T4 MotB protein, a DNA binding protein, boosts the level

of T4 late gene expression

Jennifer Patterson West1, L. Iyer2, and Deborah M. Hinton1

1Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK,

National Institutes of Health, Bethesda, Maryland 20892, USA, 2Computational Biology Branch,

National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20892, USA

E-mail: [email protected]; [email protected]

Condensation and organization of genomic DNA is crucial in all cells for orderly replication and

gene expression. In bacteria, histone-like proteins such as the abundant H-NS and its less abundant

homolog StpA, are DNA binding proteins that form higher-order nucleoprotein complexes needed

for DNA condensation. H-NS targets AT-rich DNA sequences and condenses genomic DNA

through a proposed looping mechanism that typically represses transcription at the affected region.

As phage genomes and xenogeneic sequences acquired from horizontal gene transfer often display

a high AT content, H-NS can protect bacteria from the expression of foreign genes by

preferentially binding these sequences.

Bacteriophage T4 (65.5% AT) is a lytic virus that infects E. coli, (45% AT) resulting in cell lysis

after ~20 min. The T4 genome is expressed temporally from early, middle and late promoters. The

T4 late promoter, TATAAATA, is strikingly similar to the H-NS binding motif, TCGATAAATT.

Thus, it is not surprising that T4 may need to overcome H-NS repression. T4 Arn, a protein that

structurally mimics DNA has been shown to bind H-NS, preventing its interaction with DNA and

formation of higher order structures. However, Arn is not essential.

The T4 motB gene encodes a highly conserved T4 early protein whose function has not been

characterized previously. We find that expression of plasmid-borne motB is highly toxic to E. coli,

resulting in decondensation of host DNA, cell lengthening, significant reduction in actively

dividing cells compared to a vector control, and cell lysis. MotB co-purifies with DNA, H-NS, and

StpA. Electrophoresis mobility shift assays indicate that H-NS and MotB bind similar AT-rich

sequences. However, MotB binds with 10-100-fold higher affinity than H-NS depending on the

sequence. Although a T4 motB amber mutant has no noticeable phenotype in a plaque assay, the

mutant has a 2-fold reduction in burst size and RNA-seq analyses indicate that the expression of

several T4 late genes are significantly reduced. Expression of early and middle transcripts are not

reduced. These findings are consistent with MotB relieving H-NS binding to and repression of late

promoters and/or a direct activation of late gene expression by MotB. We hypothesize that the

interaction of MotB with either H-NS (and StpA), DNA, or both is part of a mechanism used by T4

to disrupt H-NS dependent repression leading to optimal expression of its late genes. Determining

why and how MotB is toxic can provide paths to the formation of anti-bacterials that work by a

mechanism not previously targeted by antibiotics.




The regulation and mechanism of the bacteriophage T4 MR complex

(gp46/47)

Scott Nelson

Iowa State University, Ames, IA


Bacteriophage T4 gp46 (Rad50 homolog) and gp47 (Mre11 homolog) are central players in DNA

repair and are implicated in the processing of DNA ends caused by DNA double strand breaks

(DSBs). Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing

DSBs. Due to difficulties in the expression and purification of the full-length Rad50 protein, a

detailed mechanistic understanding of the MR complex has been lacking. We have established a

tractable system for the biochemical characterization of T4 Mre11 and Rad50. Rad50 enhances the

nuclease activity of Mre11, while DNA and Mre11 in combination stimulate the ATPase activity of

Rad50. The structural basis for the cross activation of MR complex has been elusive. Recent

crystal structures of truncated forms MR complex suggest that several conformational

rearrangements may occur during its ATP hydrolysis and nuclease cycle. These conformational

changes likely transmit signals between active sites and appear to affect the dimer interface of

Mre11 and the coiled-coil domain of Rad50 to the greatest extent. We have used a combination of

biochemical and biophysical experiments to explore the connections between these large-scale

movements and the various activities of the MR complex. These results have been incorporated

into a comprehensive model that describes the entire catalytic cycle of the MR complex during

processive nuclease activity.




Hot Tales of T4’s Transition from Host to Phage Metabolism

Elizabeth Kutter



Much of what we know about phage-host interactions was originally discovered by introducing

radioactively labeled molecules into T4-infected E. coli. While biologists today have access to

tools like RNAseq, the old standby of radiolabeling is still uniquely useful in exploring some

details of the process.

Shutoff of host protein synthesis: 14C-labeled mixed amino acids were used to show that

synthesis of all host proteins ceases within 1-3 min after infection, while 2-D gels showed the

detailed pattern of synthesis of each protein. The very extensive and well-studied collection of T4

amber and deletion mutants facilitated extensive identification of many of the genes.

Effects on host lipid synthesis: Measurements involving 14C – acetate incorporation showed that

T4 quite surprisingly stimulates phosphatidyl glycerol (PG) synthesis, while phosphatidyl ethanol

(PE) continues to be made at the pre-infection rate. Both the stimulation of PG and the ongoing

synthesis of PE and PG are under specific control of genes lying in the deletable region between

rIIB and gp52, which encodes one component of the topoisomerase; thus, it is probably not simply

a passive coincidence for the phage.

Host DNA breakdown and non-canonical nucleotide use explored using 3HdT: For many

years, our year-long, full-time “Molecule to Organism” program as well as the phage lab used

radiolabeled dT to explore consequences of the phage infection process through looking at phage

DNA synthesis and host DNA breakdown, in a very safe and highly reproducible manner teaching

many aspects of phage biology and learning about the principles and practice of working with

radionucleotides.

A “hibernation” mode of T4 infection of stationary-phase E. coli: When T4 infects cells that

have been growing at least 24-48 hours, it generally kills the host it is attacking quite efficiently,

but produces no progeny phage until after nutrients are added. We have determined that the host

DNA is largely broken down to mononucleotides over just a few hours and the nucleotides are

effectively re-incorporated into phage DNA, indicating that the infection proceeds at least through

production of the phage’s “new” phage nucleotide complex, but no new phage are made until

nutrient addition at 24 or 48 hours – then rapidly producing and releasing nearly 1011 phage per ml.




Superinfection exclusion systems as molecular drivers of lactococcal 936

phage evolution Jennifer Mahony1, 3, Angela Makumi1, Irma van Rijswick1, Christian Cambillau2 and Douwe van

Sinderen1, 3.

1School of Microbiology, University College Cork, Western Road, Cork; 2 Architecture et Fonction

des Macromolécules Biologiques, UMR 7257 CNRS & Aix-Marseille University, Campus de

Luminy, Case 932, 13288 Marseille Cedex 09, France; 3Alimentary Pharmabiotic Centre,

Biosciences Institute, University College Cork, Western Road, Cork, Ireland.

Bacteriophages have an overpowering abundance in the biosphere having a profound impact on the

conduct and ecology of their hosts. The two best described and understood routes in phage

propagation includes the (i) lytic cycle, which can affect the structure of microbial corporation as

well as the host mutation rates and (ii) the lysogenic cycle in which the phage genome is integrated

into the host chromosome (prophage). Such prophage integration leads to an expanded genome

providing a further potential increase in host adaptive abilities and fitness. Furthermore, bacterial

hosts may benefit from the protection conferred by prophage-encoded immunity and/or phage-

resistance against homologous and heterologous phages. One such group of phage-resistance

systems, termed superinfection exclusion (Sie) systems, provide protection against certain

members of heterologous phages. In this study, we have focused on the characterization of Sie

systems derived from dairy lactococcal strains with potential implications for the dairy

fermentation industry. Among the ten known groups of lactococcal phages, members of the so-

called 936 phage species are the most frequently encountered and are among the most problematic

to such fermentations.

Here, we present data that demonstrates that these Sie systems are powerful evolutionary drivers of

members of the 936 phage species. Mutants of representative members of the 936 phage species

capable of bypassing the Sie system exhibited an extensive set of mutations in the gene that

specifies the tail tape measure protein (TMP), highlighting not only the role of TMP in evading the

action of these Sie systems, but also its possible involvement in the DNA injection process of 936

phages. To scrutinize this further, we also present data at single cell level bringing such elusive

insights within reach, shedding light on the regulation and modus operandus involved in the Sie

system.




Host recognition by podoviruses G7C and Alt

Nikolai S. Prokhorov1,2,3, Sergey Nazarov2, Cristian Riccio2, Ricardo Guerrero-Ferreira2, Alla K.

Golomidova3, Evelina L. Zdorovenko4, Yuriy A. Knirel4,

Andrey V. Letarov3 & Petr G. Leiman1,2

1Department of Biochemistry & Molecular Biology,

University of Texas Medical Branch, Galveston, Texas 2École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

3Winogradsky Institute of Microbiology,

Research Center of Biotechnology, RAS, Moscow, Russia 4Zelinsky Institute of Organic Chemistry, RAS, Moscow, Russia

The O22 E. coli strain 4s populates the lower intestine of a horse. It is a host to a number of

bacteriophages that compete and, possibly, help each other to survive in this complex and dense

microbiological environment. N4-like phages G7C and Alt are close relatives with essentially

identical genomes, except for a single gene the product of which is responsible for host recognition

and forms a tailspike on the phage particle. We used a combination of X-ray crystallography and

cryo-electron microscopy to determine the structure of G7C tail in atomic details. It carries twelve

copies of two different tailspikes that form a branched structure. The longer tailspike - gp66 (1063

aa) - contains a phage binding domain, a flexible linker, a branched domain responsible for the

attachment of the other spike, and a C-terminal ß-helical receptor-binding domain. This protein is

identical in both phages. The shorter spikes of G7C and Alt - gp63.1 (851 aa) and gpA63.1 (854 aa)

- have nearly identical N-terminal domains that interact with gp66 and strikingly different C-

terminal receptor-binding domains. The short spike of Alt is a lyase. It degrades the E. coli 4s O-

antigen into small fragments. The short spike of G7C acts differently. It keeps the backbone of the

E. coli 4s O-antigen polymer intact but removes one O-acetyl group per its repeating unit. Thus,

G7C and Alt utilize non-homologous domains and different types of enzymatic activities to

recognize and bind to the same cellular receptor molecule. Functional analysis of host recognition

by G7C, Alt, and their tailspike mutants revealed that O-antigen binding and modification are

critically important for productive infection. We found that the kinetics of O-antigen processing by

the tailspikes appears to control the transition from primary host recognition to irreversible

adsorption and subsequent DNA release. Our findings demonstrate that phages with identical

particle structure and tail architecture can utilize strikingly different biochemical processes to

efficiently recognize, bind, and infect their host cells.

Prokhorov, N.S., C. Riccio, E.L. Zdorovenko, M.M. Shneider, C. Browning, Y.A. Knirel, P.G.

Leiman & A.V. Letarov, (2017) Function of bacteriophage G7C esterase tailspike in host cell

adsorption. Mol Microbiol. doi: 10.1111/mmi.13710.




Phages that farm: insights into the phage-carrier state dynamics

Ines Staes1, William Cenens1, Nick de la Croix1, Angela Makumi1, Sanne Wolput1, Rob Lavigne2,

Abram Aertsen1

1Laboratory of Food Microbiology, Department of Microbial and Molecular Systems (M2S),

Faculty of Bioscience Engineering, KU Leuven, Belgium, 2Laboratory of Gene Technology,

Department of Biosystems, Faculty of Bioscience Engineering, KU Leuven, Belgium


Bacteria are condemned to an eternal battle with their viral predators, bacteriophages, as the latter

can only multiply and spread their progeny at the cost of their bacterial host. Phages with a strict

lytic lifestyle inexorably kill the infected cell, consequently leading to a drastic reduction of

available hosts. A safer approach is opted by temperate phages that insert their DNA into the

bacterial chromosome and replicate along with it, although this protection comes at the expense of

an impairment in spread of progeny. A possible solution by which a phage can circumvent this

dilemma, is the formation of a phage carrier state in which a delay of the integration event allows

the growth of a phage free subpopulation that is transient resistant to superinfection. This strategy

might contribute to a stable phage-host co-existence in nature as it allows these farming phages to

foster a reservoir of bacterial cells that afterwards can be “harvested”. In the temperate phage P22 –

Salmonella Typhimurium model system, we previously observed that phage carrier cells

overproduce the P22 pid ORFan gene, which leads to derepression of the host’s dgo-operon. In the

search for phage-encoded factors involved in pid-regulation, expression or function, a screening

was performed using a library of DES-mutagenized P22-phages. This yielded interesting

mutations in the secondary immunity region of P22 that provide further insight into its infection

dynamics.




Virus Interactions inside the Cell: Competition or Cooperation?

Lanying Zeng

Department of Biochemistry and Biophysics, Center for Phage Technology, Texas A&M

University, 300 Olsen Blvd, College Station, TX 77843


Living cells make fate-determining decisions based on signals from their environment.

Understanding the decision making process is essential for unveiling the mysteries of life and for

improving human health. I will discuss our recent study of a paradigmatic system of cell-fate

determination, the bacterium E. coli and its virus – phage lambda, using a live-cell 4-color system

combined with computational modeling at the single-virus/single-virus-DNA resolution. The

textbook picture is: upon infection by phage lambda, E. coli undergoes one of two alternate

pathways - lytic (violent) or lysogenic (dormant). With the high-resolution studies, this

paradigmatic system has been revealed to be more complicated than previously thought. Our study

suggests that individual phages vote and interact within the cell: they cooperate during

lysognization, compete among each other during lysis, and confusion or coexistence between the

two pathways occasionally occurs. I will also discuss the sources/mechanisms for phages to make

different decisions and utilize variable strategies for their development.




Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a

single maturation protein and the genomic ssRNA in situ

Junjie Zhang

Center for Phage Technology, Department of Biochemistry and Biophysics, Texas A&M

University, College Station, TX 77843

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only

the structures of the capsids and their associated protein components have been resolved to high

resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice

of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature

virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus.

Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without

symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously

seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals

A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a

twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity,

which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA

release during infection. We observe a fixed pattern of gRNA organization among all viral

particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA

directly contacts every copy of the coat protein, with one-third of the interactions occurring at

operator-like RNA hairpins. These RNA–coat interactions stabilize the tertiary structure of gRNA

within the virion, which could further provide a roadmap for capsid assembly.



74 Section VIII: Open Section

What factors determine phage penetration in animals and humans? Systematic

and critical review of reports published in the years 1940-2016

Krystyna Dąbrowska

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of

Sciences, R. Weigla 12, 53-114 Wrocław, Poland


The efficacy of medical applications of bacteriophages, including antibacterial therapy,

vaccines and other types of nanocarriers rely on phage ability to reach target organs or tissues. This

ability of phages to “travel” inside human or animal body is variable. Reports from multiple human

and animal studies present differentiated or even contradictory conclusions on how a phage may

penetrate in vivo. Many factors that may potentially determine phage penetration, e.g. the route of

administration, phage type including phage morphology and host range, dose and application

schedule, characteristics of treated individuals (species, age), presence or lack of sensitive bacteria,

and many others. Mostly due to the multiplicity of these factors, their true impact on phage

penetration is still unclear.

Here I present a systematic review of phage-related literature available in PubMed (up to

Dec 31st 2016) focusing on in vivo experiments that involved phage penetration into blood or

tissues, and gut transit of phage. Systematic review was further developed by a critical review of

related mechanisms. Criteria for the systematic search were: ((phage) OR bacteriophage) AND

((per os) OR (gut) OR (intestine) OR (fecal recovery) OR (route of administration) OR (oral) OR

(inhalation) OR (intranasal) OR (intraperitoneal) OR (intramuscular) OR (subcutaneous) OR

(intracranial) OR (intravenous) OR (parenteral) OR (experimental infection) OR (skin penetration)

OR (wound) OR (transdermal) OR (penetration) OR (cutaneous) OR (circulation)), followed by

pre-defined inclusion and exclusion criteria.

The search resulted in 3279 titles that were analyzed in two-steps; eventually 216

publications were selected for full-text analysis; 119 individual experiments were extracted for

phage penetration analysis and 84 individual experiments for phage gut transit analysis. They were

quantified for the strength of the effect and investigated factors were categorized as follow: route of

administration, phage morphology, phage host, dose, species of treated individuals, their age,

presence/lack of sensitive bacteria. The most explicit correlations of phage penetration as revealed

by the statistical analysis of sample distribution in groups were:

1. route of administration, with the lowest efficacy of oral administration (96.3%) and with

equal efficacy of others (i.p., i.v., s.c., i.m., intranasal)

2. phage type, with the better penetration of podoviruses and microviruses (weaker

penetration: myoviruses, siphoviruses and inoviruses)

3. presence of a sensitive bacterial host in the system.

No important impact was noted for the factors like bacterial host type, animal species or age. Once

entering the system, phage was able to arrive to the most of organs and tissues, including spleen,

liver, kidneys and urinary bladder, thymus, hearth, lungs, muscle, and even brain and bone marrow.

Gut transit of phage depended mostly on the presence of a sensitive bacterial host in situ.

Surprisingly, no marked impact of stomach acidity neutralization was revealed. Applied phage

dose correlated to phage recovery level.

This work was supported by the National Science Centre in Poland, grant UMO-

2012/05/E/NZ6/03314 and by Wroclaw Centre of Biotechnology, programme The Leading

National Research Centre (KNOW) for years 2014-2018



Section VIII: Open Section 75

Anti-Pseudomonal activity of phage PEV20 in a dry powder formulation

in a murine lung infection model

Yoon-Kyung (Rachel) Chang1, Ke Chen2, Jiping Wang2,3, Jian Li3, Sandra Morales4, Elizabeth

Kutter5 and Hak-Kim Chan1*

1Faculty of Pharmacy, University of Sydney, Sydney, Australia, 2Monash Institute of

Pharmaceutical Sciences, Monash University, Melbourne, Australia, 3Department of Microbiology,

Monash University, Melbourne, Australia, 4 AmpliPhi Biosciences AU, Sydney, Australia, 5The

Evergreen State College, Olympia, USA


Objectives: To evaluate the efficacy of a dry powder formulation of Pseudomonas aeruginosa

phage PEV20 in a murine lung model of infection

Methods: Highly purified phage PEV20 with high titre was obtained from AmpliPhi Biosciences

AU. Phage PEV20 was co-spray dried with 17 mg/mL of lactose and 8 mg/mL of leucine in sterile

ultra-pure water (pH 7.4) using Büchi 290 spray dryer coupled with a conventional two-fluid

nozzle for atomization. Stability of phages in spray dried formulation was tested using the standard

double layer plaque assay. Immunocompromised Swiss mice (4 mice per group) were challenged

with 2 x 105 colony forming units (CFU) P. aeruginosa PSA ADL 01 strain using intratracheal

instillation. After 2h post-bacterial administration, 1mg of phage powder was intratracheally

delivered using dry powder insufflator; no treatment was given to the control group. At 4h and 24h

post-phage treatment, the lung and plasma were collected for bacterial and phage counting.

Results: Spray dried phage PEV20 powder was produced at 2 x 107 plaque forming units

(PFU)/mg. Phage-treated group showed 5-log10 reduction in bacterial load (105 CFU) in the lung

after 24h compared to untreated (1010 CFU). All the phages administered were recovered in the

lung (107 – 108 PFU) at 4 h with a 1-log10 titre increase after 24h (108 – 109 PFU) compared with

the actual phage dose given. The concentration of phage PEV20 in plasma was higher at 24h than

at 4h.

Conclusions: Stable PEV20 spray dried formulation was generated at a therapeutic dose for

murine lung infection model. A significant killing effect was observed against a clinical P.

aerugonisa strain in vivo. Both phage and bacterial load stayed consistent after 2h post phage-

administration. After 24h, significant reduction in the pathogen number conveyed phage replication

and bacteriolysis in the lung, which was verified by the increase in phage titre. This is a proof-of-

concept study demonstrating the potential use of phage PEV20 dry powder formulation for

treatment of P. aeruginosa lung infection. Powder formulations provide easy storage, transport and

administration over liquid formulations.




CRISPR-Cas and phage-host interactions in Streptococcus mutans

Cas Mosterd and Sylvain Moineau*

Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie,

Groupe de recherche en écologie buccale, Faculté de médecine dentaire,

Université Laval, Québec, Canada, G1V 0A6


Streptococcus mutans is a member of the natural oral microbiota, where it is the main cause of

dental caries. Phages are also abundant in the oral cavity. To evade viral predation in this

environment S. mutans likely encodes several anti-viral mechanisms. One of these mechanisms

may be the CRISPR-Cas system. CRISPR is an acronym for clustered regularly interspaced short

palindromic repeats and this locus is often located close to CRISPR-associated (cas) genes. Each

CRISPR locus consists of short repeat nucleotide sequences interspaced by variable nucleotide

sequences (spacer). Upon the discovery that spacer sequences often matched plasmid and phage

sequences, it was experimentally confirmed to work as a defence mechanism, specifically as an

adaptive microbial immune system. The spacer sequences provide specificity to the system by

targeting foreign DNA sequences (proto-spacers), while the cas genes were found to encode a

variety of proteins that each have a role within the system. In fact, CRISPR-Cas systems show

remarkable diversity of Cas proteins, effector complex structure, locus architecture and

mechanisms of adaptation, crRNA processing and DNA/RNA interference. CRISPR-Cas systems

belong to two classes, with multi-subunit effector complexes in Class 1 and a single-protein

effector in Class 2. Within each class, a number of types and subtypes can also be found.

In this study it is demonstrated that S. mutans strain SMQ-1336 possesses a Type II-A CRISPR-

Cas system. When the wild-type strain SMQ-1336 is challenged with a virulent phage,

bacteriophage-insensitive mutants (BIMs) can be readily obtained. Analysis of the CRISPR array

of these BIMs revealed the acquisition of new spacers. Acquisition of multiple spacers was

observed in several BIMs. Sequence analysis of the new spacers indicated that they were derived

from the infecting phage genome. Surprisingly, all the BIMs were shown to also prevent phage

adsorption, indicting a probable mutation in the unknown phage receptors. Experiments in which a

repeat-spacer-repeat unit targeting the genome of the same virulent phage was introduced into S.

mutans SMQ-1336 on a plasmid did not result in phage resistance. However, plasmid constructs

harbouring a sequence matching one of the spacers present in the CRISPR array of S. mutans

SMQ-1336 could not be transformed in the wild-type strain, confirming the interference activity of

the CRISPR-Cas system.

Taken altogether, the CRISPR-Cas system of S. mutans SMQ-1336 does not seem to protect

against phage infection but prevent plasmid transformation. The exact role of the CRISPR-Cas

system in S. mutans and the other mechanism(s) involved in phage resistance are under

investigation.




Mucosal Vaccine Delivery for Peptide Antigens via Lambda Display Phage

Patricia González Cano1, Lakshman N., A. Gammage1,2, Connie Hayes3, Karthic Rajamanickam3

Scott Napper1,4, Sidney Hayes3* and Philip J. Griebel1,5

1VIDO-InterVac, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3; 2 Current

Address: CFIA, 534-East Cordova St, Vancouver, BC, Canada. V6A 1L7; 3Department of

Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK,

Canada S7N 5E5; 4Department of Biochemistry, University of Saskatchewan, Saskatoon,

Saskatchewan, Canada S7N 5E5; 5School of Public Health, University of Saskatchewan,

Saskatoon, SK, Canada S7N 2Z4


There is increasing interest in the use of bacteriophage as protein/peptide and DNA vaccine

delivery vehicles. The safety, stability, ease of production, and immunogenicity of bacteriophages

have been characterized but little is known about their capacity to induce mucosal immune

responses in the small intestine. Whole body imaging confirmed that within 24 hours orally

delivered lambda bacteriophage (LP) was distributed through the gastro-intestinal tract of mice.

Targeted delivery of LP to specific sites in the small intestine of newborn calves confirmed LP

were immunogenic in a dose-dependent manner. LP were taken up by Peyer’s patches (PPs) and

induced LP-specific IgA and T cell responses within the mucosa-associated lymphoid tissue. A

lambda display phage (LDP) was constructed to present three immunogenic peptide epitopes

(YYR, YML, RL) fused to lambda coat protein D to display the disease specific epitopes (LDP-

DSE). Delivery of purified LDP-DSE to surgically constructed intestinal segments induced IgA

antibody-secreting cell (ASC) responses to the three displayed peptide epitopes within the targeted

PPs. Further, delivery of bacteria expressing D-DSE or bacteria containing assembled LDP-DSE

also induced epitope-specific IgA-ASC responses PP. These are the first studies to report the use of

LDP to induce mucosal immune responses in the small intestine and identify Peyer’s patches as an

important site for uptake of LP. Furthermore, these responses were observed in the absence of

exogenous immune stimulating compounds or adjuvant. Therefore, we conclude that LDP have

substantial potential to be used as an oral vaccine delivery vehicle for immunogenic protein/peptide

vaccines.




Immunophage synergy is essential for eradicating pathogens that provoke

acute respiratory infections.

Dwayne Roach1, Chung Leung2,3, Marine Henry1, Eric Morello1, Devika Singh2, James Di

Santo4,5, Joshua Weitz2,3, and Laurent Debarbieux1*

1Department of Microbiology, Institut Pasteur, Paris 75015, FR 2School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA 3School of Physics, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

4Innate Immunity Unit, Department of Immunology, Institut Pasteur, Paris 75015, FR 5Inserm U1223, Paris 75015, FR

*E-mail: Laurent. [email protected]

Pseudomonas aeruginosa is an important cause of life-threatening nosocomial pneumonia and

prone to multidrug resistance. Especially at risk are patients with weakened immune systems and

chronic respiratory disorders. Considering the scarcity of new antibiotics, the use of bacteriophage

(phages) as an alternative therapy has re-emerged. While several recent animal studies have

demonstrated the therapeutic potential of phages for treating pseudomonal lung infection in healthy

immunocompetent animals, it remains unknown whether phages are effective in the setting of

immunodeficiency. Here, we provide a systematic in vivo and mathematical investigation of host

innate immune status on the efficacy of monophage curative and prophylactic therapies for

pneumonia caused by P. aeruginosa in mouse. We show that the ability of inhaled phage therapy to

cure acute pneumonia and provide prophylaxis was host neutrophil dependent. In contrast, phage

therapy efficacy was innate and adaptive lymphocyte-independent in this setting. Mathematical

modelling the dynamics between host innate immunity, bacterial infection and phage replication,

we show that both phage lysis and neutrophil activity work in concert to reduce both phage-

sensitive and phage-resistant bacteria in the lungs. This ‘immunophage synergy’ contrasts with the

predominant view that phage therapy efficacy relies largely on the bacterial permissiveness to phage

lysis. Mathematical simulations also imply heterogeneous mixing of phage and bacterial

populations and phage saturation have important consequences on treatment efficacy. Nevertheless,

we show that phage therapy can still be efficacious in hosts with weakened innate immunity that

manifest emergence of phage resistance. In addition, there were no untoward immune effects from

high dose of phages alone in lung tissues. This study supports that phage therapy can be a safe and

effective antibacterial strategy but success is defined by immune status of the patient.




Use of bacteriophages for construction and tests of biosensors.

Marcin Łoś

Phage Consultants, Partyzantow 10/18, 80-254 Gdansk, Poland

Department of Bacterial Molecular Genetics, University of Gdansk, Wita Stwosza 59, 80-309

Gdansk, Poland


Pathogenic viruses and bacteria are very often a natural target for development of detection and

rapid diagnostics methods. The need for rapid detection and identification of pathogens spans

widely from the healthcare systems through veterinary to military use in e.g. battlefield diagnostics.

Increase in the threats of use of biological warfare against civilians were for a long time a driving

force in development of automated rapid detection devices, which could minimize a risk for

population. The aim is to build a test, which is easy, cheap, sensitive, fast and reliable. Usually it is

impossible to meet all these requirements at the same time. However, the constant increase in our

abilities to miniaturize and to build more and more complex electronics allow us to build more and

more sophisticated sensing elements able to detect even very small changes in the environment.

Despite this, in the construction of the biosensors also biological part plays very important role. My

favorite group of viruses - bacteriophages, appeared to be an ideal to play a role as a detection

target, but also as a biological part of a sensing elements. They posses many desirable

characteristics common with pathogenic viruses, but they are also very efficient in interactions with

bacterial cells. Because of that their use may in many cases appear to be superior to the use of more

traditional ways of detection employing e.g. antibodies in interactions with bacterial pathogens.

During this presentation I will summarize over 15 years of a struggle to use phages in both of those

roles - as a model and as a part of a sensing element and our approach to integrate them with non-

biological part of biosensors.




Getting the enemy to help you out: Elucidating the specificity determinants of

anti-phage genomic island excision in Vibrio cholera

Amelia McKitterick, Kimberley Seed*

University of California, Berkeley, Berkeley, California.


Vibrio cholerae is the causative agent of cholera and thrives in both aquatic environments and the

human intestinal tract. In both of these environments V. cholerae is susceptible to predation by

bacteriophages, the most prevalent of which is ICP1. In order to combat ICP1 predation, V.

cholerae has evolved a unique defense system referred to as the PLE (Phage-inducible

chromosomal island Like Element), a genomic island that is found integrated into the bacterial

chromosome. Five PLE variants have been identified in clinical isolates of V. cholerae and are

characterized by the ability to specifically block ICP1 production, although the mechanism(s)

underpinning this activity are not understood. PLEs are activated and respond specifically to ICP1

infection by excising from the chromosome and circularizing. PLE 1 encodes Int, a putative large

serine recombinase that drives recombination between attachment sites. Integration is catalyzed

solely by Int, however, excision requires a recombination directionality factor (RDF) that directs

Int-mediated recombination between the integrated attachment sites allowing for excision. As PLE

excision and circularization are observed only after ICP1 infection, we hypothesize that a phage

encoded RDF is necessary for directing PLE excision. We show that Int is necessary for PLE 1

excision, and that Int is the only PLE-encoded factor required for the recombination event in

response to ICP1. Additionally, we identified an ICP1-encoded gene product (Ind1), that is

necessary and sufficient for Int-mediated PLE excision, indicating that it is the RDF. As such, the

integrase and Ind1 required for PLE excision are encoded by separate entities, which helps to

define the molecular specificity of PLE activity. Variability in int is seen in between PLEs,

indicating that PLEs have evolved in response to changing ICP1 gene products, furthering our

hypothesis that PLEs are engaged in a molecular arms race with ICP1.




Is more better? Use of multiple hosts in phage isolation

Paul Hyman

Ashland University, Ashland, Ohio, USA


A bacteriophage’s host range is an important characteristic with implications for phage ecology as

much as applications of phages including phage therapy. In our work isolating bacteriophages, and

in the literature, there is indication that isolation methods may bias the host range of novel isolated

bacteriophages. Specifically, it appears that isolating bacteriophages with a mixture of host strains

favors broader host range phages. We have previously applied this in isolating Enterococcus

faecalis phages. Independently isolating two phages using two strains of E. faecalis together did

produce phages with broader host range than two phages isolated on only one of the strains. Oddly

though, the broader host range phages could only infect one of the two isolation strains.

Very recently we have begun isolation of phages from soil with a single host and several

combinations of multiple hosts for the enrichment culture. Our preliminary results are that more

phages (or higher titers of phages) are obtained in the single host cultures than any of the multiple

host cultures. Spot testing shows that these phages also only infect one of the isolating hosts.

These results suggest there may be negative as well as positive effects of the use of multiple hosts

in phage isolation. They also suggest that not all strains of bacteria will be equally useful in phage

isolation.

.



82 General

POSTER ABSTRACTS

Abstracts are arranged in thematic sections. Within each thematic section, posters are

ordered alphabetically by the last name of the presenting author.

Section I: Agriculture and Food Safety

Section II: Phage-Based Biotechnology

Section III: Ecology & Evolution

Section IV: Genomics & Proteomics

Section V: Structure and Molecular Mechanisms

Section VI: Temperate Phages

Section VII: Phage Therapy

The presenting author’s name appears in bold text. The names of other authors who are

attending the meeting have been underlined.



Section I: Agriculture and Food Safety 83

Enhanced inhibition of Salmonella Typhimurium using a phage cocktail

targeting three different host receptors

Jaewoo Bai, and Sangryeol Ryu*

Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology,

Research Institute for Agriculture and Life Sciences, and Center for Food and Bioconvergence,

Seoul National University, Seoul, Korea.


Objectives: With the increasing food safety concerns about fresh produce and limitations of

current disinfectant treatments, the development of novel and safe methods is urgently required to

control the contamination of fresh produce. Bacteriophages are considered as a natural antibacterial

agent for the control of foodborne pathogens. However, the rapid development of bacterial

resistance to phage infection is the most significant barrier to practical phage application. To

overcome this problem, we developed a novel phage cocktail consisting of three virulent phages

(BSPM4, BSP101 and BSP22A) that target different host receptors, including flagella, O-antigen

and BtuB, respectively. This study provides a receptor-based strategy to develop a novel biocontrol

agent against S. Typhimurium in fresh produce.

Methods: Bacteriophages infecting S. Typhimurium LT2C were isolated from various

environmental samples. To confirm the phage receptor, complement strains for rfbP, btuB, and

flgK genes were further subjected to spotting assay. For genomic analysis, Genome Sequencer FLX

(GS-FLX) instrument was used and then assembled with Newbler v2.9. Open reading frames

(ORFs) were predicted using the Glimmer v3.02, GeneMarkS and FgenesB software programs.

The annotation and functional analysis of the predicted ORFs was conducted using the BLASTP

and InterProScan databases. The bacterial growth inhibition abilities of single phage and their

cocktails were tested using S. Typhimurium LT2C strain with an MOI of 1. Fresh iceberg lettuce

and cucumber were used for food application for the phage cocktail. Each sample was cut and

sliced into the size approximately 10 cm2 and then artificially contaminated with Salmonella (~ 1 x

105 CFU). Each sample was treated with phage cocktail at different MOI values (103 to 104) and

incubated. At the indicated time point, each sample was homogenized and spread-plated for the

enumeration of S. Typhimurium.

Results and conclusions: This study was carried out to construct a novel phage cocktail using

agent against Salmonella. For this purpose, three novel phages targeting the different receptors

(i.e., flagella, O-antigen and BtuB) of S. Typhimurium were isolated and characterized. In addition,

genomic study of three phages revealed that all three phages are strictly virulent without no toxin

or virulence-related genes in their genomes, suggesting they are safe to be applied on foods. In-

vitro challenge assay and BIMs frequency analysis revealed the enhanced effect of three phages

cocktail to inhibit the host growth and to reduce the development of resistant strains, indicating the

preparation of cocktail with phages utilizing different host targets is a powerful strategy to control

S. Typhimurium. Furthermore, the effective inhibition of S. Typhimurium growth by the phage

cocktail in food systems showed that the phage cocktail has great potential of being developed as a

biocontrol agent against S. Typhimurium in fresh produces.



84 Section I: Agriculture and Food Safety

Isolation and characterization of broad host range virulent phages for control

of Salmonella on plants

Sudhakar Bhandare, Brigitte Cadieux, Anna Colavecchio and Lawrence Goodridge*

Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore

Road, Ste Anne de Bellevue, QC, H9X 3V9.


Objectives: Salmonella is an important cause of foodborne illness in Canada and worldwide. Each

year, approximately 88,000 people become sick in Canada from consuming food that is

contaminated with Salmonella, while the global burden of Salmonellosis is also substantial.

Recently, fresh fruits and vegetables have emerged as important sources of Salmonella apart from

traditional animal origin sources. This is because fruits and vegetables are grown in soil, where

contamination due to animals and non-potable water can occur. Control of Salmonella on fresh

produce is difficult and biocontrol of Salmonella using bacteriophages is a natural and efficient

way to reduce the load of Salmonella on fruits and vegetables.

The objective of this work was the isolation and initial characterization of bacteriophages capable

of infecting a wide range of Salmonella enterica serotypes, and which could be employed in

biological control of Salmonella that contaminate plants. Isolated bacteriophages were

characterized based on host range, their morphology and their genome sizes.

Methods: Bacteriophage isolation was carried out using sewage samples (1L) collected from the

Jean-R. Marcotte Wastewater treatment plant in Montreal by adopting standard enrichment and

agar overlay techniques (Van Twest and Kropinski 2009). Thirteen target Salmonella isolates,

originally isolated for food plants, and representing thirteen serotypes were used for isolation.

Isolated phages were characterized biologically by host range profile using 43 different Salmonella

isolates from 30 serotypes and broad host range phages were also screened for their lytic spectra on

40 other Enterobacteriaceae strains. Broad host range phages were further characterised

morphologically by electron microscopy and their genomes sizes were estimated using Pulsed

Field Gel Electrophoresis (PFGE).

Results: Thirteen phages were successfully isolated for each of the 13 target strains. Phages Φ3

and Φ6 could lyse 88.3%; while phage Φ9 lysed 86% and phage Φ13 lysed 83.7% of the 43 host

strains used for lytic spectra. These four broad host range phages could lyse some of the 40 other

Enterobacteriaceae strains. Φ3 and Φ6 could lyse 4 strains each while Φ9 could lyse 8 and the Φ13

could lyse 14 Enterobacteriaceae strains. Electron Microscopy revealed that phage Φ3 belongs to

the Myoviridae family while phages Φ6, Φ9 and Φ13 belonged to Siphoviridae family. Estimated

genome sizes of these phages were ~ 12.5 kb for Φ3 and ~ 30 kb for Φ6; while Φ9 and Φ13 were ~

48.5 kb in size.

Conclusions: The present study revealed that Φ3, Φ6, Φ9 and Φ13 have broad lytic ability for

Salmonella strains and could also lyse some of the other Enterobacteriaceae strains. After complete

characterisation of these four phages, they can be utilized for phage biocontrol of Salmonella on

plants.




PD_RS08885 is required for phage infection and pathogenicity of Xylella

fastidiosa

Tushar Suvra Bhowmick1, 2, Mayukh Das1, 2, Tram T Le1, 2, Ry Young1, 3, Carlos F. Gonzalez1, 2

1Center for Phage Technology, 2Department of Plant Pathology and Microbiology, 3Department of

Biology and Biophysics, Texas A&M University, College Station, Texas.

Xylella fastidiosa (Xf) is the causal agent of Pierce’s Disease (PD) of grapevines. Our laboratory

has isolated and propagated phages active against Xf, and confirmed that Type IV pili (T4P) are the

primary receptor for Xf virulent phages. We have previously reported seven in vitro selected Xf

phage resistant mutants that are non-pathogenic, retained twitching motility and exhibited an

absolute adsorption defect for cocktail phages. Genome analysis of two in vitro selected Xf phage

resistant mutants identified a frameshift in PD_RS08885 (accession number WP_004089904.1).

The PD_RS08885 product is a membrane protein with six predicted transmembrane domain

helices. The selected phage resistant frameshift mutant exhibited a truncated protein with only two

predicted transmembrane domain helices. To confirm the observed results, an in-frame deletion

(XF-15 ΔRS08885) was constructed by replacing the PD_RS08885 open reading frame (ORF)

from nucleotides 1964420 to 1965178 (Xf Temecula 1 [accession number NC_004556.1]) with the

kanamycin resistance cassette using site-directed gene disruption. Complementation of XF-15

ΔRS08885 was accomplished by introducing a wild-type copy of PD_RS08885 using a

chromosome-based complementation system. The XF-15 ΔRS08885 was not sensitive to phages

Sano, Salvo, Prado or Paz, whereas the complemented derivative exhibited sensitivity to all four

phages. Light microscopy studies confirmed twitching motility in both the deletion mutant and

complement. Both the deletion mutant and complement formed biofilms in vitro. Furthermore, XF-

15 ΔRS08885 is non-pathogenic in grapevines but the complement caused development of typical

PD symptoms, whereas both have the ability to colonize and move in grapevines. Bioinformatics

analysis showed PD_RS08885 encoded a transmembrane protein, which has a PAP2 superfamily

domain. Our results indicate that a PAP2 superfamily domain containing PD_RS08885 is required

for Xf phages infection and may be involved in the PD pathogenic process in grapevines.




Staphylococcus aureus Biocontrol Strategy by Yeast Engineering

- Displaying Phage Endolysin on Yeast

Jihwan Chun, Jaewoo Bai, Sangryeol Ryu*

Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology,

Research Institute for Agriculture and Life Sciences, and Center for Food and Bioconvergence,

Seoul National University, Seoul, Korea.


Objectives: Pathogenic bacteria has long been a threat to human civilization. With the emergence

of multi-resistant bacteria, the development of novel approaches for eradication without the use of

conventional antibiotic agents has become very urgent. Bacteriophages have been explored for a

long time to resolve this issue, even though its application in food industry is still very limited. In

current study, yeast surface display system was selected as a novel endolysin application platform

to develop safe and promising biocontrol mechanism against pathogenic bacteria.

Methods: Staphylococcus aureus ATCC 13301 strain was selected as a target host and S. aureus

phage SA11 endolysin, LysSA11, was examined as a counterpart. Shuttle vector pCTCON, which

carries Aga2 anchor protein involved in yeast surface display system, was ligated to construct

pCTCON::LysSA11. The vector construct was cloned into replication host, Escherichia coli DH5α,

and furthermore, to the expression host, Saccharomyces cerevisiae EBY100 strain. Each

transformation product was selected on antibiotic supplemented media and confirmed by PCR and

sequencing. Cultivation and expression induction of yeast were conducted on synthetic media with

2% of glucose and galactose, respectively (SD-CAA, SG-CAA). Yeasts displaying LysSA11 (A:

8.0x108 CFU/mL, B: 1.0x109 CFU/mL) were applied to S. aureus in early exponential growth (A:

3.4x103 CFU/mL, B: 9.9x103 CFU/mL) at RT. (Group A: 16-hour induction / Group B: 21-hour

induction) Then, the mixtures were cultivated on Baird-Parker media for S. aureus enumeration

and assessment of inhibitory effect.

Results: Phage endolysin LysSA11 was successfully incorporated to pCTCON vector, which was

successfully transferred to E. coli DH5α and S. cerevisiae EBY100. Transformed yeast cells were

cultivated, induced, and LysSA11-displaying yeast cells were treated to S. aureus ATCC 13301

cells. In group A (16 h induction), 1.19 log CFU of S. aureus cells were reduced in 2 hours, and the

viable count was reduced by 3.53 log unit, which means complete inhibition, after 4 hours.

Meanwhile, in group B (21 h induction), S. aureus cells were inhibited by 0.75 log unit in 1 hour,

and 2.90 log unit in 2 hours, implying enhanced lytic activity with elongated induction.

Conclusions: We have constructed a novel yeast surface display system facilitated with

bacteriophage endolysin to successfully regulate pathogenic bacteria, S. aureus. The results suggest

that introduction of phage endolysin in eukaryotic system could express active form of proteins for

inhibition of bacterial pathogen.




Storage of Staphylococcus aureus phages intended for food safety

applications

Eva González-Menéndez1, Lucía Fernández1, Diana Gutiérrez1, Ana Rodríguez1, Beatriz Martínez1

and Pilar García1*

1 Dairy Research Institute of Asturias (IPLA-CSIC). Paseo Río Linares s/n. 33300- Villaviciosa,


Bacteriophages have been proven as effective biocontrol agents for the food industry. However,

their extensive use requires the development of improved methodologies for the storage of

bacteriophages, and formulations compatible with their final application as biopreservatives. To

this aim, we determined the stability of four Staphylococcus aureus phages by using different

sample preparation techniques and storage conditions. First, we tested stability during medium- and

long-term storage at different temperatures (4°C, -20°C, -80°C, -196°C) and during lyophilization

(freeze drying) using different stabilizing additives (disaccharides, glycerol, sorbitol and skim

milk). Interestingly, phages belonging to the Siphoviridae family (phiIPLA88 and phiIPLA35)

turned out to be more stable at 4°C and -20°C than Myoviridae phages (phiIPLA-RODI and

phiIPLA-C1C). At lower temperatures (-80°C and -196°C), both siphophages and myophages

showed good infectivity after 24 months regardless of the stabilizer. Differences between families

were also observed after lyophilization. For instance, the addition of skim milk yielded a more

stable dry powder containing the phages, while sorbitol generally caused the highest reduction in

Siphoviridae phage titer after 24 months, in contrast to Myoviridae phages with similar stability in

both additives. Remarkably, storage of phages as lyophilized infected cells proved to be a suitable

option for unstable phages such as phiIPLA-C1C. As an alternative to facilitate storage and

transportation, we also explored phage encapsulation. Phage phiIPLA-RODI encapsulated in

alginate capsules retained high viability when stored at 4°C for 6 months and at room temperature

for 4 weeks. Considering the future application of phages as food biopreservatives, we also

explored the use of the spray-dryer technique to obtain encapsulated phages. Phage phiIPLA-RODI

retained their viability (<5 log units reduction) for 6 months at room temperature and phiIPLA88

up to 12 months in the presence of trehalose (<3 log units reduction). These results suggest that

dried powders might be a suitable way to deliver phages to food.




Impact of the receptor preference on host range distribution of Salmonella-

infecting bacteriophages

Y. Emre Gencay*, Michela Gambino, Tessa From Prüssing, Lone Brøndsted




Salmonella enterica consist of more than 2400 serotypes, yet major foodborne infections in Europe

are often associated serotypes Typhimurium and the monophasic variant (4,[5],12:i:-), Enteritidis,

Infantis and Derby. One of the major contributor of this diversity is the O-polysaccharide (O-PS)

units attached to conserved core sugars of the lipopolysaccharide. Salmonella phages are known to

target either conserved or serotype specific receptors such as repeating units of O-PS, conserved

core sugars, or outer membrane proteins. Except certain model phages, the extent of the host range

of phages that can infect wild type food-associated Salmonella has been poorly described so far. In

this work, we have isolated, sequenced and determined the target receptor and the host range of a

large number of phages in order to associate phage families and the target receptors with the host

range potential of the Salmonella-infecting phages isolated from the environment.

With an attempt to isolate diverse phages we have used 10 different isolation hosts (7 serotypes)

and isolated 50 phages from samples either by direct spotting or after selective enrichment with the

isolation hosts. Plaque formations were determined in a large host range analysis using 71 Danish

Salmonella pork isolates of different serotypes often implicated in foodborne illness in Europe to

reflect a snapshot of the wild isolates. Using defined prophage cured S. Typhimurium LT2 mutants

we found that 9 phages were dependent on vitamin B membrane transporter (BtuB), whereas the

majority (n: 31) was targeting O-PS. While the receptor of three phages were found to be either

partially or totally masked by O-PS, the receptors of the remaining 7 phages could not be

determined.

Subsequently, 40 of these phages were sequenced using MiSeq platform, allowing us to group 31

distinct phages into phage families and associate these with host ranges. O-PS-dependent phages

showed three distinct host range pattern in themselves. Members of Jerseyvirus infects only up to

12 strains, whereas four temperate phages can infect slightly more strains. Among the O-PS-

dependent phages, three phages of Vi1virus infected the most strains (up to 20). Interestingly, none

of the O-PS-dependent phages could infect all strains of a certain serotype, indicating the absence

of serotype specificity. The BtuB-dependent phages were identified as T5virus had the broadest

host range, infecting up to 37 different strains, whereas a few T5virus dependent on an unknown O-

PS-masked receptor showed a more limited host range.

Statistical analyses of the host range showed that the target receptor is the foremost influencer of

the host range compared to isolation host or sample or assigned family. BtuB-dependent phages

belonging to T5virus genus have higher potential to cover the diversity of food-associated

Salmonella strains, making them better candidates for biocontrol purposes.




Comparative study of phage antimicrobial proteins against Staphylococcus

aureus

Diana Gutiérrez1*, Silvia Portilla1, Lucía Fernández1, Beatriz Martínez1, Pilar García1 and Ana

Rodríguez1

1 Dairy Research Institute of Asturias (IPLA-CSIC). Paseo Río Linares s/n. 33300- Villaviciosa,

Asturias, Spain.

* E-mail: [email protected]

The rapid rise and dissemination of multi-drug resistant (MDR) pathogens represents a major

global long-term threat to human health. Recently, the World Health Organization (WHO)

published a list of antibiotic-resistant bacteria for which new antibiotics are urgently needed, where

Staphylococcus aureus, was classified as “Priority 2: High”, being of special interest S. aureus

resistant to methicillin (MRSA). Moreover, this bacterium is one of the major bacterial agents

causing foodborne diseases in humans due to their ability to produce enterotoxins. Likewise, the

complete elimination of this bacterium is a difficult task, in part because it can form biofilms on

biotic and abiotic surfaces. In this regard, the use of phage lytic proteins: endolysins and virion-

associated peptidoglycan hydrolases (VAPGHs) has shown a potent antimicrobial capacity as

therapeutic agents, biopreservatives against a range of pathogens and as antibiofilm agents. Herein,

the potential of two novel S. aureus endolysins, LysRODI and LysA72, derived from the

Myoviridae phiIPLA-RODI and the Siphoviridae phage phiA72, respectively was evaluated. In

addition, the lytic activity of the chimeric protein CHAP-SH3b, derived from the fusion of the

catalytic domain (CHAP) from HydH5 (VAPGH from phage phiH5) and the cell wall binding

domain of lysostaphin, was also compared. To do this, the three proteins were cloned into the

pET21vector, expressed in E. coli BL21 and purified by Nickel Affinity Chromatography. The

lytic activity of the proteins against S. aureus was compared by death kinetics measured by

turbidity assays and MIC. LysRODI showed the highest specific activity followed by CHAP-SH3b

and LysA72, meanwhile the lowest MIC was observed for CHAP-SH3b (16±0.2 µg/ml) and the

highest for LysA72 (79 ± 0.5µg/ml). The results obtained, allowed to stablish a protein

concentration to test their sensitivity to different environmental parameters such as pH, temperature

and salt concentration. Moreover, the effect of the presence of certain ions over the activity of the

proteins was tested, revealing that the Zn++ and Mg++ cations might negatively affect their activity.

The lytic spectrum of the proteins was tested against a collection of S. aureus strains, including

MRSA. Besides, the antibiofilm potential of the proteins was screened using the novel technology

‘xCelligence real-time cell analyzer’ (RTCA)’ and parameters such as MBEC50 (protein

concentration that removes 50% of the biofilm), LOABE (lowest protein concentration to observe

an antibiofilm effect) and specific antibiofilm activity were calculated. The results obtained so far

will be used to study the antimicrobial activity of these proteins in vivo using a zebrafish embryo

infection model.




Viral diversity in rumen microbial communities of Colombian cattle and its

variation under dietary interventions.

Hernández, R1,2., Jiménez, H2., Caro-Quintero, A2., Reyes, A1*

1 Laboratory of Computacional Biology and Microbial Ecology BCEM, Universidad de los Andes.

2 Corpoica, Sede Tibaitatá.


Bovine animals are ruminants that consume fiber-rich diets based on grasses and small shrubs,

however with the aim to increase the production of milk and meat, diets rich in carbohydrates have

been incorporated, causing changes in the regular metabolism of food digestion and even some

diseases such as acidosis. The aim of this project is to describe the diversity of bacteriophages in

the rumen of Colombian cows that feed on tropical grasses, and how this diversity changes when a

carbohydrate rich food source like corn is introduced in the diet. High concentration of

carbohydrates from corn leads to a change in the pH values inside rumen, which in turns leads to

changes in the microbial community and potentially their phages. The ruminal fluid of five female

cows with similar characteristics (weight, age, etc ...) will be sampled with a cannula. Phage

diversity will be evaluated at different times. The first time will occur when cows feed on grass, a

second time at the moment of introduction of a change in the diet with corn and lastly, when the

original diet is re-established. Viral particles will be concentrated using ultracentrifugation or by

precipitation with polyethyleneglycol. DNA will be extracted using phenol-chloroform method. If

the concentration of DNA obtained is very low, random amplification of viral nucleic acids will be

performed. Shotgun sequencing approach will be used to characterize the viral-derived reads. The

diversity of phage before and after the change in diet will be established by bioinformatic analysis.

We hope to find interactions between the phage and rumen bacteria as they change in response to

the diet switch.




Efficacy and safety of a Staphylococcus aureus phage cocktail in a murine

model of bovine mastitis

Koen Breyne1, Ryan W. Honaker2, Zack Hobbs2, Manuela Richter2, Maciej Zaczek2, Rebecca Lu2,

Anika Kinkhabwala2, Bruno Marchon2, Evelyne Meyer1, Lucia Mokres2

1Ghent University, Department of Pharmacology, Toxicology, and Biochemistry, Merelbeke,

Belgium 2EpiBiome, Inc. South San Francisco, CA

The aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of

bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were

selected based on their performance in a series of in vitro assays designed to test various phage

characteristics. Phages were tested individually and as cocktails to determine the most optimal

cocktail composition, a process referred to herein as phage scoring. The phage score incorporates

quantitative measures of killing efficiency in broth, time to resistance, host range, and other factors

predictive of a successful cocktail. The highest scoring phages were further tested for efficacy and

resistance suppression in broth and raw milk with and without supplemental IgG, and significant

decreases in CFU were observed. Based on these in vitro results the cocktail was then purified for

testing in the in vivo model.

Mammary glands were experimentally infected with S. aureus N305 (ATCC 29740), a clinical

bovine mastitis isolate that has been widely used for experimental infection of dairy cows and

mice. Lactating mice mammary glands were inoculated by intramammary infusion with S. aureus

and the phage cocktail treatment was applied via the same route four hours post infection. Treated

mammary glands were graded for gross pathological appearance and excised for bacterial load

(CFU) and phage load (PFU) quantification, and were compared to both sham- or antibiotic-treated

(the current standard of care) infected glands and non-infected controls.

Observation of clinical and gross macroscopic changes as well as quantification of CFU

demonstrated that the phage cocktail treatment significantly improved gross macroscopic clinical

observations, and significantly decreased intramammary bacterial loads. PFU indicated that the

tested phage cocktail treatment was able to maintain high local phage titers in the mammary gland

without spreading systemically. Further analyses (such as inflammatory cytokine profiling and

histopathology) will document the safety and efficacy of this candidate novel curative phage

cocktail mastitis treatment in the elegant preclinical model. These results present promising data

for phage therapy as an alternative to antibiotics for the treatment of bovine mastitis.

The first two and the last two authors contributed equally to this work.




Search of lytic phages, an alternate for control multi drag resistant Pseudomonas

infections in trout

K. Porchkhidze, T. Khukhunashvili, E.Jaiani, G. Tsersvadze, M. Tediashvili, N. Janelidze

George Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia

Cold water fish breeding is one of the most rapidly developing sectors of Georgian

Aquaculture, however the presence of bacterial pathogens leads to fish death or damage of aquaculture

products and finally the huge economic losses. Some species, most commonly P. fluorescens and P.

putida, have been reported as fish primary pathogens in the high intensity fish aquaculture systems.

Emergence of drug resistant bacteria in aquaculture industry due to unrestricted use of antibiotics

clearly indicates the need in more sustainable and environmental friendly strategies for controlling fish

infections, such as bacteriophages.

The main goal of present work was to study the antibiotic susceptibility of Pseudomonas

strains isolated from trout farms and to isolate specific bacteriophages against target pathogens. More

than 200 strains of presumptive Pseudomonas spp. were collected from water and sick fish samples.

Based on the results of conventional phenotypic identification in combination with API 20NE

biochemical systems (Biomerieux, France) and genetic identification by Species Specific PCR 28

strains of P. fluorescens and 31 – of P. putida have been revealed. The susceptibility of identified

isolates of both Pseudomonas species to 18 antibiotics most commonly used in Georgian aquaculture

was studied by Kirby-Bauer disc diffusion susceptibility test.

The results of antibiotic susceptibility testing demonstrated the highest sensitivity (94-90%) of

P. putida isolates to Moxifloxacin, Amikacin and Kanamycin and considerably less to Tobramycin

(87%), Cefepime (84%), Colistin sulphate (84%) and Gentamicin (84%). P. fluorescens strains

showed similar effectiveness to same antibiotics with relatively higher sensitivity to Ciprofloxacin

(86%). All Pseudomonas strains demonstrated complete resistance to β-lactams, Furazolidon, Tylosin

and Metronidazol. Obtained data correlated with results of our studies on presence of Antibiotic

Resistance Genes (ARG). Namely, 41 % strains were found to be positive for the blaTEM-1 gene and

31.8% to sul1gene; none of them were found to possess qnrS and TetM genes. The multidrug

resistance and presence of ARG in tested Pseudomonas strains shows the pathogenic potential of these

isolates.

Four bacteriophages isolated from water environments of Georgia were selected out of 20

primary phage isolates. By virion morphology the newly isolated phages were attributed to Myoviridae

and Siphoviridae families and showed characteristics of lytic phages, particularly short latent period

and high burst size, also high survival rate in fresh water microcosms. Broad host range was one of the

main criteria for selection of candidate phages. The phage PpML11 lytic to fish isolate P. putida 1-51

expressed activity to 90% of tested P. putida strains, also in the spot test lytic zones were registered

on the lawns of 85% of P. fluorescens isolates.

Work on selection of therapeutic phage candidates for aquaculture and their detailed

characterization is on-going. For evaluation of antibacterial efficacy of selected bacteriophages small

scale in vivo experiments will be conducted on experimental fish Danio rerio.




Chitosan Nanoparticles Loaded Bacteriophage Reduces Bacterial

Colonization and

Shedding in the Faeces of COBB Broiler Chicks

A. A. Kaikabo1,2*, M. S. Abdulkarim1 and F. Abas1

1Faculty of Food Science and Technology, University Putra Malaysia, 43300 UPM Serdang,

Selangor,

Malaysia 2Bacterial Research Department, National Veterinary Research Institute, PMB 01 Vom, 931001,

Plateau State, Nigeria


Avian pathogenic Escherichia coli O1:K1:H7 (APEC) causes infection in poultry leading to great

economic losses to poultry production globally. APEC contaminates finished poultry products thus

a problem to food safety, security and public health. The pathogen has been associated with urinary

tract infection in human and neonatal meningitis in children. It resists antibiotic therapy.

Alternative biological control measures using Bacteriophage has been explored in this work.

Bacteriophage(s) were isolated and characterized and a candidate phage has been selected based on

its characteristics and used in the biological control experiment against avian pathogenic

Escherichia coli O1:K1:H7. Oral application of phage therapy is affected by numerous factors such

as inactivation by gastric acids, enzymes and bile. We have synthesized and characterized chitosan

nanoparticles and loaded candidate phage (C-Φ NPs) to be protected and delivered. The C-Φ NPs

were applied in the in vivo experiments to assessed role in bacterial colonization in gastrointestinal

tract and shedding in the faeces of challenged birds. Microbiological culture of faeces from the

infected C-Φ NPs treated showed a decreased of viable bacterial counts from 1 to 3 weeks post

challenge compared with untreated groups. Similarly, at week 4 post challenge when the

experimental birds were euthanized culture of the intestinal scrub showed a decreased in viable

bacterial cell counts in C- Φ NPs treated group compared with untreated control respectively (p ≤

0.05). These results showed that C- Φ NPs is beneficial in reducing bacterial colonization and

shedding of APEC in the faeces of the infected birds. Thus, useful to public health, food safety and

security.




Isolation of Vibrio phages from the environmental waters in Kenya and their

application as biocontrol agents against Vibrio cholerae

Alice Nyambura Maina1,2, Francis Mwaura1, Mirriam Jumba1, Samuel Kariuki3

1University of Nairobi, Kenya, 2 Technical University of Kenya, 3Kenya Medical Research Institute


Background: To date Kenyans’ continue to experience Epidemics of Cholera more frequently than

before. In 2014 Cholera outbreak was reported in Kenya and out of the 47 Counties it spread to 30

of them. 15000 cases were reported and about 250 deaths. In May 2015 a total of 3301 cases had

been reported and 65 deaths occurred with case Fatality rates of 2%. In March 2016, 216 deaths

took place and 13 000 admitted to Hospital following Cholera outbreak that affected many in the

country. Since the December 2014 outbreak Cholera has been recurring in Kenya. In May this year

(2017) three people died of Cholera after eating food contaminated with Vibrio cholerae in a

wedding ceremony and seven admitted in the hospital. Cholera outbreaks have also been reported

in refugee and internally displaced people camps. As a developing country, bacteriophages lytic to

Vibrio cholerae can be a cheaper and a novel approach to control cholera outbreaks in Kenya.

Objective: of this research is to isolate bacteriophages lytic to Vibrio cholerae from the

environmental waters of Kenya and apply them as biocontrol agents against the pathogen. The

phages will be disseminated in the water bodies contaminated with the pathogen in regions that

experience cholera epidemics every year.

Methods: A total of one hundred and twenty two environmental water samples used for domestic

purposes were collected from two regions: Lake Victoria and Coast between March 2015 and

October 2016. Water samples were collected from ponds, lakes, rivers, wells and boreholes. Vibrio

cholerae and other bacteria were isolated from these environmental waters. Plaque assay technique

was used to isolate the phages that were purified and amplified. The host range of the Vibrio

phages was tested using bacteria isolated from the same environment.

Results: Pathogenic Vibrio cholerae O1 Eltor was isolated from the environmental waters of the

two regions. The Vibrio phage isolated was lytic against Vibrio cholerae and two more bacteria

E.coli 083 and Providentia sneebia.

Conclusions: The environmental waters of Lake Victoria and Coast regions were found to be

contaminated with pathogenic Vibrio cholerae O1 biovar Eltor. The Vibrio phage was lytic against

Vibrio cholerae, E.coli 083 and Providentia sneebia. The Vibrio phage had broad spectrum and

hence it can be used as a cocktail to control these bacteria. More work is ongoing to extract phage

DNA, determine the genome size of the vibrio phage, TEM and concentrate them for use in water

decontamination.




Isolation, characterisation and delivery of giant bacteriophages infecting the

pig enteric pathogen Salmonella enterica

Anisha Thanki1*, Nathan Brown1, Andrew Millard2, Gurinder Kaur Vinner3, Danish Malik3,

Charlotte Evans4 and Martha Clokie1.

1Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, England; 2Unit of Microbiology and Infection, Warwick Medical School, University of Warwick, Coventry,

England; 3Department of Chemical Engineering, Loughborough University, Loughborough,

England; 4AHDB Pork, Stoneleigh Park, Warwickshire, England.

*Email: [email protected]

Salmonella can infect and cause diarrhoea in pigs at any stage of their growth, from newborns to

finishing pigs. The diarrhoea in turn then accelerates transmission in farms, and can ultimately lead

to Salmonella entering the human food chain and causing food poisoning. It is estimated that pigs

cause 11.7% of salmonellosis each year. Antibiotics are used to treat these infections and they are

also used as general growth promoters in some countries. This has led to a surge in antibiotic

resistant Salmonella strains responsible for numerous outbreaks on pig farms. As a consequence,

regulatory incentives worldwide have been introduced in an attempt to reduce antibiotics in the

food chain.

Bacteriophages are a promising alternative to treat Salmonella infections and their ‘Generally

regarded as safe’ (GRaS) status granted by the FDA means that currently their use as a food

additive is compliant with current regulation in the USA. The aim of our research was to isolate a

panel of bacteriophages that could be developed therapeutically to treat UK pigs and data will be

presented on the progress we have made to date.

We liased with farmers to collect pig faecal, boar faecal, and slurry samples, from which 22

myoviruses were isolated. All phages could infect multiple antibiotic resistant strains from the most

prevalent Salmonella serotypes associated with pigs, with significantly similar efficiency of

plating. Further genetic analysis revealed that all of the phages had what is referred to as ‘giant’

genomes at approximately 240 kbp. According to predicted protein similarity they cluster together

with two other genomes in the NCBI database from Salmonella phages previously isolated in

Korea. No phages contained predicted integrases or other genes that would obviously prevent their

being used to remove Salmonella from pigs.

To investigate which phages would be optimal for therapeutic use, in vitro ‘killing assays’ were

conducted with different phage combinations. The most effective formulations were two phage

combinations, which could reduce Salmonella by 4-logs in 2 hours when they were applied at an

MOI of 100. These optimal phage combinations were characterised in terms of stability and most

phages were stable at temperatures up to 60°C for one hour, whilst one phage was stable at 90°C.

Therefore phages remained viable after the spray drying process (converting phage lysate to

powder) and powdered phage preparations will increase the shelf-life and versatility of application

of phages in the pig industry. We are currently collaborating with farmers, vets and end users to

determine the optimum intervention point at which phages could provide maximum benefit in

reducing Salmonella in pigs.




Optimisation of phage therapy against leek blight

Wagemans, J., van Charante, F., Rombouts, S., Volckaert, A., Pollet, S., Venneman, S., Maes, M.,

Van Vaerenbergh, J. and Lavigne R.

Every year, agriculture suffers from huge economic losses due to plant infectious diseases like

bacterial blight in leek. This bacterial infection is caused by Pseudomonas syringae pv. porri

(Pspo). Until recently, infected crops were still treated with copper or antibiotics like streptomycin.

However, since bacteria are becoming more and more resistant to these chemical treatments and

because of their risk for nature, alternatives are necessary. One possibility would be phage therapy.

Therefore, we first isolated five phages KIL1 to KIL5. These phages were characterized both

genomically and microbiologically (Rombouts et al., 2016). Moreover, one host range mutant

KIL3b was created, that infects 36 different isolates of Pspo. All phages were subsequently used in

preliminary field phage therapy trials in leek. A significant reduction (e.g. from 63% to 38.5%

symptomatic leek plants) of the disease symptoms was observed after application of the cocktail,

which is promising.

However, to be more effective, further research is needed. First of all, the application method has

to be optimized (so far, only one method was tested) and the composition of the phage cocktail has

to be further improved. One other important shortcoming that makes phage therapy commercially

less attractive is the fact that the identified phage cocktail often cannot be produced in an

industrially applicable way. Therefore, we optimized the production of the phage cocktail by using

an automated anion-exchange chromatography to purify the phages on CIM disks (BIA

Separations). This resulted in a fast method for the purification which is also suitable for large scale

production. Up to 80% recovery of the phages could be observed.



Section II: Phage-Based Biotechnology 97

Bacteriophages as Tools for Precision Microbiome Therapeutics

Robert J. Citorik1*, Timothy K. Lu1,2

1Microbiology Program, 2EECS, MIT, Cambridge, MA


The various microbial populations residing on and within us are known to have intricate ties to

human health. Metagenomic and metabolomic analyses, in addition to laboratory research, are

revealing potential keystone members, groups, or pathways implicated in preserving or damaging

the health of the human host. Future translation will require the ability to validate and to effect

changes in microbiota based on knowledge gained from these studies, necessitating the creation of

next generation therapeutics with programmable specificity. Additionally, these antimicrobials

could prove pivotal in the battle against increasingly drug-resistant pathogens, which can also

constitute members of the microbiome.

Previously, we have shown the pairing of CRISPR-Cas9 with bacteriophages or conjugative donor

bacteria to create sequence-specific antimicrobials. These programmed phages selectively killed E.

coli harboring resistance or virulence determinants, while leaving “unarmed” E. coli unharmed.

Antimicrobial activity is based on genetic signatures, enabling other potential targets such as

metabolic pathways implicated in disease or therapeutic outcomes.

In addition to rational payload design, work has continued on phage scaffolds, which determine the

diversity of targetable bacteria. The opportunistic pathogen Klebsiella pneumoniae is a major

health concern and can be armed with extensive arrays of resistance genes. This microbe is a prime

candidate for phage therapy to treat infection, given its resistance potential, as well as prophylactic

therapy, since it can preexist in a healthy gut before spreading within or beyond the carrier to a

susceptible site or patient. Using recently developed phage engineering strategies, we are

characterizing a pipeline of bacteria-derived or newly isolated temperate and lytic phages against a

clinical panel of carbapenem-resistant K. pneumoniae for testing in a murine gut decolonization

model. Of particular importance for this group of organisms are varieties of phage enzymes aimed

at recognition and penetration of the extensive and variable capsules that typically protect the

bacteria from environmental and immune insults. The arsenal of capsule types that may be

employed by bacteria like K. pneumoniae presents a challenge in the development of targeted

phage-based therapies, which we seek to address through both analysis of our new isolates and

engineering efforts. Ultimately, we hope these efforts will not only yield phage(s) or scaffolds

optimized for eliminating the pathogen, but will also inform design rules for the rational creation or

modification of novel engineered derivatives.

Given appropriate knowledge and engineering technologies, including genome modification,

rational payload programming, and phage scaffold tuning, bacteriophages could prove pivotal in

the next generation of antimicrobials and precision microbiome therapeutics.



98 Section II: Phage-Based Biotechnology

Phage endolysins: putative Clostridium perfringens therapeutic

Stephen M. Swift1, D. Treva Rowley1, Jerel Waters1, Brian Oakley2, David M. Donovan1*

1ABBL, ARS, USDA, Beltsville, MD.

2College of Veterinary Medicine Western University of Health Sciences, Pomona, CA


Clostridium perfringens is a major necrotic enteritis causing bacterial pathogen in poultry, and a

source of food poisoning and gas gangrene in people. C. perfringens can also cause mild to severe

enteritis in pigs. In the EU, the occurrence of C. perfringens associated necrotic enteritis in poultry

has increased as antibiotic use has decreased. As the US moves away from use of antibiotics in

animal feed, we can expect an increase in necrotic enteritis with subsequent losses from morbidity

and, in subclinical cases, losses from decreased chicken weights. Alternatives to antibiotics in

animal feed will be needed in the near future. The genomes of 43 unique C. perfringens isolates

from chicken were sequenced, examined for peptidoglycan hydrolase enzymes by homology to

known enzymes. There were more than 450 putative peptidoglycan hydrolases identified that

clustered into 15 groups according to homology [less than 50% amino acid identity between groups

and more than 90% amino acid identity within group]. Of 17 representative lysins cloned, four

lysins were shown to have high lytic activity against all 43 of the initial poultry isolates as well as 9

porcine (all tested) in plate lysis assays but not against other Gram-positive or Gram-negative

species tested. Activity was also demonstrated in zymogram and turbidity reduction assays. The

domain architecture and relative activity of the four lysins has been determined.




Discovery and Biochemical Characterization of PlyA54, PlyN74, and

PlyTB40 - Bacillus Specific Endolysins

Irina Etobayeva1,2, Sara Linden1, Farhang Alem3, Philip D. Mosier4, Allison A. Johnson5, Louise

Temple-Rosebrook6, Ramin H. Hakami3, and Daniel C. Nelson1,2,*

1Institute for Bioscience and Biotechnology Research, Rockville, MD

2Department of Veterinary Medicine, University of Maryland, College Park, MD 3National Center for Biodefense & Infectious Diseases, Biomedical Research Laboratory, George

Mason University, Manassas, VA 4Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA

5Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond,

VA 6Department of Integrated Science & Technology, James Madison University, Harrisonburg, VA


Three Bacillus spp. specific endolysins, designated PlyA54 (ORF 54 of phage Anthos), PlyN74

(ORF 74 of phage Nigalana), and PlyTB40 (ORF 40 of phage Tsar Bomba), were identified,

cloned, purified, and characterized for their antimicrobial properties. Bioinformatic analysis

revealed that all three endolysins have very different N-terminal enzymatic activity domains

(EADs) but similar C-terminal cell wall binding domains (CBDs). PlyA54 has a Peptidase-M15-4

EAD with a conserved SxHxxGxAxD metal binding motif, PlyN74 contains an Amidase-2 EAD,

and PlyTB40 possesses a MurNAc-LAA EAD. Both, PlyA54 and PlyN74, share 94% homology in

their CBD sequences, identified as a SH3b domain. In contrast, the PlyTB40 CBD only shared

~50% homology to the PlyA54 and PlyN74 CBDs, although it also displayed specificity toward the

Bacillus peptidoglycan. All three endolysins showed relatively broad antimicrobial activity against

Bacillus species with the highest lytic activity against B. cereus, with moderate but less effective

lysis noted for B. megaterium, B. pumilus, B. thuringiensis, and B. amyloliquefaciens. Surprisingly,

none of the enzymes demonstrated any measurable activity against several B. anthracis strains

despite high activity against closely related B. cereus species. Lytic activity of the EADs alone was

significantly diminished compared to that of the full-length endolysins, suggesting a necessary role

for the CBDs. Biochemical characterization showed that optimal lytic activity for all three

endolysins is in the physiological pH range (pH 7.0-8.0), over a broad temperature range (4ºC-

55ºC), and in the absence or low concentrations of NaCl (<50 mM). Out of all three endolysins,

only PlyA54 showed sensitivity to inhibition by EDTA. Dialysis of inhibited PlyA54 in buffer

supplemented with an excess of divalent metal ions (Ca2+, Mg2+) restored its lytic activity to ~80%

of the pre-inhibited level. PlyA54 showed much stronger lytic activity than PlyN74 and PlyTB40

on identical strains. These properties make PlyA54 a good candidate for further antimicrobial

development as well as bioengineering studies.




Phage display as a powerful tool to catch specific antibodies against ion

channels

Angela Holguin1, Wei Ye1, Fred Fellouse1

Department of Molecular Genetics, University of Toronto

The aim of this poster is to present a pipeline for isolation of new antibodies using synthetic

libraries against complex antigens as ion channels related to pain. There is a high interest to

develop a non-addictive non-opioid treatment for chronic pain as cancer pain, and different

antigens have been described as optimal targets. These targets could be used to fish antibodies with

phage display. This selection technique is a key tool in health science for the isolation of new

specific antibodies that can be used as treatment. There are critical factors that interfere in the

successful of the selection; for instance, the presentation of antigen. Many antigens as ion channels

are problematic at the presentation step due the difficulty to simulate the desire structural

conformation as in nature the are several changes of the protein. Ion channels, change the structural

conformation depend on the state of the channel, active state or inactive state. Here, we discuss the

phage display pipeline, problems along the process, and troubleshooting when working with

complex antigens as ion channels.




Vibriophage endolysins as a new antibacterial control strategy

Lina A. Zermeño-Cervantes, Sergio F. Martínez-Díaz, César S. Cardona-Félix, Carlos O. Lomelí-

Ortega*

Instituto Politécnico Nacional (IPN), Av. IPN s/n, Col. Playa Palo de Sta. Rita, La Paz, B.C.S.,

México. C.P. 23096. Phone 6121234658 ext. 81565


Vibriosis is a set of diseases of high incidence in the aquaculture sector, responsible for massive

mortalities and economic losses worldwide. Recently, multiple Vibrio parahaemolyticus strains

carrying pVA1 plasmid that encodes a binary toxin called PirAB were identified as the causal agent

of acute hepatopancreatic necrosis disease also known as early mortality syndrome

(AHPND/EMS). AHPND has generated significant economic losses in the major shrimp producing

countries since 2009, spreading rapidly to Asia and Mexico. However, the latest reports about the

presence of pirA and pirB genes in other Vibrio strains apart from V. parahaemolyticus strains

(VPAHPND) are concerning, since the problem is more serious and complex than estimated.

Attributed mainly to gene transfer versatility, the problem is exacerbated by the high degree of

bacterial resistance recorded in VPAHPND strains and the lack of new control strategies available to

address this aquaculture health problem. In this context, bacteriophage lytic enzymes (endolysins)

could be a promising antibacterial alternative. In this study, the recombinant endolysin of a marine

vibriophage called LysVPMS1 was obtained and its lytic activity against different VPAHPND strains

and other Vibrio species was evaluated. Interestingly, LysVPMS1 does not resemble any reported

endolysin. Furthermore, LysVPMS1 has a lytic effect against virulent strains of VPAHPND, V.

alginolyticus, V. harveyi, V. campbelli, and other non-Vibrio Gram-negative bacteria. Its optimum

lytic activity is around pH 6, and increases with Ca2+ ions. The enzyme retains its lytic activity after

being exposed at 50°C for 30 min. These findings show that it would be feasible to use it in

aquaculture due its broad-spectrum activity against various Vibrio strains. The next step will be to

evaluate their ability to prevent or control vibriosis under in vivo conditions. Currently there are

severe restrictions on the use of antibiotics, thus lytic enzymes encoded by bacteriophages could be

a novel promising and safe class of antibacterial alternative to reduce the risks in this industry.




Phage transduction for improvement of bacterial strains

Barbara Marcelli*, Jan Kok, Oscar P. Kuipers

Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute,

University of Groningen


L. lactis is one of the most studied species of the Lactic Acid Bacteria (LAB); it is widely used in

the food industry in starter cultures for the production of a wide variety of cheeses and butter milk.

The consumers’ demand for improved products constantly pushes industries to develop even better

products with enhanced characteristics in terms of, for example, quality, taste or shelf life.

Bacteriophage transduction can be used as a naturally occurring tool to transfer genetic material

from one L. lactis strain to another, or in some cases even between LAB species, in order to

introduce desirable traits into industrially relevant strains.

A large number of bacteriophages has been isolated from the dairy environment, as the starting

material for this project: their genomes have been sequenced and annotated as a first step. A

subgroup of the phageshas been further characterized to get more insights into their nature and life

cycle. Finally, a number of the bacteriophages have been tested for their ability to transfer either

plasmid or chromosomal DNA among different LAB strains. The frequency of transduction, the

amount and the nature of bacterial DNA transferred have been examined to evaluate for the

applicability, in the industry, of phage-mediated horizontal gene transfer.




Anti-Biofilm Activity of the Endolysin PlyGRCS against Multi-Drug

Resistant Staphylococcus aureus

Sara B. Linden1 and Daniel C. Nelson1,2,*

1Institute for Bioscience and Biotechnology Research, Rockville, MD

2Department of Veterinary Medicine, University of Maryland, College Park, MD


The ability of Staphylococcus aureus to form biofilms represents a major virulence factor and

contributes to its ability to cause chronic infections. Furthermore, S. aureus can form biofilms on

medical devices, leading to colonization of areas of the body where it would not normally be able

to persist. Biofilms are notoriously difficult to eradicate, as they are resistant to antibiotics and are

resilient against the host immune system. Currently, the treatment for S. aureus biofilm infections

is debridement or removal of the affected implant, which is not a desirable outcome, and as such,

alternative therapies are needed. PlyGRCS, an endolysin which has shown antimicrobial activity

against planktonic S. aureus, represents such an alternative option. In this study, we investigate the

use of PlyGRCS against staphylococcal biofilms. PlyGRCS displays the ability to remove biofilms

from abiotic surfaces at concentrations much lower than its minimum inhibitory concentration. In

addition to its ability to disperse biofilms, as shown by crystal violet staining, in vivo imaging, and

confocal microscopy, PlyGRCS also kills bacteria within biofilms. PlyGRCS removes S. aureus

biofilms formed under dynamic conditions in medical-grade catheters, mimicking an in vivo

infection. Finally, PlyGRCS possesses the ability to kill bacteria within biofilms grown on

epithelium, without harming eukaryotic cells. In summary, we show that PlyGRCS has a potential

application an anti-biofilm therapeutic.




Structure-Guided Mutagenesis of the Bacteriophage Endolysin PlyC Cell

Binding Domain: From Octamer to Monomer

Xiaoran Shang1 and Daniel Nelson1,2,*

1Institute for Bioscience and Biotechnology Research, Rockville, MD 2Department of Veterinary Medicine, University of Maryland, College Park, MD


Objectives: The bacteriophage endolysin PlyC possesses potent lytic activity towards specific

streptococcal hosts. Molecular characterization of the plyC operon reveals that PlyC is composed

of two separate gene products, PlyCA, which contains the catalytic domains and PlyCB, which

serves as the cell binding domain. Additionally, the X-ray crystal structure indicates PlyC is a

holoenzyme consisting of a single PlyCA polypeptide tethered to a ring-shaped assembly of eight

PlyCB subunits. Our aims were study the binding mechanism and create new chimeric endolysins

through engineering the PlyCB monomer.

Methods: The crystal structure of PlyCB octamer was analyzed for residues that are involved in

intra-subunit interactions via hydrogen bonding and salt bridges. Systematic mutations to these

residues were generated through use of the Change-IT mutagenesis kit, and the structure (i.e.

octamer or monomer) of each mutant was analyzed by analytical gel filtration. Fluorescence

microscopy was used to assess binding of PlyCB and its mutants to streptococci.

Results: Mutations to Lys40, Asp41, and Glu43 were chosen for construction in seven

combinations. All of the PlyCB mutants were successfully expressed and purified. However, only

one combination, Lys40 and Glu43, indicated a monomer of PlyCB via gel filtration. Furthermore,

this mutant retained the ability to bind streptococci.

Conclusions: Structure-guided mutagenesis of PlyCB was successfully used to create a PlyCB

monomer. This PlyCB-derived cell binding domain retained its binding specificity for streptococci,

which will allow for additional bioengineering studies.




Evaluation of the tumor-limiting capacity of a phage-display selected peptide

motif in a human papillomavirus tumor model

Lanre Precieux Sulaiman1*, Ana Paula Lepique2 and Luisa Lina Villa1.

1. Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

2. Instituto de Cinecias Biomedicas, Universidade de São Paulo, São Paulo, Brazil.


Objective: Women infected with high-risk human papillomaviruses (HPV) have an increased risk

for developing cervical neoplasia. To better understand the role of immune responses to these

viruses and disease development, we used phage display-based combinatorial selection to isolate

peptide ligands to antibodies present in the sera of HPV-positive women. Some peptides were

significantly enriched from a phage library displaying between 108 and 109 unique random

sequences. The most enriched motifs show different levels of homology to the L1 protein of both

low and high-risk HPV types. We want to see if the selected peptides had the capacity to limit

tumor growth in an HPV-model and if so, try to understand the mechanism of action.

Methods: We tested recombinant M13 phages containing an insert of the selected peptide - in both

prophylactic and therapeutic manners – against TC-1 tumor graft (an HPV model) in C57Black

immune-competent and knock-out mice. Mice were grafted with the tumor and then treated with

the phage injections (with or without adjuvants), or they were first immunized with the phage

injections and then grafted with the tumor. Thereafter, we analyzed tumor growth and kinetics by

pachymeter measurement, cellular infiltrate response by flow-cytometry and humoral response by

both phage-ELISA and peptide-specific ELISA.

Results: The mice immunized with the recombinant phage had reduced tumor growth when

compared with the controls and the mice that were treated at certain time-points after tumor

grafting also had varying degrees of tumor growth reduction. In all the cases, there seems to be

peptide specific stimulation of cytotoxic CD8 cells while also showing detectable peptide-specific

antibody production. This was further confirmed with the use of CD8 knock-out mice where

immunized mice lost the protection observed in the wild-type immune-competent specimens. There

thus seems to be a TH1 type response to the selected peptide that helps to reduce the rate of tumor

growth.

Conclusion: Phage-display is here proven again as a useful tool in selecting possible antigenic

epitopes in cancer and may offer complementary treatment options in HPV-positive cancers. While

our peptide may not offer full protection against the tumor, there may be a possibility for its

usefulness in a combination therapy. Further work is necessary to completely understand the

mechanism of action of the selected peptide.




Standardization of protocols to isolate phages against petroleum-souring,

sulfate-reducing bacteria

Santiago Hernández Villamizar, Martha Vives F.*

Universidad de los Andes, Bogotá D.C., Colombia

*E-mail: [email protected] , [email protected]

The oil industry is one of the largest income source in Colombia and is pillar of the world

economy. The souring is the increase of sulphide concentrations in the oil, consequence of the

reducing metabolism of anaerobic bacteria classified as sulfate reducing bacteria (SRB). The

souring affects the quality of the oil and the downstream process are more expensive. It also affects

the infrastructure of the industry, since the biocorrosion process deteriorate the material of the

pipelines and storage tanks. Therefore, the souring of oil and the biocorrosion generate economic

losses in this industry. At present these issues are controlled with biocides but it is necessary to find

other solutions because of its environmental impact, and the bacterial resistance to biocides.

The use of bacteriophages to control the populations of SRB is an alternative. This work seeks to

standardize the techniques to isolate effective bacteriophages against SRB since anaerobic bacteria

requires special conditions to thrive. An initial approximation was tested with Desulfovibrio

vulgaris as a host. Four treatments were carried out to assess the optimal growth of anaerobic

bacteria in solid medium. The best treatment was the traditional double layer method using 6 mL of

agar soft in the second layer. Moreover, waste samples and injection water used in oil recovery

were employed to isolate bacteriophages using direct and enrichment methods in anaerobic

conditions. Through the enrichment method was possible to visualize clear halos, which could

indicate the presence of bacteriophages in the samples. However, these results could not have been

replicated over time. The few Desulfovibrio sp. bacteriophages known in the literature are

temperate, which could be the reason for the no-replication of the said halos.

To test the reproducibility of the methodology of isolation of phages against SRB described in the

current work, new experiments with several SRB will be run. Besides, additional samples will be

tested in order to isolate lytic bacteriophages. The importance of the present work is based in the

fact of the little research made in the SRB bacteriophages; probably, for the demanding conditions

that require the anaerobic bacteria to thrive. Consequently, the result of this work will contribute to

the understanding of the anoxic bacteriophages and will provide clues to use bacteriophages in the

anoxic world.



Section III: Ecology & Evolution 107

My Sabbatical (Spring, 2017)

Stephen T. Abedon

Dept. Microbiology, the Ohio State University, phage.org, phage-therapy.org, archaealviruses.org


Some things new, some things old. I’ve spent 6 months checking things off my to-do list…

Abedon, S. T. 2017. Multiplicity of infection, Reference Module in Life Sciences. Elsevier.

Abedon, S. T. . 2017. Plaques, Reference Module in Life Sciences. Elsevier.

Blasdel, B. G., Abedon, S. T. 2017. Superinfection immunity, Ref. Mod. Life Sci. Elsevier.

Abedon, S. T. 2017. Phage "delay" towards enhancing bacterial escape from biofilms: a more

comprehensive way of viewing resistance to bacteriophages. AIMS Microbiology 3:186-226. (from

my talks in Liverpool and “Bacteriophage 2017”, both as available online; open access)

Abedon, S. T. 2017. Information phage therapy research should report. Pharmaceuticals 10:43.

(from my talk given in Stratford-Upon-Avon, as available online; open access)

Abedon, S. T. 2017. Commentary: Communication between viruses guides lysis-lysogeny

decisions. Frontiers in Microbiology 8:983. (based on more or less my entire career; open access)

Abedon, S. T., García, P., Mullany, P., Aminov, R. 2017. Editorial: Phage therapy: past, present

and future. Front. Microbiol. 8:981. (phage therapy review for the following; open acc.)

Aminov, R., Abedon, S. T., García, P., Mullany, P. 2017. Phage Therapy: Past, Present and

Future. See: journal.frontiersin.org/researchtopic/4111/phage-therapy-past-present-and-future

Howard-Varona, C., Hargreaves, K. R., Abedon, S. T., Sullivan, M. B. 2017. Lysogeny in

nature: mechanisms, impact and ecology of temperate phages. ISME Journal 11:1511-1520.

Alves, D. R., Abedon, S. T. 2017. An online phage therapy bibliography: separating under-

indexed wheat from overly indexed chaff. AIMS Microbiology 3:525-528. (open access)

Abedon, S. T. Post-proofs. Active bacteriophage biocontrol and therapy towards removal of

unwanted bacteria on sub-millimeter scales: from foods to microbiomes. AIMS Microbiology.

(from my Zurich talk, available online—finally got back to working on these… open access)

Abedon, S. T. Post-proofs. Bacteriophage clinical use as antibacterial "drugs": utility precedent.

Microbiology Spectrum and also in Cani, P., Britton, R. (eds.), Bugs as Drugs. ASM Press.

Abedon, S. T. Pre-proofs. Phage therapy: various perspectives on how to improve the art. Meth.

Mol. Biol. (also from my talk given in Stratford-Upon-Avon, as available online)

Abedon, S. T., Katsaounis, T. I. Pre-proofs. Basic phage mathematics. Meth. Mol. Biol.

Abedon, S. T. Pre-proofs. Why archaea are limited in their exploitation of other, living organisms.

Witzany, G. (ed.), Biocommunication of Archaea. Springer.

Abedon, S. T. Pre-proofs. Viruses of microorganisms: what are they and why should we care?

Hyman, P., Abedon, S. T. (eds.), Viruses of Microorganisms: Diversity and Applications. Caister

Academic Press. (we are desperately working on finalizing this multi-authored monograph…)

Abedon, S. T. Pre-proofs. Archaeal viruses. Ditto.

Abedon, S. T. Pre-proofs. Phage plaques. Harper, D., Abedon, S. T., Burrows, B. H., McConville,

M. (eds.), Bacteriophages: Biology, Technology, Therapy. Springer.

Abedon, S. T., Katsaounis, T. I. Pre-proofs. Phage enumeration via plaque counts. Ditto.

Dąbrowska, K., Górski, A., Abedon, S. T. Pre-proofs. Bacteriophage pharmacology and

immunology. Ditto.

Plus a whole lot of new web pages/postings, and still in preps. See abedon.phage.org for more.



108 Section III: Ecology & Evolution

Surveillance of Shigella specific phages:

Potential use in epidemiology and diagnosis of Shigellosis

Mahmuda Akter*, Mahmuda Yeasmin, Sharmin Luna, Shahin Mahmud, Arafat Oany, Rubhana

Raqib and Kaisar Talukder

International Centre for Diarrheal Diseases Research, Bangladesh, GPO Box 128, Dhaka- 1000,

Bangladesh


Objectives: Global burden of shigellosis caused by Shigella species is >160 million cases yearly,

mostly affecting under-5 children in resource poor countries. The aim of the study was to monitor

the prevalence of Shigella specific phages in the environment and to utilize this in development of

an accurate diagnostic tool to accurately estimate actual disease burden.

Methods: A total of 180 water samples have been collected from rivers, lakes, canals and ponds

at 16 different sites in and around Dhaka, Bangladesh, for isolation of Shigella-specific phages.

The collection was carried out in two phases—during February, 2006, to January, 2007, and July,

2014, to December, 2015. Standard microbiological and biochemical methods were used for

isolation of phages. Among 142 isolated phages, 85 were characterized phenotypically using 57

serotypes of Shigella including atypical and new variants (n=200), 69 different serotypes of E.

coli (n=132), Salmonella (n=12), Vibrio cholerae (n=2) and Yersinia (n=1) used for testing

susceptibility to phages. PCR and RFLP were done for extensive characterization.

Results: The highest number of serotype diversity of Shigella specific phages was found in

canals, followed by rivers, lakes and ponds. A majority of phages isolated during 2006-2007 lysed

most of the serotypes of all Shigella species although very few phages were S. flexneri specific.

However, in 2014-2015 the pattern changed; very few phages lysed all Shigella species while

maximum number of phages lysed S. sonnei and S. flexneri, always including predominant

serotypes (2a, 2b, 3a, 1c, 6). icddr,b hospital data showed that isolation rate of S. boydi and S.

dysenteriae from patients was lower in 2014-2015 compared to 2006 while isolation rate of S.

sonnei gradually increased and S. flexneri remained broadly unchanged.

Among 85 phages, 4 phages were each specific for S. dysenteriae 3, S. dysenteriae 6, S. boydii 1

and S. sonnei. Again, one phage lysed all S. flexneri serotypes, S. dysenteriae 1 and S. sonnei and

was positive for capsid gene belonging to T4 type phage. Nine phages lysed S. flexneri serotypes

only, while five phages lysed combination of S. flexneri and S. sonnei.

A mixture of 9 phages lysed all serotypes of Shigella including atypical and new variants, and a

mixture of two phages lysed predominant serotypes of Shigella. These phages did not lyse other

enteric bacteria tested. A majority of the phages were different from each other as determined by

restriction pattern analysis.

Conclusions: Detection of Shigella specific phages may be used as a reliable indicator for

monitoring prevalence of Shigella in the environment and provide new insights into epidemiology

of Shigella infection. A single drop of cocktail-phage preparation may be used to diagnose any

Shigella. When applying commercially available polyvalent antisera, however, up to 16 antisera

may be needed to confirm. The isolated phages may also have therapeutic potential.




The Human Gut Virome in Lean and Obese individuals

Laura Avellaneda-Franco1,6, Jaime L Moreno1, Lance Boiling2, Katrine Whiteson3, Andrew

Heath4, Forest Rohwer2, Jeffrey I. Gordon5, Alejandro Reyes1,5,6*

1Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia; 2Department

of Biology, San Diego State University, San Diego, CA 92182; 3Departament of Biology,

University of California, Irvine, CA 92697; 4Department of Psychiatry, and 5Center for Genome

Sciences Washington University School of Medicine, St. Louis, MO 63132; 6 Max Planck Tandem

Group in Computational Biology, Universidad de los Andes, Bogotá, Colombia; 3Department of

Biology, San Diego State University, San Diego, CA 92182.


Objective: Viruses have been cataloged as the “dark matter” of biological sciences because of the

narrow knowledge that we have about them. The aim of this study was to characterize the gut

virome of lean and obese individuals and to assess their correlation with the BMI.

Methods: Fecal samples of 59 individuals with one to three sampling points (0, 3, or 12 months

after the first sampling) were obtained in part from the Missouri Adolescent Female Twin Study,

which consists of samples from female twin pairs and their mothers. Isolation of Viruses Like

Particles was performed and its DNA was extracted and amplified using MDA and sequenced

using 454 GS FLX pyrosequencing. The obtained reads were cleaned using Trimmomatic. Cleaned

reads from each individual were assembled using Newbler. Obtained contigs of all samples were

de-replicated at 90% of identity. Open reading frames (ORF) were predicted using Glimmer. Next,

the reads of each individual were mapped to a) the dereplicated contigs and b) the predicted ORF to

construct two abundance matrices, which were normalized using RPKM counts. De-replicated

contigs were annotated by comparing both the nucleotide and aminoacid sequences against the

NCBI non-redundant database. Also, α and β-diversity were estimated using QIIME from the

constructed matrices as a proxy to OTU tables. Finally, the relationship between specificity and

precision, and mutual information was used to select viral contigs that are specific markers to a

given individual, family, and/or BMI.

Results: We obtained in average 46,000 reads per sample. After the assembly, which incorporated

over 90% of the reads per sample, and de-replication, we obtained 16,223 contigs with at least 500

bp in length, we predicted that 3099 of these contigs were circular. Also, we predicted 44,885

ORFs. We found that the difference among the individuals virome can not be explained by either

the BMI or familial relationship, however, beta diversity analysis suggest that the virome within an

individual is stable over time. We identified discriminatory contigs that were characteristic at the

individual, family, and in certain BMI ranges.

Conclusion: The virome in adults is highly stable over time and its overall composition is a

signature of the individual and not their family or other physiological factor such as BMI.

However, a small set of viral contigs were identified as specific for individual families or BMIs.

Furthermore, we were able to identify viral contigs closely related to prophages contained within

sequenced bacterial genomes. In addition, certain viral contigs were identified with high abundance

and incidence among the sampled population, some of those with high identity to the crAssphage.

In summary, the human virome still poses challenges for our understanding and factors such as

BMI or family relationship do not seem to be major shapers of its diversity.




Testing the Ferrojan Horse Hypothesis: Iron-Virus Interactions in the Oceans

C. Bonnain, S. Caprara, K.N. Buck, M. Breitbart*

University of South Florida, Saint Petersburg, Florida, USA


Iron is an essential nutrient in the oceans, with the sub-nanomolar concentrations found in open

ocean surface waters often insufficient for supporting biological activity. More than 99.9% of

dissolved iron is bound to organic ligands, yet identifying the sources of these ligands in seawater

remains a major challenge. A significant portion of iron-binding ligands fall into the colloidal

fraction, which is operationally defined as the fraction collected between a 0.02 µm and a 0.45 µm

filter. Among the organic ligands in this fraction persists an extremely abundant biological

candidate: viruses. On average there are 107 viruses per milliliter of seawater, most of which are

phages (viruses that infect bacteria). The impact of viruses on ocean biogeochemistry is often

evoked purely through the act of lysing hosts and very few studies have considered the

geochemical potential of the viral particles themselves. Recent work in non-marine model systems

has revealed the presence of iron atoms within the structure of diverse phages infecting Escherichia

coli. Combined with the small size and sheer abundance of phages in the oceans, the inclusion of

iron in phage structures would translate into a major factor for cycling of this important trace metal.

In addition, iron is so critical for growth that bacteria have evolved multiple uptake systems for

assimilating iron, such as siderophores. Certain outer membrane proteins serve a dual function in

siderophore uptake and as a phage receptor, suggesting that some of the strategies utilized for iron

acquisition make bacteria vulnerable to phage infection. Given the constant arms race between

bacteria and phages to develop resistance and counter-resistance, respectively, it is not surprising

that phage would have evolved to utilize critical regions of surface-exposed proteins which are

indispensable for bacterial growth as receptors. The research presented here explores the potential

of marine phages to serve as iron-binding ligands and discusses the implications for both trace

metal biogeochemistry and marine phage-host interactions.




How diverse are coliphages in the environment?

Stephen Ahern, Michela Gambino, Cæcilie Ingerslev Staffe, and Lone Brøndsted*




Escherichia coli is a highly versatile and adaptable species, exhibiting high degree of diversity and

consisting of both harmless commensals and pathogenic variants. In fact, it has been shown that

only 20% of the genes of the E. coli genome are shared among all strains, and there are over 200

serotypes based on somatic, capsular, fimbrial and flagella antigens. The E. coli surface diversity

has fostered highly diverse phages using many different host receptors and infection strategies,

with most phages only infecting a subset of E. coli. The E. coli Reference Collection (ECOR) is a

set of 72 reference strains isolated from a variety of hosts and geographical locations, which have

been shown to represent the genetic diversity of E. coli. The diversity within the E. coli species

makes designing phage biocontrol strategies difficult.

Here we aim to identify phages able to infect diverse E. coli. Using the ECOR library as hosts, we

isolated 128 phages from a variety of environmental samples, infecting 56% of ECOR strains.

Sixty-eight phages were selected for whole genome sequencing using MiSeq. Based on sequence

homology and genome organization, 20 phages could be grouped into 8 different and unrelated

phage types, most of which have not been characterized previously. Within phage types, related

phages were isolated from multiple environments and locations. We set out to identify phage

receptors and other host genes required for infection by using positive phage selection on a library

of Tn5 transposons of phage sensitive ECOR strains. Of the phages in our collection, we identified

phage receptors including outer membrane proteins such as OmpC and FhuA, as well as several

LPS moieties. These findings are currently being confirmed using clean deletion mutants. Our

work highlights the difficulty of isolating phages able to cover a large host range, even within one

species. It also demonstrates that E. coli phages are highly diverse and that seemingly unrelated

phages have adopted similar host recognition and adsorption strategies.




Impact of a dominant temperature-dependent phage on the seasonal

population dynamics of a tropical bacterial pathogen assessed by

mathematical modelling.

Andrew Morozov, Halil Egilmez, Martha Clokie and Ed Galyov*

University of Leicester, UK


Melioidosis, a serious environmentally-acquired bacterial infection, is often characterised by a

noticeable seasonality. Burkholderia pseudomallei, the causative agent of melioidosis, is

commonly found in tropical soil and stagnant waters that represent environmental reservoirs of the

disease. Environmental populations of B. pseudomallei are known to be regulated by a variety of

biotic and abiotic factors, but the contribution of different factors to the seasonal dynamics of B.

pseudomallei population is poorly understood. Mathematical modelling has long been recognised

as a powerful, efficient and reliable research tool to predict population dynamics. Here we build

and explore a novel mathematical model to describe and predict both the daily and seasonal

dynamics of the B. pseudomallei population and associated phages. The model focuses on the size

and composition of B. pseudomallei populations that are controlled by phages in the surface water.

The model is centred on our recent findings that a particular clade of phages, which is highly

abundant in the endemic area of Thailand, differently infects B. pseudomallei depending on the

temperature conditions: phages are predominantly lytic at a higher temperature of 37oC and mainly

lysogenic at 25oC. Lysogens survive in the environment at lower temperatures, but are killed and

release their viruses when they are then put into elevated temperatures of 37 oC.

Our model explores the effects of daily and seasonal variations in the environmental temperature

that result in frequent temperature-dependent switching between the types of phage-bacteria

interactions that affect the B. pseudomallei population. The model is based on a system of

differential equations that describe the densities of bacteria in the phage-free, lysogenic and lytic

states as well as the number of free phages. The parameters of the model are informed by our

research, other scientific publications and publicly available records. Various scenarios of the

seasonal progression have been investigated. Based on our extensive computer simulations, we

explored the sensitivity of model predictions on a few key parameters. Our simulations predict the

existence of three main types of fluctuations of the size and composition of bacterial population in

the field: short-term (daily), intermediate (from 1 week to a month) and longer-term (seasonal)

fluctuations. Our striking conclusion from this work is that most of the modelling outcomes appear

to correlate with the apparent higher risk of melioidosis acquisition during the “warm and wet”

season where the phages are predominantly undergoing replication in the lytic cycle and the

bacterial populations are dominated by phage-free B. pseudomallei. The model predicts annual

blooms of phage-free bacteria, and during such periods B. pseudomallei are more likely to establish

an infection. Our data suggest that seasonality of melioidosis and potentially other environmentally

acquired bacterial infectious diseases could be explained by the action of dominant condition-

dependent phages.




New insights into crAssphage

Emma Guerin1,2*, Andrey Shoporov1 Angela McCann1 , Paul Ross1,2 , Colin Hill1,2

1 APC Microbiome Institute, University College Cork, Ireland; 2 Department of Microbiology,

University College Cork


In 2014, Bas E. Dutilh et al. discovered a previously unidentified phage with a circular genome of

~97kb with 80 predicted open reading frames. This phage was named crAssphage based on the

cross-assembly programme used for its assembly. It was found to be highly prevalent in the human

gut, making up as much as 90% of the sequencing reads in faecal viral metagenomes. Through co-

abundance analysis it was predicted that a member of the Bacteroides genus is likely to be the main

host for this phage. However, wet lab work has yet to prove these in silico predictions.

Since the publication of the crAssphage genome there is little knowledge on its significance. Much

of the knowledge generated thus far is in silico based, but given its prevalence it is likely to have a

major role in influencing the bacterial population of the gut. To further investigate in silico

predictions, wet lab experiments are key. Of particular interest is confirming whether Bacteroides

is in fact the host for crAssphage. Gaining insight into phage morphology as well as how it

interacts with and replicates itself within the host may help to shine light on how it helps to shape

the gut microbiota.

The aim of this work is to reinforce bioinformatic predictions by confirming the host for

crAssphage and generating information on its role in the gut environment, using faecal samples

known to be crAssphage rich, based on bioinformatics data. The experimental pipeline developed

for this analysis may prove useful for other applications such as the examination of phage-host

interactions and the role of phage in influencing disease states in the gut.




Monitoring Pseudomonas aeruginosa bacteriophages in the environment

Ermir Kadija

”Luigj Gurakuqi” University of Shkodra, Albania


Obligately lytic bacteriophage isolation is crucial for phage therapy. Isolation from environmental

samples is the simplest method and often times easy. However, isolation of certain phages, such as

S. aureus phages, remains difficult and challenging. For this reason we have monitored

Pseudomonas phages in environment from May - October 2012. Summer months were chosen for

being the hottest months of the year with temperature reaching 40˚C. Samples from a water

discharge that run on the river Buna were used for phage monitoring and water temperatures were

also monitored.

Environmental samples were collected monthly and delayed two days later if sites experienced

rain. They were centrifuged, filtered, and plated using a double agar layer protocol on a

Pseudomonas aeruginosa strain. The following day, plates were checked for phage plaques.

Results show a correlation between phage titer and temperature with August having 172 PFU mL-1

and the lowest phage titers were observed in May and October with 4 PFU mL-1. These results

correspond with temperature results since on August water temperature was 23˚C.

Repeating the same experiment with S. aureus we weren't able to isolate phages but a specific

phage for S. aureus was possible to isolate using an enrichment protocol with a 50 mL

environmental sample. This data corresponds with previous data with the number of phage

particles increasing proportionally with availability and density of bacteria (temperature).

However, the diversity of phages for P. aeruginosa was higher than that of S. aureus since 3

different phages for Pseudomonas were isolated with direct plating.

As the results shows that temperature is a factor of successful phage isolation, more research is

necessary in this subject since more factors must influence phage isolation like sewage chemical

composition, phage stability, burst size, bacterial growth requirements, bacterial generation time,

etc.




Isolation and characterization of two novel cyanophages from a subarctic

Canadian lake

Alice Lévesque1,2,4,5*, Antony T. Vincent2,5, Simon J. Labrie2,4, Sylvain Moineau2,4,

Warwick F. Vincent1,3 & Alexander I. Culley1,2,4,5

1 Centre d’Études Nordiques (CEN), Université Laval, Québec, Canada; 2 Dept. of Biochemistry,

Microbiology and Bioinformatics, Université Laval, Québec, Canada; 3 Dept. of Biology,

Université Laval, Québec, Canada; 4Groupe de recherche en écologie buccale (GREB), Université

Laval, Québec, Canada; 5Institut de biologie intégrative et des systèmes (IBIS), Université Laval,

Québec, Canada


Cyanobacteria are key components of arctic aquatic ecosystems, as both essential primary

producers, and as significant contributors to the biogeochemical cycling of essential nutrients such

as carbon and nitrogen. The two primary mechanisms of top-down control of cyanobacterial

populations are grazing and viral lysis. Viruses are the most diverse and abundant biological

entities on Earth with estimates of 1031 viral particles globally and 104 virotypes per mL of water. It

is now widely recognized that viruses are important drivers of host evolution, and influence

nutrients cycles and microbial dynamics through viral lysis. Cyanophages, viruses that infect

cyanobacteria, infect keystone primary producers in marine and freshwater environments, and have

been shown to harbour host-like genes that are involved in essential host metabolic processes. In

order to better understand cyanophage dynamics, genomics and ecology, we focused our research

on the isolation and characterization of cyanophages that infect polar cyanobacteria.

Water from a Canadian subarctic lake located in the vicinity of Whapmagoostui-Kuujjuarapik

(Nunavik, Canada) was sampled in August, 2015, and was filtered through 0.45µm and 0.22µm

pore size filters to remove large particles (cells, clay, etc.). The filtrate containing viruses was used

to inoculate different cyanobacterial cultures for viral amplification. Cultures presenting signs of

lysis relative to an untreated control were serially diluted to isolate a single virus. Clonal isolates

were subsequently sequenced on a NGS sequencing platform (MiSeq, Illumina).

We succeeded in isolating two novel cyanophages (B3 and B23). Electron micrographs revealed

that both isolates possessed icosahedral capsids and long contractile tails, typical features of viruses

in the order Caudovirales, family Myoviridae. B3 and B23 genome lengths were 244,930 bp and

243,633 bp, respectively. Genomic analysis and ORF annotation indicated the presence of two

host-related metabolic genes (hli and phoH) in both genomes. However, the function of only 25%

of the putative ORFs was identified using RAST based on homology with sequences in the SEED

database. Phylogenetic analysis based on alignments of the B3 and B23 primase genes and those

from phages from a representative number of taxa showed that both viruses clustered with other

cyanophages. These results suggest that B3 and B23 are highly divergent from previously

described cyanophages and that this study will result in new insight into the world of arctic

cyanophages, of which yet little is known.




Which are the host factors influencing the replication of virulent

bacteriophages in the gastrointestinal tract?

Marta Lourenço1, Matthieu Galtier1, Damien Maura1, and Laurent Debarbieux1*

1 Group Interactions Bacteriophages Bacteria in Animals, BMGE Unit, Microbiology Department,

Institut Pasteur


Virulent bacteriophages have been frequently proposed as alternative antibacterials to target

bacterial pathogens resistant to multiple antibiotics. Often, some of these pathogens are

asymptomatically carried in the gastrointestinal tract (GIT) of mammals. However, despite several

attempts using different animal models, the use of virulent bacteriophages to efficiently reduce

carriage levels have often been disappointing, achieving at best a moderate reduction. We believe

the lack of knowledge on the factors that influence the replication of bacteriophage in the GIT is

impeding the development of strategies to improve overall bacteriophage efficacy.

To address this question, we characterized nine bacteriophages isolated using three different strains

of Escherichia coli: the enteroaggregative strain 55989, the adherent invasive strain LF82 and the

uropathogenic strain AL505. Each of these E. coli strains was used to colonize the GIT of mice.

Using homogenized GIT samples from these mice, we assessed the ex-vivo replication of these

bacteriophages in different sections of the GIT. We found that some bacteriophages replicate

efficiently regardless of the GIT sections while others display variable replication efficiency.

Therefore, these data support our hypothesis that the local GIT environment is influencing the

infectivity of bacteriophages, raising questions regarding the identification of the actors involved

and how, in the future, we could design ways to improve overall efficacy of phage therapy

treatments.




Metagenomic time series reveal temporal variations within the viral

community in the Baltic Sea

Emelie Nilsson*, Daniel Lundin, Stina Israelsson, Jarone Pinhassi, Karin Holmfeldt

Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and

Environmental Science, Centre for Ecology and Evolution in Microbial Model Systems (EEMiS),

391 82 Kalmar, Sweden.


Viruses infecting microbes are numerous and play an important role in aquatic ecosystems,

influencing biogeochemical cycles, such as cycling of carbon, nutrients and energy. To increase

our knowledge regarding viruses, metagenomic studies, such as Tara Oceans and the Pacific Ocean

Virome, are important. Such studies have improved our knowledge regarding the great diversity of

aquatic viruses as well as their spatial distribution and variation. However, temporal dynamics of

viral communities is a yet unexplored area within the field. Here, we seek to address this

shortcoming by presenting data from 25 viromes taken across all seasons from 2012 to 2015 at the

Linnaeus Microbial Observatory in the Baltic Sea.

Each time-point was assembled into contigs and annotated with VirSorter and DIAMOND against

a database of all viral genomes in NCBI. A majority of the contigs had little resemblance with

known viruses, but several were large enough to indicate that they are complete or nearly complete

viral genomes. In this study, we investigated large contigs (>20 genes, 290-2449 contigs per

sample). These contigs were diverse and showed similarities to globally abundant T4-like

cyanophages and pelagiphages, but also to more rare Cellulophaga phages, isolated from the Baltic

Sea, and phycodnaviruses. By recruiting reads from each time point back to the contigs from all

time points, we were able to investigate temporal patterns of the large contigs. These showed most

similarity to Myoviridae, Podoviridae, Siphoviridae and Phycodnaviridae, but the majority of

ORFs did not show any resemblance to known viruses. To understand why the relative abundance

of the families varied over time, we selected the contigs that mainly contributed to the increases in

relative abundance and studied the temporal abundance in more detail. The contigs similar to T4-

like cyanophages and pelagiphages were present and had a relatively high abundance in all

samples. Contigs similar to Cellulophaga phage phi38:1 were also present, but the relative

abundance was low except for two samples in spring 2012, in which it rivalled the dominating

pelagiphage. An increase in relative abundance during late summer and autumn could be seen for

the Synechococcus phage S-CBS4, while other Synechococcus phages within Siphoviridae showed

no such seasonal trend.

Overall, this study shows novel temporal patterns within viral communities that indicate seasonal

variability in diversity and the next step is to discover the relationship between viral populations

and microbial succession.




Coevolution of human gut Enterococcus faecium with lytic phage V12

Andrew Oliver1, Stephen Wandro1, Tara Gallagher1, Claudia Weihe2, Whitney England1, Jennifer

B.H. Martiny2, Katrine Whiteson1*

1 Department of Molecular Biology and Biochemistry, University of California, Irvine; 2

Department of Ecology and Evolutionary Biology, University of California, Irvine


Bacteriophages are highly abundant in the human microbiota, but their effect on the composition

and diversity of resident microbes is largely uncharacterized. Phage predation can drive the

evolution of bacterial resistance, which can then drive reciprocal evolution in the phage. These

coevolutionary dynamics have been extensively studied in few ecological systems, but studies

relating to the human microbiome are lacking. We performed experimental evolution and

coevolution of isolates of Enterococcus faecium from healthy human stool with or without a single

infecting T4-like bacteriophage, V12. Quadruplicates of E. faecium and phage were grown together

in semi-continuous culture for eight days, diluting in fresh BHI media every twelve hours. In the

evolution scenario, bacteria were either grown alone or naively reintroduced every twelve hours

with an evolving phage, whereas in the coevolution scenario, both bacteria and phage were grown

in the same microcosm. At several time points during the experiment, bacterial and phage DNA

from each replicate was extracted and sequenced. Unique to the coevolution scenario, E. faecium

obtained small nucleotide polymorphisms (SNPs) in a gene related to exopolysaccharide biogenesis

and export. Phage V12 evolved SNPs in a capsid protein and obtained varying amounts of 1.8kb

duplications in the tail fiber gene when coevolving with E. faecium. Host range assays confirmed

that coevolution was directional where phage resistance and host infectivity increased over time.

This study provides insight into genes that may be involved in coevolution in this novel bacteria-

phage system.




Identifying gene signals for phage host-range prediction

Jason Shapiro1, Siobhan C. Watkins2, Catherine Putonti1*

1Loyola University Chicago, Chicago, IL; 2New Mexico Tech, Socorro, NM


Objectives: As more novel, uncultured phage genomes are published, new tools are needed for

placing these genomes in an ecological and evolutionary context. There are no universal genes

shared by all phages, and horizontal gene transfer between viruses is common. In essence, every

phage genome is a mosaic that reflects the often disparate evolutionary histories of its genes. Host

range, in particular, constrains viral ecology and evolution, and predicting a virus’ host is a key

challenge when characterizing novel, uncultured genomes. Taking a computational approach, our

work is aimed at improving methods for phage-host prediction.

Methods: We have built gene-level networks representing the co-occurrence of genes across phage

genomes. In these networks, nodes represent a set of homologous genes (across phage genome

sequences) and two nodes are connected by an edge if the genes co-occur within the same genome.

This is a departure from genome-based networks, which have previously been used for recovering

taxonomic groups of phages, in which nodes represent genomes and edges signify the presence of

homologous genes. Taking this gene-centric approach, we sought to quantify the reliability of

observed clusters within the network and to ask if subsets of genes could be used to predict a

phage’s host. We developed an evolutionary algorithm to identify the subset of genes that

maximizes the mutual information of clusters and hosts.

Results: Here, we first show that clusters of genes within this network provide a robust view of

virus genetic diversity. Gene-level networks provide a higher resolution view of phage genetic

diversity and offer a novel perspective on virus ecology – in particular viral host range. We can

predict virus host range at the genus level with over 80% accuracy for many host genera. This

exceeds the accuracy of current bioinformatic approaches for host prediction.

Conclusions: Gene-level networks can provide a high-resolution view of viral genetic diversity

and have the potential to provide meaningful insight into predicting virus-host interactions. We

expect similar methods can be applied to other aspects of viral ecology, including isolation source

(e.g. freshwater, marine, soil, leaf, gut, hospital, etc.) and abiotic or biotic factors that might vary

across these locations (e.g. temperature, pH, O2, nutrient concentrations, and available host

diversity).




Broad host range – lessons from the literature

Alexa Ross and Paul Hyman*

Ashland University, Ashland, Ohio, USA


A bacteriophage’s host range is an important characteristic. While conceptually simple, host range

determination has several technical challenges. First, it can only be tested on the species and

strains available to the tester, which is by necessity a subset of potential hosts found outside the

laboratory. As well, there are few standard sets of strains for any particular bacterial species.

Second, host range varies depending on the specific testing method used. Different methods will

show varying host ranges even when done on the same hosts by the same researchers. It is quite

common, for example, for a bacteriophage to show a different host range on a single set of hosts

when tested by spot testing versus by plaque formation. Third, the terminology of “narrow” and

“broad” host range is inconsistently used.

In our work isolating bacteriophages, and in the literature, there is indication that isolation methods

may bias the host range of novel isolated bacteriophages. Specifically, it appears that isolating

bacteriophages with a mixture of host strains favors broader host range phages. In order to test this

contention, we have surveyed the literature on newly isolated bacteriophages published over the

last 70 years on a wide variety of bacterial types. Not surprisingly, the majority of phages were

isolated on a single host, but there are a few examples of multi-host isolation. While there are not

enough examples to make definitive statements, there is certainly enough evidence to support

further exploration of multi-host isolation.

We also note that when host range is tested, even on single-host isolated phages, host range appears

to be a trait with significant diversity between bacteriophage. Both narrow and broad host range

phages are often isolated at the same time on the same host strain suggesting that both are

reasonably common. In the few cases where hosts of multiple genera are tested, as opposed to just

strains of the same species, both single-species and multiple genera (polyvalent) host-range phages

can be isolated.

Our literature review suggests that the belief that bacteriophages are generally species specific may

be less true among phages as a group, perhaps due to a narrower host-range bias resulting from

single-host isolation procedures. Already isolated phage species may also harbor greater host

range diversity that has not been identified because generally the complete potential breadth of

phage host range is never fully tested. This has implications for the evolution and ecology of

bacteriophages as well as for phage therapy applications, for which polyvalency is not adequately

considered even as broad host range on the target host species is explored.




Genome-derived insights into interactions between cyanophage Vb-AphaS-

CL131 and filamentous cyanobacteria Aphanizomenon flos-aquae

Sigitas Šulčius1,3*, Gediminas Alzbutas1, Eugenijus Šimoliūnas2, Emelie Nilsson3, Giedrius

Gasiūnas2, Inga Songailienė2, Vykintas Jauniškis2, Jolita Kemeraitė1 & Karin Holmfeldt3

1 Nature Research Centre, Vilnius, Lithuania; 2 Vilnius University, Vilnius, Lithuania; 3 Linnaeus

University, Kalmar, Sweden


Filamentous diazotrophic cyanobacteria Aphanizomenon

flos-aquae contribute significantly to community

composition and biogeochemical processes in many

temperate lakes and brackish water ecosystems, among

other locations in the Baltic Sea. Recent experimental

studies demonstrated an important role of cyanophage

infections in A. flos-aquae population dynamics, with

implications for food web structure and functioning. In

addition, metagenomic and transcriptomic surveys have

indicated that cyanophages are common and active in the

Baltic Sea. However, our understanding of the genomic

basis of phage-host interactions in these ecologically relevant cyanobacteria and their viruses

remains elusive. Therefore, in this study we performed genome sequence analysis of a cyanophage

Vb-AphaS-CL131 (CL131) and A. flos-aquae host strain 2012/KM1/D3 (D3), to better understand

the complexity of their reciprocal interactions at the genetic level.

Cyanophage CL131 has a linear dsDNA genome 112,793 bp in length. The genome contains 147

predicted protein coding genes and 2 tRNA genes. The majority of the predicted genes have no

detectable homologues in the present databases and the genome showed no co-linearity with

previously sequenced genomes of other cyanophages. The recruitments of CL131 reads from

available and local viral metagenomes are currently being performed to represent spatial and

temporal distribution of CL131 cyanophage. Neither integrase genes were found in CL131 genome

nor prophage sequences in the D3 genome, suggesting that CL131 does not enter into lysogenic

interactions with their hosts. No genes related to photosynthesis and carbon metabolism were

found, implying that CL131 replication and progeny formation strongly relies on host metabolism

and its intracellular resources. Cyanophages encode putative Class 2 type V-U4 CRISPR-Cas

system; those complexes are composed of single Cas protein and crRNA. Northern blot analysis

confirmed transcription of cyanophage CRISPR region during cyanophage infection. On the other

hand, host strain D3 encodes an I-D type CRISPR system that has a spacer that is complementary

to CL131 cyanophage genome, implying that I-D type CRISPR system may provide resistance to

the host against CL131 infection. The simultaneous genomic characterization of both cyanophage

and its host should enable better insights into possible virus-host interactions of bloom forming

filamentous cyanobacteria.




Identification and characterization of bacteriophages in global sewage

samples

Henrike Zschach1*, Barbara Lindhard1, Vanessa Jurtz1, Frank M. Aarestrup2, Morten Nielsen1,

Ole Lund1

1 Department of Bio and Health Informatics, Technical University of Denmark (DTU), 2800 Kgs.

Lyngby, Denmark; 2 National Food Institute, Technical University of Denmark (DTU), 2800 Kgs.

Lyngby, Denmark


Sewage is a major source of both human pathogens and their associated bacteriophages. Phages

control bacterial populations by predation and can act as natural reservoirs for accessory genes

such as antimicrobial resistance genes and virulence factors. However, currently limited knowledge

is available about the sequence and functional diversity of such sewage phage communities. We

here present a study of the phage communities of 81 sewage samples from 62 different countries

around the world.

The samples consist of metagenomic assemblies in which we identified phage contigs by using the

MetaPhinder tool. These contigs were subsequently screened for the presence of known virulence

and resistance genes with the VirFinder and ResFinder tools. Additionally, we performed host

prediction with HostPhinder and taxonomic classification.

Antimicrobial resistance genes were found in the phage population of 52 out of 80 samples and

virulence factors in 18 of the samples. Potential hosts were predicted for 12.7 ± 3 % of phage

contigs. Among the most common host genera were Escherichia, Caulobacter and Bacillus.

Taxonomic classifications were assigned to 0.5% of the phage contigs on average. Among the

most common taxonomic assignments is crAssphage which was identified in 74 of the samples.

In conclusion, we found that the phage communities in sewage are extremely diverse and contain

many novel sequences.



Section IV: Genomics & Proteomics 123

Comparative genomics of CEV2, a T5-family bacteriophage infecting

Escherichia coli O157:H7

María Cecilia Aristimuño Ficoseco1*, María Jose Olaya Passarell1, Elvira Hebert1, Elizabeth

Kutter2* and Raul Raya1

1CERELA (Centro de Referencia para Lactobacilos)-CONICET. Tucumán, Argentina.

2Evergreen State College Olympia, WA, USA.

*E-mail: [email protected]; [email protected]

Escherichia coli phage CEV2, a siphovirus (phages with long non-contractile tails) of the T5-like

family, possesses a 118,347 bp genome (39.3% GC) which encodes 160 ORFs and 22 tRNAs.

Phages CEV2 and T5 showed similar host ranges, but only CEV2 infects virtually all tested E. coli

O157: H7 strains. In their irreversible final step of phage binding to their hosts, both phages

recognize, through their PB5 proteins (oad gene; 99% identity), the outer cell membrane FhuA

protein as receptor. However, their long tail fiber (LTF) proteins, responsible for the reversible

interaction that occurs between the phage and the LPS (antigen O) of the bacterium, presented only

46% identity. These results could explain the difference in host range observed between these

phages. A comparative genomic study of eight T5-like phages [T5 (AY543070.1 and

NC_005859.1); CEV2; SPC35 (HQ406778.1); DT571/2 (KM979355.1); DT57C (KM979354.1);

FFH1 (KJ190157.1); AKFV33 (HQ665011.1); and APCEc03 (KR422353.1)], conducted primarily

with the programs BlastP and BlastX; Clustal Omega; Easyfig 2.1; ARAGORN and tRNAscan,

revealed a collinear organization of their genes, with identities over 80% throughout their

sequences, with phages CEV2 and FFH1 being most closely related, except in two regions: the

“LTF” and the “tRNAs” regions located, respectively, at 80-85- and 28-37-kb of their genomes.

The LFT protein of CEV2 presented a 71% identity with that of phages FFH1 and APCEc03. The

number of tRNA genes in these phages ranged from 9 to 23. CEV2 encodes 22 tRNAs, although

Gly- and Tyr-tRNA isoaccepting species were not detected. On the other hand, T5 encodes 23

tRNAs and lacks tRNAs genes for glycine (Gly) and tryptophan (Trp). The greater diversity

observed among the LFT proteins of these phages indicates their ability to adapt to different

scenarios, in particular to the different types of specific receptors located in the LPS of their host

cells. In turn, the presence of a variable number and specific tRNAs may contribute to the overall

rate of protein synthesis during phage infection.



124 Section IV: Genomics & Proteomics

Making publicly available viral sequence data easier to use

J. Rodney Brister*, Olga Blinkova, Eneida L. Hatcher, Yuri Ostapchuck, Igor Tolstoy

National Center for Biotechnology Information, National Library of Medicine, National Institutes

of Health, Bethesda, MD 20894, USA


What’s in my tube? In the age of next generation sequencing, this is an increasingly important

question in the realm of biology. Perhaps the easiest way of answering this question is to find

similar sequences, but even when a match can be found, the effectiveness of this approach is

dependent on the availability and quality of sequences used for comparison. While GenBank and

other International Sequence Database Consortium members provide public platforms for the

storage and dissemination of data, the sheer volume of currently available data makes it

increasingly difficult to use. Moreover, the archival nature of these databases all but guarantees that

some aspects of the data will become outdated. Our group is focused on understanding the ever

expanding viral sequence space and making this data easier to use. Here I will describe the various

approaches we are implementing to build a validated layer upon viral GenBank and Short Read

Archive (SRA) sequence data. These approaches include working with other groups on taxonomy

issues, defining approaches to provide better nucleotide and protein reference space, developing

processing approaches that increase sequence data value, and building easy to use interfaces that

allow people to find the sequences they want.




Hairy jumbo phages of Enterobacteriaceae

Colin Buttimer1, Yannick Borne2, Hanne Hendrix3, Hugo Oliveria4, Aidan Casey5, Horst Neve6,

Olivia McAuliffe5, R. Paul Ross5, Colin Hill7, Jean-Paul Noben8, Jim O’Mahony1, Lars Fieseler2,

Rob Lavigne3, Aidan Coffey1*

1Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland; 2ZHAW – Zürcher Hochschule für Angewandte Wissenschaften, Institut für Lebensmittel- und

Getränkeinnovation, Fachstelle Mikrobiologie, Switzerland; 3Laboratory of Gene Technology, KU

Leuven, Leuven, Belgium; 4CEB – Centre of Biological Engineering, LIBRO – Laboratório de

Investigação em Biofilmes Rosário Oliveira, University of Minho, 4710-057, Braga, Portugal; 5Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland; 6Department of

Microbiology and Biotechnology, Max Rubner-Institut, Kiel, Germany; 6APC Microbiome

Institute and School of Microbiology, University College, Cork, Ireland; 8Biomedical Research

Institute and Transnational University Limburg, Hasselt University, Diepenbeek 3950, Belgium.


Myoviridae have the most sophisticated virion design, possessing a tail capable of contracting on

infection and generally having the largest genomes when compared to the other families. However,

only a small number of the known Myoviridae phages have genomes greater than 200 kbp; and

these are often referred to as “giant” or “jumbo” phages. These are not often isolated by

conventional methods, and this is believed to be due to their larger virion structures. As a result,

they have received less attention than the smaller members of the family. Examples of the jumbo

phages are Enterobacteria phage vB_PcaM_CBB (CBB) and Erwinia amylovora phage Y3; and

both are unusual in that they possesses atypical whisker-like structures along the length of their

contractile tails. With a genome of 355,922 bp, excluding a predicted terminal repeat of 22,456 bp,

phage CBB is the third largest phage sequenced to date, sharing homology with the Rak2-like

phages. Phage Y3 possesses a much smaller genome of 261,365, sharing homology with

Pseudomonas phages Lu11 and PaBG as well as Ralstonia phage RSL1. This work presents the

insights gained from the genomic and proteomic analysis of these atypical jumbo myoviridae

phages.




Host-phage interactions between novel jumbo phage PA5oct and

Pseudomonas aeruginosa

Katarzyna Danis-Wlodarczyk1*, Ho Bin Jang1, Bob Blasdel1, Yves Briers2, Jean-Paul Noben3,

Zuzanna Drulis-Kawa4, Rob Lavigne1

1 Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21, 3001 Leuven, Belgium 2 Laboratory of Applied Biotechnology, Ghent University, Valentin Vaerwyckweg 1, 9000 Gent,

Belgium 3 Biomedical Research Institute and Transnational University Limburg, Hasselt University, 3950

Diepenbeek, Belgium 4 Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology

Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77,

51-148 Wroclaw, Poland


The PA5oct, a novel jumbo myovirus, has a giant linear A+T-rich double-stranded DNA genome

(287,182 bp), making it the third largest sequenced Pseudomonas phage genome. Based on DNA &

RNA sequencing, 462 genes could be confirmed, of which only a small number could be

functionally assigned. The number of genes was further supported by structural mass spectrometry

analysis. The genetic relationships between PA5oct and other jumbo bacteriophages and other

Caudovirales viruses, was constructed using a protein-sharing network. The PA5oct, Escherichia

phages 121Q/PBECO4, Klebsiella phage vB_KleM-RaK2, Klebsiella phage K64-1, and

Cronobacter phage vB_CsaM_GAP32 presented closer relationships based on shared conserved

core genes. Furthermore, PA5oct, PBECO4, GAP32, T5, FelixO1 appear to be distantly diverged

members of Tevenvirinae.

To examine phage transcription across the temporal phases of infection cycle at single-nucleotide

resolution as well as the phage- host interactions, RNA-seq was performed based on three selected

infection time points (5, 15 and 25 min). Reads originating from the phage and the host at each

stage of infection were mapped to the phage and host genomes respectively, revealing that PA5oct

progressively dominates host transcription. Indeed, PA5oct transcripts represent 21% of total non-

rRNA transcripts within 5 minutes and eventually proceeds to 69% then 92% by middle and late

infection, respectively. The PAO1 host-mediated transcriptional response to PA5oct infection is

similar to 14-1, PEV2, YuA, and LUZ19 infection response. Interestingly, PA5oct progressively

dominates the mRNA and sRNA environment of the cell with strong transcription, as well as the

expression of an RNase-H-like protein putatively involved in degrading host transcripts. This shift

from host transcripts to phage transcripts reduces all host transcripts relative to the total in the cell.




Comparative genomics of rhizobiophages from western Canada

K.M. Damitha Gunathilake1*, Anupama P. Halmillawewa1, Marcela Restrepo-Córdoba1,

Benjamin J. Perry2, Christopher K. Yost2, Michael F. Hynes1

1University of Calgary, Calgary AB, Canada, 2University of Regina, Regina, SK Canada,


Bacteriophages that infect the rhizobia, bacteria that form nitrogen-fixing nodules on the roots of

legumes, have been studied for decades, and have been suggested as a means of manipulating

rhizosphere rhizobial populations. Genomic studies on rhizobiophages are relatively recent, and

only a few complete genome sequences are available in the databases. We have isolated a large

collection of phages that infect Rhizobium leguminosarum, Mesorhizobium loti, and R. gallicum

from rhizosphere soils from a variety of legumes in western Canada, and characterized them by

examining host range, lysogeny, morphology, and genome composition. Complete genome

sequences have been produced for six of them: vB_RleS_L338C genome: accession no:

KF614509, vB_RglS_P106B (KF977490), vB_RleM_PPF1 (KJ746502), vB_RleM_P10VF

(KM199770) and vB_MloP_Lo5R7ANS (KM199771). The completed genome of M. loti phage

Cp1R7ANS-C2 is also available but has not been submitted to a public database. Draft genomes

for a further five R. leguminosarum phages P9VFCI, V1VFA, AF3, P11VFA, L338G and M. loti

phage Cp1R7A-A1 have also been produced. Phages isolated using R. leguminosarum as host all

belonged to the Myoviridae or the Siphoviridae, whereas phages that infected M. loti were mostly

members of the Podoviridae, with one example of a probable Siphovirus. The majority of the

phages had genomes that were modified and resistant to restriction digestion with many common

enzymes. On the phages with larger genomes, such as P10VF, L338G, AF3, and P9VFC1, many

genes encoded predicted proteins with no significant homology to anything in the databases. We

have performed genome comparisons between our isolates, and comparisons and phylogenetic

analysis in the context of all rhizobiophages sequenced so far. Rhizobium phage P10VF shows no

significant similarities to 16 other whole genome sequences of Rhizobium, Mesorhizobium and

Sinorhizobium phages available in the NCBI database. However, it shows significant similarities

and phylogenetic relationship to Sinorhizobium meliloti Phage ΦM9 for which the complete

genome is now available. Furthermore, the draft genomes of Rhizobium phages P9VFCI and AF3

show similarities to P10VF, although both have genomes larger than P10VF. Another Rhizobium

phage, V1VFA, has a part of its genome identical to P10VF. In spite of being isolated from

different geographic locations, from soils planted to different legumes, and being isolated using

different strains, all three of phages P9VFCI, AF3 and V1VFA are related to P10VF as indicated

by the phylogenetic analysis with amino acid sequences of putative terminases and capsid proteins.

Siphophage P11VFA shows significant similarity to L338C, with identical terminase and capsid

proteins, but also contains large sections of DNA similar to a Sinorhizobium plasmid.

Mesorhizobium phages Cp1R7AS-C2 and Lo5R7ANS (Podoviridae) have similar genome

arrangements and sizes, and share some properties with T7-like phages. Mesorhizobium phage

Cp1R7A-A1 has a much larger genome with no strong similarities to anything so far sequenced.




Lytic bacteriophages present in the lower female genital tract targeting E. coli

Anna-Ursula Happel1, Shameem Z. Jaumdally1,2, Hoyam Gamieldien1, Heather B. Jaspan1,3,

Smritee Dabee,1 Shaun Barnabas,1,4 Linda-Gail Bekker MD PhD,1,4 Jo-Ann S. Passmore1,2,5, Remy

Froissart6*

1Institute of Infectious Disease and Molecular Medicine (IDM), University of Cape Town, South

Africa; 2DST-NRF Centre of Excellence in HIV Prevention, 3Seattle Children’s Research Institute,

University of Washington, Seattle, Washington, USA, 4Desmond Tutu HIV Centre, University of

Cape Town, South Africa, 5National Health Laboratory Service, Groote Schuur Hospital,

Observatory, Cape Town, South Africa, 6UMR 5290 MIVEGEC, CNRS-IRD-University of

Montpellier, Montpellier, France Desmond Tutu HIV Centre, University of Cape Town, South

Africa


Background: As in other human mucosal niches, bacteriophages are present in the lower female

genital tract (FGT). Little is known about the origin and transmission of bacteriophages in the FGT,

their bacterial targets and their effect on commensal bacterial communities in this niche over time.

Understanding the pathogenic interaction between bacteriophages and their specific bacterial hosts

in important since they may be a cause of dysbiosis in the FGT, typified by commonly occurring

bacterial vaginosis (BV). Several lines of evidence suggest the presence of bacteriophages in the

lower FGT viral sequences related to Caudovirales have been found in the lower FGT, and

temperate bacteriophages have been demonstrated within commensal or anaerobic pathogenic

bacteria from the FGT; in addition to these genital bacterial isolates having CRISPR loci and Cas-

genes. To our knowledge, no lytic phages have ever been isolated from human FGT and

characterized phenotypically. The aim of this study was to screen for and isolate lytic

bacteriophages from the lower FGT and test experimentally how these are involved in the etiology

of vaginal dysbiosis within human FGTs.




Isolation and Analysis of Cluster Q Phage Ein37 and F1 Phage Plumbus

Daniel Devonshire, Sophie Harrison*, Cody Allison, Samantha Altheide, Brianna Bordwell,

Mandy Chen, Adrienne Chitwood, Tokala Christensen, Carollyne Coulson, Nichole Criss, Melinda

Dauley, Christopher Davis, Kyle Dobbs, Jacob Durham, Michael Espy, Jarred Figlar-Barnes, Parke

Funderburk, Evan Furrer, Brianna Gage, Daniel Gilberts, Andrew Glassbrook, Kyle Grosten, Jenny

Green, Annelise Hooper, Casey Irwin, Coleman Kaestner, Katherine Kallus, Nolan Knaus, Tamara

Koledin, Randolph Kraus, Stacia Krause Ledbetter, Tessa LaLonde, Madesyn Lockwood, Anthony

Lopez, Kody Ludwig, Nacia Magass, Trevor McCliment, Bryanne McNamara, Robert Munoz,

Micah Needham, Melyssa Nocis, Taylor Nowotny, Luke Ogden, David Parker, Habib Quaraishi,

Olivia Parks, Kaden Phillips, Rian Plastow, Justin Roberts, Shayna Rossiter, Breck Roundtree,

Candace Saunders, Marissa Scoville, Alexea Seil, Petal Shohadai, Keelan Smith, Sean Staub,

Charles Thompson, Steven Vananzo, Amber Vidal, Jenny von Henkelman, Crystal Whitford,

Chandler Williams, Clarissa Dirks, Alberto Napuli, James Neitzel

*E-mail: [email protected] (The Evergreen State College)

The student participants in our third cycle of the SEA-PHAGE project were again members of the

interdisciplinary program Introduction to Natural Sciences, a year-long, full-time learning

community with integrated instruction in biology, chemistry, and science process skills. During the

fall quarter students collected and purified phages using enrichment cultures of Mycobacterium

smegmatis mc² 155 at 37 C. This year 23 phages were isolated from local soils, purified, and

entered into the PhagesDB collection. Compared to prior years, we had a lower percentage of

successful isolations. This set of phages had their DNA purified and analyzed by restriction

enzyme digestion and gel electrophoresis. Successful DNA extractions were completed for most of

the phages, although some difficulty in dealing with DNA stability was encountered. DNA quality

and restriction enzyme experiments were used to select genomes for sequence analysis. Phages

were also analyzed by transmission electron microscopy after negative staining with uranyl acetate.

This resulted in clear images of all of the isolated phages, which appeared to be Siphoviridae. The

phages sequenced and analyzed were Ein37 and Plumbus. DNA from these phages was sequenced

using the Illumina process at the Pittsburg Bacteriophage Institute. The sequence of Ein37 revealed

a 53,748 bp linear double stranded DNA genome with a sticky fourteen bp 3’ overhang and with a

GC content of 67.4 %. Analysis of the sequence of this phage confirmed that it was a Siphoviridae

in the Q cluster. BLASTn results showed that Ein37 had a 99% sequence identity with the other 7

members of cluster Q, indicating a high degree of similarity in this group, with almost half of the

predicted genes being Phams unique to this cluster.The genome of Plumbus is a 54,468 bp dsDNA

with a sticky ten bp 3’ overhang and a GC content of 61.1%. BLASTn results indicated its closest

relative was Cluster F1 phage Kimberlium. Both genomes were analyzed for potential protein

coding open‐reading frames using Glimmer and GeneMark, and protein functions were predicted

by BLASTp and HHPred, as well as examining synteny with related phages. Preliminary results

suggest the presence of 86 protein coding genes in Ein39 and 106 in Plumbus. We identified no

tRNA or tmRNA genes in either phage. Further work is being conducted to identify and confirm all

protein coding regions and to identify functions for predicted protein products.




Klebsiella podovirus KP32 produces two polysaccharide depolymerases,

promising anti-virulence tools

Agnieszka Latka1,2, Grazyna Majkowska-Skrobek1, Flavia Squeglia3, Rita Berisio3, Yves Briers2

and Zuzanna Drulis-Kawa1*

1Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology,

University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland 2Laboratory of Applied Biotechnology, Department of Applied Biosciences, Ghent University,

Valentin Vaerwyckweg 1, 9000 Gent, Belgium 3Institute of Biostructures and Bioimaging, National Research Council, Via Mezzocannone 16,

I-80134 Naples, Italy


Bacteriophages (phages) as natural enemies of bacteria have a huge ecological impact on the

dynamics of bacterial populations. To overcome the capsule barrier, some phages are equipped

with a virion-associated depolymerase – highly specific enzymes depolymerizing capsular

polysaccharides (CPS), thus also responsible for host cell recognition and receptor binding.

Bacteriophages possessing proteins with such activity are also able to better diffuse in the biofilm,

gaining access to the present microcolonies, and infecting embedded bacteria. Depolymerization of

CPS results in the increasing susceptibility to chemical and physical agents as well as to host

defences such as phagocytosis or complement-mediated killing. Although depolymerases do not

kill the bacterium, they have special interest as anti-virulence compounds that disarm the

bacterium, reducing or even avoiding the infection process.

In the presented study two genes encoding putative depolymerases have been identified in the

genome of K. pneumoniae specific KP32 podovirus. According to in silico prediction (PHYRE2),

the β-helical structure is highly probable for both proteins. Selected gene products have been

produced as recombinant proteins. Zymographic analysis using the crude exopolysaccharides

extracted from bacterial strains, confirmed their enzymatic activity. Both depolymerases, one of

which has been annotated as a tail fiber with similarity to T7 tail fiber region, while the other as

hypothetical protein, recognize and cleave CPS of different Klebsiella serotypes. They possess

mainly β-conformation, according to circular dichroism (CD) spectroscopy, what explains their

stability at high temperature and wide range of pH. The oligomeric state of all depolymerases is

trimeric, according to multi-angle static light scattering (MALS) spectroscopy. Microbiological

results showed that these depolymerases by modifying the phenotype of the bacterial cells without

affecting their viability, make the bacteria easier to eradicate by immune response mechanisms.




Characterization of Temperature Sensitive Mutants of Bacteriophage K

Justin Leavitt* and Jason Gill

Department of Animal Science, Texas A&M University, College Station, TX 77843, USA

Center for Phage Technology, Texas A&M University, College Station, TX 77843, USA.


Bacteriophage K is a Twort-like virus that infects many clinically important strains of

Staphylococcus aureus. Many of the genes in the phage K genome have unknown functions, and

many details about the phage K lifecycle require further study. Because Bacteriophage K has

therapeutic potential in treating staphylococcal infections, there is added incentive to learn more

about its biology. Using a classical genetic screen, a large number of temperature sensitive mutants

were isolated. These mutants were sequenced and further characterized by their ability to lyse the

host cell, make complete phage particles, and infect other host cells at the non-permissive

temperature. The phenotypes of these mutants and corresponding genetic changes will be

discussed.




Characterization of Bdellovibrio bacteriovorus phage

Kema Malki1*, Mark Martin2, Bentley Fane3, Mya Breitbart1

1College of Marine Sciences, University of South Florida, Saint Petersburg, Florida, USA

2Biology Department, University of Puget Sound, Tacoma, Washington, USA 3Department of Immunobiology, School of Plant Sciences, University of Arizona, Tucson, Arizona,

USA


Bdellovibrio are small, parasitic bacteria that are best known for their ability to attack and lyse

gram-negative bacterial cells. These δ-Proteobacteria were originally isolated from soil in the

1960s during a search for bacteriophage (phage). Since then, different strains of Bdellovibrio with

varying host ranges have been discovered from diverse environments including freshwater,

seawater, soil, and sewage. Though originally thought to be obligate parasites, strains of host-

independent Bdellovibrio have also been isolated and cultured in laboratory settings. Due to their

capability to lyse bacteria, Bdellovibrio have important ecological implications and have even been

proposed as potential antimicrobial agents for human health applications.

The purpose of this study is to expand our knowledge of phage capable of infecting Bdellovibrio

bacteriovorus. Several studies in the early 1970s isolated Bdellovibrio phage and described their

morphology and infection parameters. However, to date, genomic information is only available for

two Bdellovibrio phage, the small ssDNA phage ΦMH2K and the dwarf myovirus Φ1402, which is

the smallest autonomous myovirus. These phage genomes suggest that Bdellovibrio phage may

represent a large reservoir of unexplored diversity, particularly regarding phage with reduced

genome sizes. In this study, phage capable of creating plaques on the host-independent,

Bdellovibrio bacteriovorus strain HI 109 were isolated from sewage samples, plaque purified, and

are currently being characterized based on their morphology and genome sequences. The

availability of well-characterized Bdellovibrio phage systems in the lab represents a critical step

towards future work examining the net ecological impact of Bdellovibrio phage on environmental

bacterial communities.




Genomic Changes in a Host Range Mutant of Staphylococcus aureus

Bacteriophage Sb-1

K.V. Sergueev1, A.A. Filippov1, J. Farlow1, A.D. Reddy1, L. Kvachadze2, N. Balarjishvili2, M.

Kutateladze2, M.P. Nikolich1*

1 Walter Reed Army Institute of Research, Silver Spring, MD, USA; 2 G. Eliava Institute of

Bacteriophages, Microbiology and Virology, Tbilisi, Georgia.


Infections with methicillin-resistant Staphylococcus aureus (MRSA) is a major and growing

multidrug resistance problem in global medicine. Bacteriophage (phage) therapy is one of

promising approaches for treatment of MRSA infections in concert with antibiotics. S. aureus-

specific bacteriophage Sb-1 was isolated in the Georgian Republic (USSR) in 1977 and has been

widely used for treatment of various human S. aureus infections. Sb-1 has a very broad host range

within S. aureus that includes MRSA strains, and its host range can be further expanded. We

tested the activity of Sb-1 phage manufactured at the Eliava Institute against a panel of 25 diverse

US Military MRSA isolates and showed that it was able to lyse 23 of them. A host range mutant of

Sb-1 designated Sb-1M was then isolated to address the two additional strains in the military

MRSA panel, and then it was tested in comparison with the parental phage. Sb-1 was active

against 79 out of 94 (84%) diverse global S. aureus isolates, while eight additional strains (87/94 =

92.6%) were susceptible to Sb-1M. In an effort to better understand the genetic basis for this host

range expansion, we sequenced the complete genomes of Sb-1 and Sb-1M. Comparative genomic

analysis revealed a complex hypervariable repeat structure in the Sb-1 genome in which a distinct

allele correlated with the expansion of host range. This hypervariable region appears to be a novel

host range determinant that had not previously been characterized in Twort-like phages.




Comparative genomics of fully sequenced Cp8viruses, with reference to the

Irish Campylobacter phage, vB_CjeM_Los1

L. O’Sullivan1,3, H. Neve2, D. Bolton3, O. McAuliffe4, and A. Coffey1

1 Department of Biological Sciences, Cork Institute of Technology, Co. Cork, Ireland; 2 Max

Rubner-Institute, Federal Research Institute of Nutrition, Hermann-Weigmann-Strasse 1, Kiel,

Germany; 3 Food Research Centre, Teagasc, Ashtown, Co. Dublin, Ireland; 4 Biotechnology

Department, Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland.


Campylobacter jejuni is a commensal of up to 83.1% of Irish broiler chickens

and it has the ability

to cause severe gastrointestinal infection in humans. Phages against C. jejuni have been previously

isolated and proven effective in reducing C. jejuni numbers in live poultry and contaminated

carcass skin. The lytic C. jejuni Myovirus vB_CjeM_Los1 (hereafter Los1) was isolated from a

poultry faecal sample in a Cork slaughterhouse and it has proven the ability to lyse C. jejuni strains

isolated from both poultry and clinical origin. Well-studies Campylobacter phages are

representative of two genera, Cp220viruses and Cp8viruses as proposed by the ICTV. To date, 8

Campylobacter phages have been sequenced all with similarity to Cp8 (regarding size and whole-

genome sequence), including Los1 and this poster details some of the comparative analyses

performed using the eight genomes, whose phages have a diverse range of origins.




Broad host-range T5-like Yersinia enterocolitica phage R2-01

Maria I. Pajunen1*, Lotta J. Happonen2, Jin Woo Jun1, Ayesha Nawaz1, Laura Mattinen1 and

Mikael Skurnik1*

1Department of Bacteriology and Immunology, Medicum, Research Programs Unit,

Immunobiology, University of Helsinki, Helsinki, Finland. 2Lund University, Department of Clinical Sciences Lund, Infection Medicine, Lund, Sweden.

*E-mail: [email protected], [email protected]

Here we report the complete genome sequence and morphological characterization of the T5-like

siphovirus vB_YenP_R2-01 (in short R2-01) infecting Yersinia enterocolitica, a zoonotic food-

borne pathogen causing yersiniosis in humans and animals. Bacteriophage R2-01 encodes for 152

open reading frames (ORFs) and 19 tRNA-molecules on its 122,696 bp-long double-stranded DNA

genome including 9,901 bp terminal repeats. A total of 115 of the ORFs are similar to genes of the

well-characterized bacteriophage T5, including all genes involved in phage morphogenesis. The

genome of R2-01 is mostly syntenic to that of T5, and the major differences between the genomes

reside in areas with genes encoding for hypothetical R2-01 proteins. Other phages similar to R2-

01 are the Salmonella enterica serovar Typhimurium phage Stitch, the Escherichia phage EPS7,

the Salmonella phage Shivani, the Escherichia phage vB_EcoS_FFH1, the Enterobacteria phages

DT57C and DT571/2, the Escherichia phage AKFV33, and the Salmonella phage SPC35. The host

range of the phage is broad among Y. enterocolitica and it infects strains of both pathogenic and

non-pathogenic serotypes. It does not infect strains of Y. pseudotuberculosis, Y. nurmii, Y.

pekkanenii, Y. mollaretii, Y. frederiksenii, Y. intermedia, Y. ruckeri, Y. bercovieri, Y. kristenseni

(except one strain), and Y. aleksiciae. The phage also failed to infect Escherichia coli and Shigella

strains. Isolation of phage-resistant mutants from a transposon-insertion library revealed that the

phage receptor is the BtuB protein.




Development of a random mutagenesis protocol for marine cyanophage

Tamsin Redgwell1*, Andrew Millard 2, Dave Scanlan1

1 School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK. 2 Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK.

*E-mail: [email protected] Objectives:

Viruses are the most abundant biological entities on the planet, with viruses specifically infecting

cyanobacteria termed cyanophage. Marine cyanobacteria of the genera Prochlorococcus and

Synechococcus are key oceanic primary producers, and through their lysis following cyanophage

infection large amounts of dissolved organic matter are released into the environment directly

affecting marine carbon cycling. The discovery of auxiliary metabolic genes (AMGs) in

cyanophage genomes has altered our understanding of the role viruses play in marine ecosystems.

These AMGs are widespread in nature and whilst their specific roles are poorly understood it is

thought they play a key role subverting host metabolism to increase infection success. The

development of a random mutagenesis protocol for cyanophage would allow us to assign function

to these phage AMGs, and aid in the discovery of more. Through development of a chemical

mutagenesis system I hope to develop a protocol that not only is applicable to marine cyanophage

but can also be applied to freshwater cyanophage systems.

Methods:

A 24-hour period of hydroxylamine mutagenesis was conducted on both a coliphage (control) and

cyanophage S-PM2d, to create single nucleotide polymorphisms (SNPs). Time points were taken

every 6 hrs and dialysed to remove the mutagen. The time point at which there was a 3-log drop in

phage titre (99.9% killing) was used to identify the length of incubation required to create 1 SNP

per genome. To confirm this 15 individual plaques per time point were picked, and DNA extracted

and sequenced on an Illumina MiSeq using the Nextera XT library prep kit, for each phage. Reads

were trimmed with Sickle, assembled with SPAdes, and SNPs were called using VarScan.

Results:

In the coliphage control 99.9% killing did correspond to 1 SNP per genome. However, this was not

the case for cyanophage S-PM2d, where the number of SNPs per genome does not appear to be

related to the length of incubation with mutagen. Here, the number of SNPs is initially and

consistently higher than in the control. The types of mutation caused are also not limited to the

transition changes known to be generally attributed to hydroxylamine mutagenesis.

Conclusions:

It is evident that cyanophage possess auxiliary metabolic genes that likely affect the behaviour of

the host during infection. Elucidation of the function of these genes has been limited by the lack of

a cyanophage genetic system. Initial results show that hydroxylamine is capable of creating SNPs

in cyanophage genomes, but at a rate much higher than in a coliphage control. Further work will

aim to determine whether this behaviour is specific to this particular cyanophage, as well as

characterise mutants of interest.




Digging in the dirt for diversity - Genomic and molecular analysis of a

diverse set of E.coli phages

Imke Schmidt1, Christine Rohde1, Boyke Bunk1, Cathrin Spröer1, Manfred Rohde2 and Johannes

Wittmann1*

1Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures,

Braunschweig, Germany; 2Helmholtz Centre for Infection Research, Braunschweig, Germany


Escherichia coli is among the most common pathogens in poultry and the causative agent of

colibacillosis. This frequent disease creates significant economic losses and is commonly treated by

antibiotics, which in turn promotes the selection of multidrug resistant (MDR) bacteria. A high

incidence of MDR bacteria is problematic not only for animal health but also because of the

potential for zoonotic transfer to humans through contaminated food. Therefore, there is a high

demand to develop new strategies as alternatives to antibiotic therapies. In accordance with the

One-Health concept this project aims to isolate and characterize phages to fight E. coli infections in

broilers.

Apart from generating a suitable phage mixture for a field trial with broilers, one further aim of this

study is to get further insight into the diversity of E. coli phages followed by enhanced work on

taxonomic issues in that field.

Thus far, we isolated phages from sewage (21), surface water (4), manure (34), poultry (18)

respectively horse dung (1), and hospital wastewater (5). Morphological characterization revealed a

diversity of morphotypes (77% Myo-, 17% Sipho-, and 6% Podoviridae), genome sequencing

resulted in genomes sizes from 44 up to 370 kb. Annotation and comparison with databases

showed similarities in particular to T4- and T5-like phages. Host range analyses were performed by

spot tests on 72 E. coli strains with different resistance patterns.

We identified E28 as suitable strain for in vivo experiments, 11 of the isolated phages infect E28

with varying efficiency. In order to avoid the development of phage resistance we composed a

mixture of virulent phages that differ in host coverage, genotype, growth parameters, and inhibit

the growth of E28 in vitro.



138 Section V: Structure and Molecular Mechanisms

Investigating immunity mechanisms of an antiphage island in Vibrio cholera

Zachary Barth and Kimberley Seed*

University of California, Berkeley


The lytic myovirus ICP1 is engaged in an evolutionary arms race with its host, pandemic Vibrio

cholerae. V. cholerae strains possessing a phage inducible chromosomal island-like element (PLE),

are able to resist infection by ICP1. The V. cholerae PLE shares several functional similarities with

phage inducible chromosomal islands, a class of satellite phages found in Gram positive bacteria.

These functional similarities include excision of the island from the host chromosome upon phage

infection, followed by replication of the island, as well as mobilization and integration into other V.

cholerae cells. While the PLE does not prevent death of infected cells, it is able to completely

block production of ICP1 from infected cells, protecting V. cholerae on a population level. A

precise mechanism through which the PLE blocks ICP1 production remains unknown. Previously,

the PLE was found to decrease ICP1 DNA replication during infection, and to alter cell lysis

kinetics. Here, we further investigated the PLE’s impact on processes necessary for ICP1

replication in order to understand the mechanism(s) of PLE conferred immunity. We investigated

PLE effects on ICP1 gene expression, and tested for potential links between PLE replication and

impairment of ICP1 replication. Our efforts to elucidate the mechanisms underpinning PLE

mediated immunity will provide an improved understanding of phage-host interactions.



Section V: Structure and Molecular Mechanisms 139

Spacer capture and integration by a type I-F Cas1:Cas2-3 CRISPR adaptation

complex

Robert D. Fagerlund1, Max E. Wilkinson1,2, Oleg Klykov3, Arjan Barendregt3, F. Grant Pearce4,

Sebastian N. Kieper1, Howard W.R. Maxwell1, Angela Capolupo3, Albert J.R. Heck3, Kurt L.

Krause2, 1,5, Richard A. Scheltema3, Raymond H.J. Staals1 and Peter C. Fineran1*

1Department of Microbiology and Immunology and 2Department of Biochemistry, University of

Otago, PO Box 56, Dunedin 9054, NZ; 3Biomolecular Mass Spectrometry and Proteomics, Bijvoet

Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, and

Netherlands Proteomics Center, Utrecht University, 3584 CH Utrecht, NL; 4Biomolecular

Interactions Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800,

Christchurch 8020, NZ; 5Otago Centre for Electron Microscopy, University of Otago, PO Box 56,

Dunedin 9054, NZ


CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and

plasmids and integrate them as spacers into CRISPR arrays. In most CRISPR-Cas systems,

adaptation is driven by Cas1:Cas2 complexes. Type I-F systems however, contain a unique fusion

of Cas2 with the type I effector helicase and nuclease for invader destruction, Cas3. Here, we

present a structural model of the 400 kDa Cas14:Cas2-32 complex from Pectobacterium

atrosepticum bound to protospacer substrate DNA. The two Cas3 domains flank Cas2 of the

Cas1:Cas2 core and form a groove where the protospacer binds. We developed a sensitive in vitro

integration assay and demonstrated that Cas1:Cas2-3 catalyzed spacer integration into CRISPR

arrays via the integrase domain of Cas1 and this was independent of Cas3 activities. Integration

required at least partially duplex protospacers with free 3’-OH groups and leader-proximal

integration was stimulated by integration host factor (IHF). In a coupled capture and integration

assay, Cas1:Cas2-3 processed and integrated protospacers independently of Cas3 activity. These

results provide insight into the structure of protospacer-bound type I Cas1:Cas2-3 adaptation

complexes and their integration mechanism.




Imprecision during spacer acquisition by type I CRISPR-Cas systems

increases CRISPR diversity in bacterial populations

Simon A. Jackson1, Nils Birkholz1, Corinda Taylor1 and Peter C. Fineran1, 2


New Zealand. 2Bio-Protection Research Centre, University of Otago, PO Box 56, Dunedin 9054,

New Zealand.


Type I CRISPR-Cas systems use protospacer adjacent motifs (PAMs) to discriminate between

target and non-target sequences. PAMs are also cues for the Cas1-Cas2 complex that trims pre-

spacer substrates prior to their integration into CRISPR arrays. The Cas1 protein possesses a PAM-

sensing domain and cleaves pre-spacer substrates adjacent to PAMs (or within PAMs for type I-E).

This ensures crRNAs produced from spacers are compatible with the PAM-sensing domains of

interference complexes. The Pectobacterium atrosepticum type I-F CRISPR-Cas system has a

PAM-proximal spacer acquisition fidelity of greater than 93% in vivo. Most of the remaining

spacers result from cleavage within one nucleotide of the canonical position. This imprecision is

termed ‘slipping’. Here, we show that slipping typically results in spacers that are not functional for

interference. Instead, slipped spacers strongly stimulate primed CRISPR adaptation (priming),

resulting in increased CRISPR diversity within bacterial populations. We propose that slipping-

induced priming pre-empts escape mutations in phage and mobile genetic elements. Thus, the

diversity-generating characteristics of slipping might account for why higher fidelity Cas1-Cas2

activity has not arisen.




Quorum sensing controls adaptive immunity through multiple CRISPR-Cas

systems

Adrian G. Patterson1, Simon A. Jackson1, Corinda Taylor1, Gary B. Evans2, George P.C. Salmond3,

Rita Przybilski1, Raymond, H.J. Staals1 and Peter C. Fineran1,4


New Zealand; 2Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Rd,

Lower Hutt 5010, New Zealand; 3Department of Biochemistry, University of Cambridge, Tennis

Court Road, Cambridge CB2 1QW, UK; 4Bio-Protection Research Centre, University of Otago, PO

Box 56, Dunedin 9054, New Zealand.


Bacteria commonly exist in populations at high cell density, making them prone to viral predation

and horizontal gene transfer (HGT). To combat these invading elements, bacteria possess defence

strategies, such as the CRISPR-Cas adaptive immunity. These systems capture short sequences of

genetic material from invading nucleic acids (termed ‘spacers’) which are incorporated into

genomic CRISPR arrays, forming a genetic ‘memory bank’ of previous exposure. When expressed,

these provide RNA-guides for Cas protein complexes to identify specific cognate sequences and

mediate their destruction. Since their discovery, a plethora of research has been directed at

understanding CRISPR-Cas function, while comparatively little is known about their regulation

within natural hosts. Many bacterial populations coordinate their behaviour as cell density

increases, using a signalling mechanism called quorum sensing (QS). Here we show that QS

upregulates CRISPR-Cas activity to protect bacterial populations when they would be most

vulnerable to HGT and the spread of viruses. Strains unable to communicate were less effective at

defending against invading elements targeted by the CRISPR-Cas systems. Additionally, the

acquisition of new spacer sequences, required for subsequent identification of foreign targets, was

impaired in the absence of QS signalling. These results demonstrate that bacterial populations use

QS-mediated communication to regulate CRISPR-Cas activity, thereby modulating the efficacy of

community level defence.




Characterization of bacteriophage CPS1 infecting

Clostridium perfringens

Eunsu Ha1 and Sangryeol Ryu1,2

1Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and

Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea 2Center for Food and Bioconvergence, Seoul National University, Seoul 08826, Korea

Objectives: Clostridium perfringens is a Gram-positive, anaerobic, and spore forming bacterium

widely distributed in the environment. It is one of the most common causes of foodborne illnesses.

Bacteriophages are regarded as one of the alternatives to control pathogenic bacteria without

causing antibiotics resistance problem. However, the number of reported C. perfringens phages are

small and nothing much is known about their receptors. In this study, we isolated C. perfringens

virulent bacteriophage CPS1 and identified its receptor.

Methods: CPS1 was isolated from animal feces. CPS1 morphology was examined by transmission

electron microscopy. Phylogenetic tree was constructed with MEGA7 by the neighbor-joining

method. C. perfringens ATCC 13124 random transposon mutant library was constructed using Ez-

Tn5 (Epicentre) and screened for CPS1 resistant mutant. The gene disrupted by Tn5 insertion was

cloned in pET28a plasmid and expressed in E. coli BL21 (DE3). The protein was purified by Ni-

NTA chromatography and the histidine tag of the protein was removed by thrombin kit (Novagen).

Glucose oxidation assay and Morgan-Elson reaction were done with the purified protein to

elucidate the activity of the protein. Cell wall associated polysaccharides were quantified by

phenol-sulfuric acid analysis. Capsule staining was done with Anthony’s method.

Results: Analysis of full genome and morphology of CPS1 showed that CPS1 belongs to

Picovirinae subfamily. Screening of Ez-Tn5 random transposon mutant library of C. perfringens

ATCC 13124 revealed that C. perfringens ATCC 13124 CPF_0486::Tn5 had CPS1 resistance. The

gene was initially annotated as UDP-glucose 4-epimerase (galE). However, glucose oxidase assay

and Morgan-Elson reaction showed that CPF_0486 encoded protein had both UDP-glucose 4-

epimerase and UDP-N-acetyl glucosamine 4-epimerase activities, suggesting that the gene is gne.

According to the previous reports (Bernatchez et al., 2005; Valiente et al., 2008), gne but not galE

is required for synthesis of capsular polysaccharides (CPs). These results indicate that CPs are

possible receptors of the phage. Comparison of the amount of CPs produced in the wild type C.

perfringens and the mutant by phenol-sulfuric acid analysis revealed that the amount of CPs was

lower in the mutant. Capsule staining also showed that the wild type produced larger amount of

CPs than the mutant. These results support that CPS1 phage receptor is CPs.

Conclusions: C. perfringens bacteriophage CPS1 was isolated from environmental samples.

Analysis of morphology and genome of CPS1 revealed that CPS1 belonged to Picovirinae

subfamily. The CPS1-resistant mutant of C. perfringens was found to have mutation in the

CPF_0486 gene associated with biosynthesis of CPs, suggesting that CPs are the receptor of CPS1

phage.




Characterization of a potential toxin/anti-toxin system within Vibrio

cholerae’s anti-phage immunity island

Kristen N LeGault*, Kimberley D Seed

University of California, Berkeley, Berkeley, CA


Bacteriophage predation has driven the evolution of several anti-phage immunity mechanisms in

their bacterial hosts. One such system is the phage-inducible chromosomal-like element (PLE) of

Vibrio cholerae, recently shown to block infection by specific lytic phage (O’Hara et al. 2017).

The mechanism of phage-immunity conferred by the PLE is not yet known, as the PLE

encompasses a number of open reading frames (ORFs) of unknown function. Two of these ORFs

possess features of a toxin/anti-toxin system. Here we show that overexpression of the putative

toxin causes cell filamentation and a loss of viability, while co-expression of the putative anti-toxin

restores normal growth. However, bacterial adenylate cyclase two-hybrid (BACTH) assay suggests

that these genes do not interact with each other at the protein level. Two determine what these

proteins are interacting with, co-immunoprecipitation with the tagged toxin and antitoxin will be

down during phage infection. The role of the toxin/anti-toxin system within the PLE is not known,

yet we hypothesize that the toxin/anti-toxin may help maintain the PLE within the superintegron.

To investigate this hypothesis, the toxin and anti-toxin were expressed on plasmids in a PLE minus

background, either by themselves or together. Plasmid loss was lower when the toxin and anti-

toxin were co-expressed compared to when the anti-toxin was expressed on its own. Preliminary

results from a subsequent experiment suggest that PLE loss is higher when the toxin has been

deleted. Further molecular studies will improve our understanding of how the unknown ORFs of

the PLE function to prevent productive phage infections.




Septimavirus genus bacteriophage vB_PaeS_PS9N – virion DNA structure

and post-infection bacterial phenotypes

Aleksandra Głowacka a, Agnieszka Bednareka, Kamil Dąbrowski a, Jan Gaworb, Wioleta Woźnicaa,

Monika S. Hejnowicza, Beata Weber-Dąbrowskac, Anna Baraniakd , Marek Gniadkowskid, Robert

Gromadkab, Andrzej Górskic, Małgorzata Łobocka a,e,*.

aDepartment of Microbial Biochemistry, bLaboratory of DNA Sequencing and Oligonucleotide

Synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw,

Poland, cLaboratory of Bacteriophages, Ludwik Hirszfeld Institute of Immunology and

Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland, dDepartment of

Epidemiology and Clinical Microbiology, National Medicines Institute, Warsaw, Poland, eAutonomous Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw

University of Life Sciences, Warsaw, Poland


Pseudomonas aeruginosa bacteriophages are considered to be alternatives to antibiotics in curing

infections by antibiotic resistant P. aeruginosa strains. However, only phages that are obligatorily

lytic and cannot transfer DNA between bacteria by transduction can be considered as possible

candidates for therapeutic applications. Here we characterize a newly isolated, obligatorily lytic P.

aeruginosa siphovirus, vB_PaeS_PS9N (PS9N). Its genomic sequence appears to be highly

homologous to the sequences of Septima3virus genus phages (1). Certain of them in addition to

infecting P. aeruginosa strains, can also infect certain strains of Stenotrophomonas maltophilia (2).

The distribution of homologies between PS9N and its relatives is mosaic, with no single phage

being the most closely related over the entire genome length. However, based on homologies of

PS9N tail structures to the tail structures of selected Septima3virus genus representatives, we

predicted that the specificity of PS9N could also cover strains of two different bacterial families.

Indeed, of 13 clinical S. maltophilia isolates one was sensitive to PS9N. Despite the narrow range

of strain specificity against P. aeruginosa and S. maltophiliaI, PS9N could infect certain

carbapenem sensitive as well as carbapenem resistant P. aeruginosa strains. Surprisingly, the

dramatic lysis of susceptible cells infected with PS9N was followed after a few hours by the

overgrowth of phage resistant cells. Moreover, when the infecting phage was propagated in

meropenem resistant P. aeruginosa cells, cells that could grow on meropenem supplemented

medium appeared among the progeny of phage resistant cells. Whether these cells represent

transductants or spontaneous mutants whose selection is favored in phage resistant cells remains to

be found. We show that PS9N DNA is packed to phage heads by a headful packaging mechanism

and identify the PS9N pac site. Analysis of the PS9N genome indicates the lack of genes encoding

enzymes that degrade DNA of infected bacterium. Headful packaging and the lack of genes

encoding host DNA degrading enzymes are characteristic features of transducing phages. Thus, a

possible transducing potential of PS9N and other phages of Septima3virus genus should be taken

into consideration in the use of this phages for the therapeutic purposes.

1.Krupovic M, et al., 2016. Taxonomy of prokaryotic viruses: update from the ICTV bacterial and archaeal viruses

subcommittee. Arch Virol. 161:1095-1099.

2. Peters DL, et al., 2015. The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages

capable of cross-taxonomic order infectivity. BMC Genomics.16:664.

This work was supported by funds from the Operational Program ‘Innovative Economy, 2007-2013’ Development

Project No. POIG 01.03.01-02-003/08, and from the statutory funds for the Institute of Biochemistry and Biophysics of

the Polish Academy of Sciences




Capsid localization of the AFN38122.1 (A2), AFN38181.1 (A4),

AFN38152.1 (A8) proteins on staphylococcal phages 676Z and A3R.

Zuzanna Kaźmierczak1, Katarzyna Hodyra-Stefaniak1, Joanna Majewska1, Sylwia Nowak2,

Marek Harhala1, Paulina Miernikiewicz1, Katarzyna Zielonka1, Dorota Lecion1, Andrzej Górski1,

Krystyna Dąbrowska1, Marta Mazurkiewicz-Kania2, Andrzej Górski1, Barbara Owczarek1, Anna

Kłopot1, Karolina Wojtyna1

1Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12,

53-114 Wroclaw, Poland, 2Institute of Experimental Biology, University of Wrocław, Sienkiewicza

21, 50-335 Wrocław, Poland

Objectives: Bacteriophages A3R and 676Z infect Staphylococcus aureus strains pathogenic to

humans. Recently both phages were a subject of an extensive studies. Genomes of A3R and 676Z

were sequenced in 2014. The size of genomes are 141018 bp and 148564 bp, respectively. Both

phages are used in the Phage Therapy Unit in Wrocław, Poland. Phages were morphologically

described and sequenced. The protein structure of phages is not known.

Interests: In these studies we analyzed genomes of A3R and 676Z and annotations of hypothetical

capsid proteins. We have selected several genes encoding hypothetical capsid proteins. In further

research we cloned and expressed genes to obtain selected proteins in order to investigate whether

they are present on phages capsids.

Methods: C57Bl6/J mice were given purified phage proteins (A2 or A4 or A8) in PBS,

subcutaneously in day 0, 20, 40. Control group was given PBS s.c. After 40 days blood samples

were collected and evaluated for the presence of anti-protein IgG antibodies. Antibody levels were

measured by ELISA immunoassay. Phages were purified and concentrated. Next, phages were

incubated with an appropriate antibody conjugated with gold nanoparticles (5nm in diameter),

deposited on a girds, fixed and stained. Phages were examined by TEM.

Conclusions: These data show that proteins A2, A4, A8 are present on capsids of both

staphylococcal bacteriophages A3R and 676Z. A2 which was annotates as hypothetical major

capsid protein, occurs on both heads of staphylococcal phages. Protein A4 is helically present on

the tail of both phages. The base plate of A3R and 676Z phages is co-built by A8 protein.

Acknowledgements: This work was supported by the National Science Centre in Poland grant

UMO-2012/05/E/NZ6/03314 and by the Wroclaw Centre of Biotechnology, programme The

Leading National Research Centre (KNOW) for years 2014–2018.




Natural deletion mutant LysF1 of Kayvirus endolysin with a broad range of

lytic activity and characterization of its SH3b cell wall binding domain

Martin Benesik1, Jiri Novacek2, Lubomir Janda2, Radka Dopitova2,3, Marketa Pernisova2,3, Katerina

Melkova2, Jiri Doskar1, Lukas Zidek2,3, Jan Hejatko2,3, Roman Pantucek1*

1 Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, CZ-

61137 Brno, Czech Republic, 2CEITEC – Central European Institute of Technology, Masaryk

University, Kamenice 5, CZ-62500 Brno, Czech Republic, 3National Centre for Biomolecular

Research, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech

Republic


Bacteriophages with a broad host range are suitable candidates for fighting unwanted bacteria in

medicine, food industry, biotechnology and agriculture. Polyvalent ”Twort-like” bacteriophages of

the family Myoviridae and genus Kayvirus are used for the lysis of the serious bacterial pathogens

of the genus Staphylococcus. Their genomic, proteomic and structural characterization suggests

they are safe for phage therapy.

In the Czech Republic, the spontaneous broad host-range mutants 812F1 and 812K1/420 derived

from polyvalent phage 812, belonging to family Myoviridae and genus Kayvirus, are used for

phage therapy of staphylococcal infections. Their endolysin, designated LysF1, has a deleted

middle amidase (Ami-2) domain compared to wild-type LysK endolysin. This two-domain

enzyme, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase

(CHAP) domain and C-terminal SH3b cell wall-binding domain (CBD) has never been tested for

its antimicrobial activity. In this work, LysF1 and both its domains were prepared as recombinant

proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus

species of the 43 tested, while phage 812F1 was only able to lyse 14 of them. CBD had a crucial

role in binding the enzyme to the bacterial cell wall, since the lytic activity of the truncated variant

LysF1 containing the CHAP domain alone was decreased.

The results of a newly designed co-sedimentation assay of CBD with peptidoglycan (PG) showed

that it was able to bind to three types of purified staphylococcal PGs 11.2, 11.3 and 11.8, which

differ in their peptide bridge, but also to the PG of Streptococcus uberis (PG group 11.5), and this

capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy.

Using several different approaches, including NMR, we have not confirmed the previously

proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell-wall.

The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic

spectrum and therefore is an appropriate enzyme for practical use as an enzybiotic against most

frequent Staphylococcus pathogenic species, such as S. aureus, S. epidermidis, but also against

other significant pathogens such as S intermedius, S. lugdunensis, S. haemolyticus, S.

saprophiticus, and S. warneri. The broad binding spectrum of SH3b domain is a promising feature

for creating new chimeric endolysins by combining it with more effective catalytic domains.

Supported by the Ministry of Agriculture (QJ1510216) and Ministry of Health of the Czech

Republic (16-29916A).




Campylobacter bacteriophage DNA modifications are conserved in each

phylogenetic group

Jessica C. Sacher1, 2, Yan-Jiun Lee3, James Butcher4, Annika Flint4, Ian F. Connerton5, Alain

Stintzi4, Peter R. Weigele3, and Christine M. Szymanski1,2

1Department of Biological Sciences, University of Alberta, Edmonton, Canada, 2Complex

Carbohydrate Research Center and Department of Microbiology, University of Georgia, Athens,

Georgia, USA, 3New England BioLabs, Ipswich, Massachusetts, USA, 4Department of

Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada, 5Division of Food Sciences, University of Nottingham, Nottingham, UK


The refractory nature of Campylobacter bacteriophage DNA to manipulation has been cited for

several years, leading to the speculation that phages targeting this organism encode heavily

modified DNA. Campylobacter phages fall into three phylogenetic groups, all within the family

Myoviridae: Cp220likeviruses (group II), Cp8unalikeviruses (group III) and the as-yet

understudied group I phages. These groups were originally designated based on capsid and genome

size. However, the genomes of several phages from groups II and III have since been sequenced,

which has provided phylogenetic support for these groupings. It has also recently been

demonstrated that phages of groups II and III separate based on their host recognition properties,

with group II phages requiring motile host flagella and group III phages recognizing capsular

polysaccharide receptors.

To characterize the DNA modifications in Campylobacter phages, we analyzed genomic DNA

from one representative group I phage, two group II phages and two group III phages by liquid

chromatography/electrospray ionization mass spectrometry and restriction endonuclease profiling.

Remarkably, we found that phages from groups II and III display complete substitution of

deoxyguanosine residues for modified bases, while group I phage DNA displays partial base

substitution. We found the modifications to be conserved within, but not between, phylogenetic

groups. To gain insights into the biosynthesis of these non-canonical bases, we analyzed the

genome of phages from each of groups II and III. We found several putative group II modification

biosynthetic genes encoded on the group II phage genome, but a clear pathway for biosynthesis of

the group III-specific modification was not apparent. To identify candidate genes involved in

synthesizing this modified base, we examined total host and phage gene expression during group

III phage infection using RNA-Seq. Interestingly, we observed phage-induced up-regulation of

several C. jejuni purine biosynthesis genes as well as several genes involved in nucleic acid

modification and deoxyribonucleotide synthesis. Further studies are required to elucidate the extent

to which these or other phage-induced host genes contribute to the biosynthesis of modified phage

DNA.

Together our results point toward three separate mechanisms for Campylobacter phage DNA

modification, which may have evolved as a method of protecting DNA from enzymatic digestion

upon host infection. In addition to elucidating the biosynthetic pathways for synthesis and

incorporation of these modifications, our current and future studies are focused on the properties

that allow for their accommodation by phage DNA polymerases as well as the biological

implications of these modifications on phage-host interactions.




In silico analysis of phage proteins based on machine learning

Patryk Waśko


Introduction: Mammalian immune system is designed to protect them against potential pathogens.

For the needs of bioinformatics, these potential pathogens could be simplified to particles that do

not belong to the organism and are therefore recognized as foreign. However, some proteins trigger

more intense immune response than the others and the intensity of immune response can be

determined by a variety of biological and physical factors.

Objectives: Machine learning is a promising approach for an out of the box analysis of complex

sets of data with many dependencies. I applied the principles of machine learning to investigate

phage proteins with respect to their potential interactions with mammalian immune system. In the

analyses, I chose to focus on the induction of immune response based on major histocompatibility

complex epitopes.

Methods: In each iteration I analyzed a model with two sets of proteins: one that contains proteins

with high probability of being immunogenic and the second one with “normal” ones that reflect

standard distribution of proteins. Each protein in both sets was mapped to its epitopes with suitable

score from epitope database. Those were used to train the classifier that will be used in the second

phase of the project to analyze other – in this case phage – proteins.

Discussion: One of the features that comes with machine learning is that the results are self-tested.

When sets of data are used to train the classifier, random part of the set is picked to check the

accuracy of the method and logic. Self-testing also allows for determining which of the data have a

real impact on the investigated attribute.



Sections VI: Temperate Phages 149

Intact, widespread, and mosaic prophages of the prominent gut symbiont

Bacteroides

Danielle Campbell*, Patrick Degnan

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL


Objectives: The human gut harbors a dense and diverse microbial community that plays a

significant role in human health and development. The bacterial genus Bacteroides is one of the

most abundant bacterial genera in the human gut microbiota, playing important roles in complex

carbohydrate digestion and immune modulation. Despite this, little is known about the phages that

infect it. Characterized here is BVΦ01, a novel temperate phage infecting the Bacteroides.

Methods: Genetic modification of phage and host genomes was performed by allelic exchange

using a counter-selectable vector system. Release of prophages from bacterial hosts was tested by

PCR of cell and phage fractions. In silico comparative analyses of related prophages and genes was

accomplished using a combination of homology, alignment and phylogenetic approaches.

Results: Phage BVΦ01 was first identified as a prophage on the chromosome of the genetically-

tractable host B. vulgatus ATCC 8482. The bacterial attachment site (attB) utilized by phage

BVΦ01 is widely conserved across gut isolates of the family Bacteroidales. This attachment site is

used by several intact BVΦ01-like prophages as well was as several divergent prophages in other

Bacteroidales. Moreover, regions of the BVΦ01 genome have been observed in human gut-

associated viromes.

Prophage BVΦ01 is not inducible with well-known phage-inducing agents that cause DNA stress,

however it is constitutively released from the host at low levels under laboratory conditions. Phage

BVΦ01 harbors three integrase-like genes that are not only highly divergent from each other, but

also from other known phage integrase-like sequences. Here, we’ve demonstrated that one of these

integrases is required for BVΦ01 excision from the host genome. Cell lysis is predicted to be

caused by a conserved, putative holin-lysin-spanin operon in BVΦ01. Heterologous expression of

this operon is sufficient to cause death in both Bacteroides BVΦ01-lysogens and non-lysogens. Co-

culture experiments show that phage BVΦ01 confers context-dependent growth advantages for its

bacterial host.

Conclusions: Phage BVΦ01 is an intact prophage infecting the gut symbiont Bacteroides. We

have validated the activity of 4 highly divergent genes encoding essential functions for BVΦ01

lysis and integration. BVΦ01 has homology to a small cluster of closely related BVΦ01-like

phages and also a broader group of phages utilizing the same attB suggesting that BVΦ01 is a

representative of a broad group of mosaic phages infecting gut symbionts.



150 Sections VI: Temperate Phages

Insights into phage-mediated mechanisms of Mycobacterial host immunity

Catherine M. Mageeney*1, Marta Dies*2, Javier Buceta2, Vassie C. Ware1

1Department of Biological Sciences, 2Department of Chemical and Biomolecular Engineering

Lehigh University, Bethlehem, PA 18015 (*contributed equally to work)

Corresponding Author: [email protected]

Objectives: Mycobacteriophage Butters (cluster N) has one of the smallest known annotated

mycobacteriophage genomes: 41,491 bp with 66 ORFs and 4 novel genes (gp30, gp31, gp33, gp34)

with no mycobacteriophage homology called orphams, found within the central “variable” region

of the genome where nucleotide and gene content differences are most apparent between N cluster

phages. Work presented here is aimed at characterization of Butters orphams gp30 and gp31. First,

we have explored the cellular localization of both proteins: GP31 is predicted computationally to

be a 4-pass transmembrane protein while GP30 has no predicted membrane domains and is likely

cytoplasmic. Secondly, we have utilized M. smegmatis strains expressing gp30 or gp31 alone or

together to probe immunity mechanisms related to prophage-mediated defense against viral attack.

Methods: Butters GP30 and GP31 were tagged with a tetracysteine tag and expressed in E. coli.

Imaging of fluorescent proteins was completed using a microfluidics fluorescence microscopy

system. Immunity testing was completed using a series of phages from various clusters, excluding

other N cluster phages. Phages were spotted onto lawns of M. smegmatis expressing gp30 or gp31

alone or in tandem. Changes in phage infection for wildtype M. smegmatis, a Butters lysogen, and

strains expressing Butters proteins were monitored over a 48-hour period and efficiency of plating

was recorded.

Results: Mycobacteriophage Butters GP30 is localized in the cytoplasm while GP31 is localized to

the membrane, co-localizing with an E. coli membrane marker. With dual expression of GP30 and

GP31 from the same vector, GP30 localization changes and appears to be sequestered at the

membrane, suggesting a potential interaction between GP30 and GP31. Immunity results differ

depending on which genes are expressed. M. smegmatis strains expressing only gp30 mount a

defense against A3 phage PurpleHaze. Defense against viral attack is not observed when gp31 is

expressed alone but is abrogated when gp30 and gp31 are expressed together.

Conclusions: Taking the imaging and immunity data together, we propose that GP30, in its

cytoplasmic state, confers defense against specific viral attack in the lysogen. Additional factors

contributing to the defense mechanism are unknown, but may include GP30 release from

membrane sequestration (facilitated by GP31). Other Butters variable region genes (expressed in

the lysogen) may influence the putative release of GP30 from membrane association and confer

defense against other phages. A model for Butters defense against viral attack involving GP30 and

GP31 will be presented.




Lysogenic conversion of Stenotrophomonas maltophilia D1585 by temperate

phage DLP4

Danielle L. Peters*, Paul Stothard and Jonathan J. Dennis

Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.


Objectives: The use of bacteriophages to treat antibiotic resistant bacterial infections requires the

characterization of potential therapeutic phages. Lifestyle determination and genome annotation for

each phage must be conducted as temperate phages can produce lysogenic conversion of their

bacterial host. Bacteriophage DLP4 was isolated from asparagus soil collected in Edmonton,

Alberta, Canada using the highly antibiotic resistant bacterium Stenotrophomonas maltophilia

strain D1585. Characterization of DLP4 revealed it is a temperate phage capable of establishing a

lysogenic cycle in D1585. Due to the temperate nature of DLP4, genomic analysis and

identification of important moron genes was imperative to determine how suitable DLP4 is for

inclusion in a therapeutic phage cocktail.

Methods: Morphology was determined with a 4% uranyl acetate stain of DLP4 lysate on a copper

grid and viewed with a Philips/FEI transmission electron microscope. Host range analysis was

conducted with 27 clinical S. maltophilia and 11 Pseudomonas aeruginosa strains. Restriction

fragment length polymorphism analysis was performed using 36 FastDigest enzymes and separated

on a 1% agarose gel. To date, two moron genes of particular interest have been identified and are

being investigated for their functionality using complementation testing and observation of

phenotypic and transcriptomic changes between the DLP4 lysogen and wild type D1585.

Results: DLP4 is a member of the Siphoviridae family. Host range analysis indicated that DLP4

infects 14 of 27 clinical S. maltophilia isolates tested. All 36 restriction enzymes tested failed to

digest the genomic DNA. DLP4 was assembled into a 63,945 bp contig that was confirmed with

PCR and Sanger sequencing. DLP4 is predicted to encode 83 ORFs, one tRNA and an attP site.

Two moron genes currently under investigation are folA (BIT20_024) and ybiA (BIT20_065). The

folA gene encodes dihydrofolate reductase (DHFR), an enzyme responsible for trimethoprim

resistance. I-TASSER analysis of DLP4 DHFR predicts it is structurally similar to Bacillus

anthracis DHFR. Trimethoprim resistance in Escherichia coli (E. coli) DH5α increases 260-fold

with DLP4 folA expressed from the pBBR1-MCS plasmid compared to the empty vector control.

The ybiA gene encodes N-glycosidase YbiA, which is required for the swarming phenotype of E.

coli K-12. Complementation of E. coli BW25113 ybiA- with DLP4 ybiA shows partial

complementation, with swarming ability greatly improved over the ybiA- pBBR1-MCS control.

Conclusions: Although DLP4 is not suitable for therapeutic use due to the presence of moron

genes, these findings are the third documented incidence of lysogenic conversion in S. maltophilia.

More research into S. maltophilia temperate bacteriophages could help elucidate the role of phages

in S. maltophilia infections. Future research using reverse transcription PCR will identify whether

ybiA and folA are expressed in the lysogenic cycle so their potential impact on host cell

pathogenicity and antibiotic resistance can be determined.



152 Sections VI: Temperate Phages

Activation of a Bacillus subtilis prophage encoded on the pBS32 plasmid

releases defective particles

Jolene Ramsey, Bat-Erdene Myagmarjav, Melissa A. Konkol, Suchetana Mukhopadhyay, Daniel

B. Kearns

Indiana University Bloomington, Bloomington, IN


Objective: The ancestral Bacillus subtilis strain 3610 contains several prophages in the

chromosome, including PBSX and SPβ, that can be induced to excise and generate particles. The

ancestral strain also harbors an 84-kb plasmid called pBS32 that was lost during domestication of

commonly used laboratory derivatives. pBS32 encodes the protein ComI, which is a potent

inhibitor of DNA uptake by natural transformation, and the genes of a putative prophage. We

hypothesized that the coexistence of the prophage genes and competence inhibition were linked to

lysogeny and therefore sought to characterize the phage.

Methods: After induction with the DNA damaging agent mitomycin C, we monitored plasmid-

dependent host growth and lysis. Supernatant from lysed host cells was assayed for phage particles

and the ability to transfer plasmid DNA. Isolation of phage particles for transmission electron

microscopy was attempted using precipitation and various gradients.

Results: When chromosomal prophages were deleted, we observed pBS32-dependent cell killing

upon addition of mitomycin C. The plasmid copy number increased by nearly 100 fold, and was

protected in supernatant from DNase treatment by a heat-labile factor. A complex including the

ZpbH capsid protein from the pBS32 prophage sedimented in CsCl gradients, but particles were

not observed by electron microscopy, nor were naïve B. subtilis killed by addition of these purified

complexes. Killing activity was linked to expression of a plasmid-encoded sigma factor, ZpdN.

Conclusion: The extrachromosomal plasmid pBS32 of B. subtilis encodes a prophage that, when

activated, kills the host. We hypothesize that pBS32-mediated cell death releases a phagelike

particle that is defective and unstable.




Genomics of Staphylococcus sciuri siphophages

Michal Zeman1*, Ivana Mašlaňová1, Adéla Indráková1, Marta Šiborová2, Pavel Plevka2, Veronika

Vrbovská1, Jiří Doškař1, Roman Pantůček1

1Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611

37 Brno, Czech Republic. 2Central European Institute of Technology, Masaryk University,

Kamenice 5, 625 00 Brno, Czech Republic.


Objectives: Staphylococcus sciuri is a bacterial pathogen associated with infections in animals and

humans, and represents a reservoir for the mecA gene encoding methicillin-resistance in

staphylococci. Even though there have been 15 genomes of S. sciuri in the public databases, no

S. sciuri siphophages or prophages were known until now.

Methods: Genomes of S. sciuri strains P575 and P879, and two phages induced from these strains

were sequenced by Ion Torrent™ PGM. Phages were subjected to electron microscopy, adsorption

experiments, their genomes were in silico analysed for presence of pathogenicity related genes and

compared to publicly available prophage genomes. Twenty-one S. sciuri prophages were manually

located in S. sciuri genomes obtained from the public database. Phage genomes were compared by

Mauve and ClustalW.

Results: Transmission electron microscopy and cryo-electron microscopy confirmed that phages

φ575 and φ879 belong to Siphoviridae family. Cryo-EM revealed spike at the base plate of the both

phages. Phages φ575 and φ879 were able to adsorb to various hosts even from different

staphylococcal species but they were unable to propagate on non-S. sciuri strains. Reversible

adsorption to Staphylococcus aureus suggested a two-stage adsorption mechanism. Whole genome

sequencing revealed that both phages have genomes with cos ends, and the size and modular

structure of their genomes is similar to other staphylococcal siphoviruses. Phage φ575 encoded

genes for staphylokinase and phospholipase in the genome and phage φ879 was able to package

plasmid mediating resistance to aminoglycoside antibiotics and staphylococcal chromosome

cassette mec from host strain. Two additional prophages were identified in the S. sciuri strain P879

(phage φ879 was induced from this host). Comparative genomic analysis distinguished 10 different

prophages and divided them into 4 distinct clusters. Phage integration sites varied in the host

genome. No known virulence factors were found however metallo-beta-lactamase or

acetyltransferase were present in the genome of some prophages.

Conclusions: The identification of bacterial virulence factors and possible antibiotic resistance

determinants encoded by S. sciuri phages and prophages, their ability to package and transmit

mobile elements and to adsorb onto the cells of other staphylococcal species show that S. sciuri

siphoviruses may contribute to the horizontal gene transfer within the Staphylococcus genus.



154 Sections VII: Phage Therapy

Biology and genomics of an historic therapeutic Escherichia coli

bacteriophage collection

Abiyad Baig1, Joan Colom1, Paul Barrow1, Catherine Schouler2, Arshnee Moodley2, Rob Lavigne3

and Robert Atterbury1*

1School of Veterinary Medicine and Science, Sutton Bonington Campus, University of

Nottingham, United Kingdom, 2ISP, INRA, Université François Rabelais de Tours, 37380

Nouzilly, France, 3Department of Veterinary and Animal Science, Faculty of Health and Medical

Science, University of Copenhagen, 4Laboratory of Gene Technology, Department of Biosystems,

KU Leuven, Belgium


We have performed microbiological and genomic characterisation of an historic collection of nine

bacteriophages, specifically infecting a K1 E. coli O18:K1:H7 ColV+ strain. These phages were

isolated from sewage and tested for their efficacy in vivo for the treatment of systemic E. coli

infection in a mouse infection model by Smith and Huggins, 1982. Transmission electron

microscopy showed that six of the nine phages were Podoviridae and the remaining three were

Siphoviridae. The four best performing phages in vivo belonged to the Podoviridae family. In vitro,

these phages exhibited very short latent and rise periods in our study.

In agreement with their microbiological profiles, characterisation by genome sequencing showed

that all six podoviruses belong to the Autographivirinae subfamily. Of these, four were isolates of

the same species (99 % identity), whereas two had divergent genomes compared to other

podoviruses. The Siphoviridae phages, which were moderate to poor performers in vivo, exhibited

longer latent and rise periods in vitro. Two of the three siphoviruses were closely related to each

other (99 % identity), but all can be associated with the Guernseyvirinae subfamily. Genome

sequence comparison of both types of phages showed that a gene encoding for DNA-dependent

RNA polymerase was only present in phages with faster replication cycle, which may account for

their better performance in vivo. These data allow a more rational evaluation of the original in vivo

data and pave the way for the selection of phages for future phage therapy trials.

The current alarming rise in levels of antibiotic resistance is an increasing threat to animal and

public health. To address this concern there is an immediate need for new alternative therapeutics.

Phages have been investigated historically and shown to be effective in controlling bacterial

infections. The significance of the present study is that it correlates the microbiological, genomic

and in vivo performance of an historic collection of phages to identify a combination of

characteristics which can be used to make an informed and rational selection of phages for use in

the treatment of systemic E. coli infections.



Sections VII: Phage Therapy 155

Isolation of phages targeting different adherent-invasive E. coli strains

Andrei Sorin Bolocan*, Siobhan McDonnell, Ciorsdan Campion, Emma Guerin, Amanda Forde,

Fergus Shanahan, Paul Ross, Colin Hill

APC Microbiome Institute, University College Cork, Cork, Ireland


The threat of an antibiotic resistance crisis has revived interest in diverse biological approaches

against infectious diseases, including the use of phages against bacterial infections. Due to its

malleable genetic character, E. coli has one of the widest spectra of disease of any bacterial species.

E. coli phages are commonly isolated from sewage, hospital waste water, polluted rivers and faecal

samples of humans or animals. The adherent invasive E. coli (AIEC) pathotype has been associated

with intestinal diseases but its prevalence in these diseases remains largely unexplored. The aim of

this study was to isolate broad host range phages from human faeces to target different AIEC E.

coli isolates.

Phages were isolated from faeces using two enrichment steps, whereby the faecal material was

mixed with multiple- or individual- E. coli strains in Luria Bertani broth (LB) supplemented with

CaCl2 and MgSO4, and incubated shaking for 24h at 37°C. Following centrifugation and filtration,

samples were analysed for the presence of the phages using standard spot and double-layer plaque

assay techniques. Of 50 plaques tested we found at least five distinct phage types on the bases of

RAPD-PCR and DNA enzymatic restriction profile analysis. Genome sequencing revealed the

virulent nature of all five phages. Eighteen AIEC strains isolated from faeces and discriminated by

the fingerprint profiles generated by rep-PCR were included for host range assay. For comparison,

15 non-AIEC E. coli strains were also tested, including representatives of the uropathogenic E.

coli (UPEC- UTI), enterotoxigenic, E. coli (ETEC), enteropathogenic E. coli (EPEC)

enterotoxigenic E. coli (ETEC) and enteroaggregative E. coli (EAggEC or EAEC). The five phages

demonstrated broad host range specificity in a spot test, especially against UPEC and AIEC. We

plan to investigate their therapeutic potential for some of the diseases in which these pathotypes are

implicated as causative agents.




Phage D29 titer reduction due to shipping, saline preparation, and

aerosolization

Nicholas B. Carrigy1, Rachel Y. Chang2, Sharon S.Y. Leung2, Melissa Harrison3, Zaritza Petrova4,

Welkin H. Pope4, Graham F. Hatfull4, Warwick J. Britton5, Hak-Kim Chan2, Dominic Sauvageau3,

Warren H. Finlay1, and Reinhard Vehring1*

1 Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada, 2 Faculty

of Pharmacy, University of Sydney, Sydney, NSW, Australia, 3 Department of Chemical &

Materials Engineering, University of Alberta, Edmonton, AB, Canada, 4 Department of Biological

Sciences, University of Pittsburgh, PA, USA, 5 Centenary Institute of Cancer and Cell Biology, and

Sydney Medical School, Royal Prince Alfred Hospital, and University of Sydney, Sydney, NSW,

Australia


Objective: To determine titer reduction when anti-tuberculosis phage D29 is shipped, diluted into

saline, and aerosolized with clinically-relevant inhalation devices.

Methods: Phage D29 was amplified and shipped from Canada to Australia using cold packs and an

insulated container. At arrival, this lysate was diluted 1:100 in isotonic saline, and the titer

measured after 18 hours of storage at room temperature. The diluted phage D29 preparation was

then input to three inhalation devices: 1) PARI LC Sprint jet nebulizer; 2) Aerogen Solo vibrating

mesh nebulizer; 3) Respimat soft mist inhaler. The titer reduction due to aerosolization was

measured by capturing the emitted aerosol on a filter and performing full-plate plaque assays in

triplicate at multiple dilution levels with Mycobacterium smegmatis. A mathematical model

predicted the number of nebulization cycles the phage underwent with the jet nebulizer, which

corresponds to the number of baffle impactions.

Results: Titer was not significantly different (p > 0.2) after 2-day shipping with cold packs, during

which the temperature increased from 5°C to 20°C, with a mean temperature of 11°C. Neither was

there significant phage deactivation due to dilution into isotonic saline over the tested time period

of 18 hours (p > 0.1). Aerosolization titer reduction was device-dependent, being 3.7 ± 0.1

log(pfu/mL) for the jet nebulizer, 0.4 ± 0.1 log(pfu/mL) for the vibrating mesh nebulizer, and 0.6 ±

0.3 log(pfu/mL) for the soft mist inhaler. On average, phage were predicted to undergo 108

nebulization cycles prior to exiting the mouthpiece of the jet nebulizer.

Conclusions: Phage D29 stable after shipping and dilution in saline at room temperature for at

least 18 hours. It survived aerosolization depending on the inhalation device used. The vibrating

mesh nebulizer may be useful for animal studies requiring high titers of active phage delivered

quickly, and the soft mist inhaler for self-administration phage therapy applications. Repeated

baffle impaction may be the cause of titer reduction with the jet nebulizer, which makes this device

a poor choice for animal studies with D29. Other phage readily survive jet nebulization, however,

showing that robustness of phages is variable and needs to be tested on a case-by-case basis.




Preparation of liposome-encapsulated bacteriophage PEV2 formulation

Sharon S.Y. Leung1, Sandra Morales2, Warwick Britton3, Elizabeth Kutter4, Hak-Kim Chan1*

1 Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia, 2AmpliPhi

Biosciences AU, 7/27 Dale Street, Brookvale, Sydney, NSW 2100, Australia, 3The Evergreen State

College, Olympia, Washington 98502, USA, 4Centenary Institute of Cancer and Cell Biology, and

Sydney Medical School, Royal Prince Alfred Hospital, and University of Sydney, Sydney, NSW

2006, Australia


Objective: Liposome-encapsulated (LE) phages have been reported to improve phage survival rate

and biodistribution in body. Conventional thin film hydration followed by extrusion was generally

employed to produce LE formulation. This study investigated the feasibility of using a microfluidic

approach to encapsulate a Pseudomonas aeruginosa phage PEV2 into liposomes.

Methods: A microfluidic setup consisted of a stainless steel cross connector (U-430, IDEX Health

& Science, USA) and a fluorinated ethylene propylene tube with an internal diameter of 200 µm

and 100 mm in length were used to encapsulate phage into liposomes. A mixture of soy

phosphatidylcholine and cholesterol at a ratio of 4:1 dissolved in absolute ethanol with a total solid

content of 17.5 mg/mL was injected through the center inlet channel of the cross mixer. Phage

suspension was injected into the cross mixer from the two side channels intersecting with the center

channel. The produced liposome was collected into an Eppendorf tube at the outlet of the mixing

FEP tube. The lipid and phage suspension flow rates were set at 80 and 160 µL/min, respectively.

The size and polydispersity index (pdi) of the produced liposomes were analyzed by dynamic light

scattering using Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). The titer reduction

due to the encapsulation process and encapsulation efficiency was determined using a surface-

droplet (Miles-Misra) technique. The phage encapsulation efficiency (EE) was calculated

according to the equation EE (%)=100−(Cfree/Ctotal) × 100, where Cfree is the titer of unencapsulated

phage, and Ctotal is the total phage titer (unencapsulated and encapsulated phages) obtained by

lysing 0.5 ml of liposome-encapsulated phage formulation with 0.5 ml of bile salts (50 mM). Phage

encapsulation using conventional method at a hydration and extrusion temperature of 40 ºC was

performed to compare the encapsulation efficiency and titer reduction upon processing.

Results: The average size and pdi of liposomes produced by the microfluidic method were 242 ±

2.6 nm and 0.33 ± 0.01, respectively, which were larger than the liposomes generated by the thin

film hydration method (size = 207.2 ± 30.1 nm and pdi = 0.22 ± 0.06). The titer reduction caused

by the encapsulation process was similar for both methods (~0.6 log). The microfluidic approach

had slightly lower encapsulation efficiency (35 ± 3%) than the thin film hydration method (46 ±

18%). The formation of liposome in the microfluidic approach relies on the diffusion of ethanol

and water across the ethanol/water interface through a “self-assembly” mechanism. Therefore,

encapsulation of phage would depend on the availability of phage at the ethanol/water interface. On

the other hand, the thin film hydration method involved agitation between the lipid film and phage

suspension to form large heterogeneous liposome before they were extruded to form homogeneous

liposomes of smaller size. This mixing step would more likely increase the contact between phage

and lipid, hence higher encapsulation efficiency.

Conclusions: This study demonstrated the feasibility of a microfluidic approach to encapsulate

phage into liposomes. The effects of process parameters, total liquid flow rate and flow rate ratio,

on the phage encapsulation efficiency and phage viability will have to be optimized.




Nebulisation effects on structural stability of bacteriophage PEV 44

Ariel Astudillo1, Sharon Shui Yee Leung1, Elizabeth Kutter2, Sandra Morales3, Warwick J.

Britton4, Hak-Kim Chan1*

1 Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia, 2 The Evergreen State

College, Olympia, Washington 98502, USA, 3AmpliPhi Biosciences AU, 7/27 Dale Street,

Brookvale, Sydney, NSW 2100, Australia, 4Centenary Institute of Cancer and Cell Biology, and

Sydney Medical School, Royal Prince Alfred Hospital, and University of Sydney, Sydney, NSW

2006, Australia


Objective: Nebulization being used for inhaled phage therapy can cause phage titer losses which

may be the result of mechanical damage to the phages. This study aimed to determine effects of

nebulization on the structural stability and bioactivity of a Pseudomonas lytic phage PEV44, which

is a myovirus consisting of a hexagonal head and a contractile tail connected by a neck.

Methods: Three nebulizers were investigated: 1) Sidestream® jet nebulizer with Porta-neb®

compressor operated at 7.5 L/min, 2) Aeroneb® Go vibrating mesh nebulizer and 3) Omron® NE22

static mesh nebulizer. A volume of 3 ml PEV44 phage suspension (5.2×1010 pfu/mL) was

aerosolized and the emitted aerosols were collected using a test tube impaction apparatus. The

nebulized aerosol was drawn into an ice-cooled test tube placed inside an aspiration flask through a

Tygon tubing connected with a 2.0 mL plastic pipette at 8 L/min. The morphologies of phages in

the stock/nebulized samples were examined by transmission electron microscopy (TEM, JEOL-

JEM 2100 microscope, Japan) using random sampling to identify the proportion of different phage

morphologies. The titer reduction due to nebulization was determined using a surface-droplet

(Miles-Misra) technique. The viable phage respirable fraction (fraction of phages delivered by

aerosols smaller than 4.5 µm) of the three nebulizers was determined using a multi-stage liquid

impinger (MSLI, Copley, U.K.) operated at 30 L/min.

Results: The morphology of the phage in the nebulized samples was categorized into two groups:

“whole” (the capsid and tail held together) and broken (the capsid separated from the tail) phages.

Within the “whole” phage group, they were divided into three groups: 1) intact, 2) contracted tail

and 3) empty capsid. In the stock phage suspension, considerable portions of broken phages (36%),

“whole” phages with contracted tail (10%) and empty capsid (19%) were detected. The fraction of

broken phages was significantly increased after nebulization, with the jet nebulizer (83%) being

more pronounced than the mesh type nebulizers (50 – 60%). While the amount of intact phage

(from 35% to 2 – 10%) and phage with contracted tail (from 10% to 0.2 – 3%) were significantly

reduced, the proportion of phage with empty capsids were not significantly different after the

nebulization process, possibly due to innate elasticity properties of the phage capsids. The loss

titres obtained by TEM quantification (94%, 75% and 70% for the jet, actively and passively

vibrating-mesh nebulizers, respectively) were comparable with those obtained by the conventional

plaque assay results. Among the three nebulizers, the Omron nebulizer achieved a much higher

viable phage respirable fraction (30%) than the SideStream (3%) and Aeroneb Go (6%).

Conclusions: This study identified various changes on the phage structure and viability, and

respirable phage dose from different types of nebulizers. Understanding these effects and the phage

tolerance to nebulization stress can potentially improve our choice of the delivery method for

inhaled phage therapy.




Bacteriophage therapy treats cholera

Sudhakar Bhandare1^, Joan Colom2^, Abiyad Baig2, Jenny M Ritchie3 Paul Barrow2, Robert

Atterbury2*

1Department of Food Science and Agricultural Chemistry, Faculty of Agricultural and

Environmental Sciences, Macdonald Campus, McGill University, 21,111 Lakeshore Ste Anne de

Bellevue, Quebec, Canada, 2School of Veterinary Medicine and Science, Nottingham University,

Loughborough, Leicestershire, UK, 3Faculty of Health and Medical Sciences, AX Building,

08AX01 University of Surrey, Guildford, Surrey, UK.

^ These authors contributed equally to the study


Cholera is a persistent threat to public health and an endemic disease in many countries. Recent

reports of emergence of antibiotic resistance in Vibrio cholerae show the need to obtain new

effective treatments of cholera. One such treatment could be the use of bacteriophages. This study

evaluates the potential of phage being used as a biocontrol for V. cholerae.

Seven phages against V. cholerae were isolated in China and five were obtained from various

sources. These twelve phages were biologically characterised and their genomes were sequenced.

Amongst the phages studied, the phage vB_VcholP_1 had the widest host range (67 % of 91 V.

cholerae strains) and lacked genes related to lysogeny or virulence (antimicrobial resistance or

toxins). The biological characterization of vB_VcholP_1 showed that this phage had a latent period

of 12 ± 0.0 min and a burst size of 43 ± 5.5 PFU/cell. This phage was selected for use in a

therapeutic trial to treat V. cholerae infections in infant rabbits.

The animals in the control group developed cholera whereas no sign of disease was observed in the

phage-treated animals. V. cholerae reductions in the caecal fluids were 3.5, 4.1 and 3.9 log10 CFU/g

(P < 0.01). Similarly, the reductions in the mid colon were 3.0, 3.1 and 4.2 CFU/g (P < 0.01).

Reductions in the counts of more proximal segments upper, middle and lower small intestine were

comparatively lower than those in the distal regions, but still significant (P < 0.01). Phage

replication was detected in all the sampling sites and the MOI was constant through the intestinal

tract with values between 1.8 and 3.5.

Furthermore, phage vB_VcholP_1 was tested as a prophylactic treatment for V. cholerae infection

in the infant rabbit model. After treatment, the animals looked healthy and significant reductions of

V. cholerae were observed along the intestinal tract. In stools reductions of 3.4, 4.3 and 2.5 log10

CFU/g were observed (P < 0.001, P < 0.01, P < 0.05). Comparable reductions of 3.8, 2.9 and 4.0

log10 CFU/g were obtained in mid colon samples (P < 0.001 and P < 0.01). Finally, in the proximal

intestinal tract, reductions were similar to those obtained in the therapeutic trials (P < 0.001, P <

0.01). In this case, the phage replication was only evident in mid colon and stools with constant

MOI values ranging from 1.6 to 2.3.

No phage resistant V. cholerae colonies were detected in any of the treated animals. In conclusion,

phage vB_VcholP_1 applied as a therapeutic or prophylactic agent prevents the death of infant

rabbits infected by V. cholerae, therefore, it may be suitable for the treatment of humans.




Faecal phageome transfer reshapes the murine bacteriome after antibiotic

perturbation

Lorraine A. Draper1,2, Feargal J. Ryan1,2, Marion Dalmasso1,2,†, Pat G. Casey1,2, Angela

McCann1,2, Vimalkumar Velayudhan 1,2, R. Paul Ross1,3, Colin Hill1,2*.

1 APC Microbiome Institute, University College Cork, Cork, Ireland, 2 School of Microbiology,

University College Cork, Cork, Ireland, 3 Teagasc Food Research Centre, Moorepark, Fermoy, Co.

Cork, Ireland, †Present address: Normandie Univ, UNICAEN, ABTE, 14000 Caen, France


Background: It has become increasingly apparent that creating and maintaining a complex and

diverse gut microbiome is fundamental to human health. Thus, there are increasing efforts to

identify methods that can modulate and influence the microbiome, especially in those who due to

disease or circumstance have had their native microbiome disrupted.

Methods: The current study describes how the administration of virus-like particles (VLPs)

influenced and reshaped the murine microbiome of test mice following microbial perturbation with

antibiotics. Subsequent to administration of penicillin and streptomycin, test mice (n=8) received a

bacteria free-VLP enriched faecal transplant(s), while control mice (n=8) received a heat-treated

nuclease treated version of the same.

Results: Mice that received VLPs, in which bacteriophages predominate, had a bacteriome (as

determined via 16S rRNA sequencing) that separated into distinct groups via principle co-ordinate

analysis (PCoA) and contained differentially abundant taxa when compared to the control group.

Additionally through metagenomic sequencing of VLP DNA, we were able to obtain a snapshot of

the putative viruses present in the gut of test and control mice at various time points. It was

observed that viral content appeared to differ in both abundance and diversity.

Conclusions: This study has validated the role of bacteriophage in gut microbiome population

dynamics. There is potential to take advantage of such, especially with the increasing trend toward

performing FMTs. Up until now these have focussed on transferring living bacteria and spores; as

bacteriophage are non-living proteinaceous entities they could form a robust, inexpensive

alternative that is susceptible to standardisation and could be delivered as a freeze dried

formulation.




Isolation of broad host range bacteriophages active against multidrug resistant

Acinetobacter baumannii

Reham Samir, Maha Omran and Marwa ElRakaiby*

Faculty of Pharmacy, Cairo University, Cairo, Egypt


The evolution of pathogenic microorganisms and their acquisition of resistance elements against

antimicrobial agents is one of the most threatening global risks to human health. A “red alert”

pathogen, Acinetobacter baumannii, poses a particular challenge to the currently used antimicrobial

agents owing to its remarkable ability to up-regulate existing genes or acquire new resistance

mechanisms. With the emergence of multidrug or even pandrug resistant A. baumannii, new

treatment strategies are required. Phage therapy is a promising treatment option because of its high

specificity and minimal side effects.

Objectives: Here, we aim to preemptively fight multidrug resistant A. baumannii, before their

possible spread, using phages isolated from the Egyptian environment.

Methods: Eighteen Acinetobacter baumannii strains were isolated from patients in Egyptian

hospitals. All strains were molecularly typed by oxa51 gene primers. Bacteriophages were isolated

from sewage samples obtained from Al-Gabal El-Asfar treatment station in Cairo governorate.

Phages were purified and propagated in Tryptic Soya Broth (TSB), according to standard

procedures. The agar overlay plaque assay was used to check the lysate for the presence of phages

and subsequent isolation of pure phage particles. Host range of the isolated bacteriophages was

assessed against the clinical isolates. Phages with broad host ranges were chosen for further

analyses.

Results: Twenty-nine phages were isolated from the sewage samples using combinations of A.

baumannii clinical strains. Five of these phages showed high degree of infectivity against the A.

baumannii isolates.

Conclusion: Bacteriophages isolated from the Egyptian environment are capable of infecting and

lysing resistant strains of A. baumannii. Candidate phages will be characterized and identified then

their therapeutic and prophylactic abilities will be tested in murine animal models.




Evaluation of the therapeutic potential of bacteriophage ZCKP1 in the

treatment of Klebsiella pneumonia isolated from Egyptian diabetic foot

patients

Omar A. Taha1, Phillippa Connerton2, Ian Connerton2 and Ayman El-Shibiny1*

1Biomedical Sciences, University of Science and Technology, Zewail City of Science and

Technology, Sheikh Zayed District, 12588, Giza, Egypt, 2Division of Food Sciences, School of

Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire

LE12 5RD, United Kingdom


The predicted growth in infection by multidrug resistant (MDR) bacteria necessitates prompt

efforts towards developing alternatives to antibiotics, such as bacteriophage therapies. Immuno-

compromised patients with diabetes mellitus are particularly prone to foot infections by MDR

Klebsiella pneumonia, which may be compounded by chronic osteomyelitis. Bacteriophage

ZCKP1ɸ, isolated from Giza, Egypt, was tested in vitro to evaluate its lytic activity against a MDR

Klebsiella pneumonia KP/01, isolated from the foot of a diabetic patient in Egypt. Characterization

of ZCKP1ɸ indicated that it belonged to the Myoviridae family of bacteriophages with an

approximate genome size of 48 kb. ZCKP1ɸ lysed a range of osteomyelitis pathogenic agents with

activity against different Klebsiella spp. strains but also against Proteus spp. and E. coli isolates.

The bacteriophage reduced viability of its hosts by ≥2 log10 CFU/ml at 25°C and remained

functional at 4°C, permitting treatment over a range of temperatures. The phage demonstrated a

significant reduction in biofilm biomass (more than 50%) and viability (up to 66%) with high MOI

(50 PFU/CFU), achieving the greatest effect. These characteristics of ZCKP1ɸ phage indicate

potential therapeutic value to treat Klebsiella pneumonia infections and warrant further

investigation.




Preliminary pharmacokinetic study of phage therapy for the control of

Salmonella in commercial broilers

Juan C. Farfan-Esquivel, Martha J. Vives Florez1

1 University of Los Andes, Colombia.

E-mail: [email protected], [email protected]

Salmonella is a common foodborne pathogen usually found in broilers and related products. Due to

the increasing acquisition of mechanisms that generates resistance to antibiotics there is a necessity

to find new methods for its control. One of these alternative methods is phage therapy, which has

been tested in animal models and germ-free chickens. Nevertheless, this approach has not been

proven in broilers in a real setting or at a production scale. The aim of this project was to perform a

pharmacokinetic analysis of a selected phage from a phage cocktail effective against Salmonella, in

broiler chickens housed in a production facility.

The first, necessary step of the project was the evaluation of the protective effect of three different

excipients to improve the stability and viability of a phage. The excipients tested were the

following: saccharose 64% (SAC), magnesium hydroxide 8.4% (MGH), calcium carbonate 30%

(CAR). Stability assays with excipients were performed in three temperatures: 4°C, 25°C and

37°C. After one month, all excipients retained similar phage titers (1010 PFU/ml) at 4°C and 25°C.

At 37°C, a decrease in phage titer was measured: SAC 106 PFU/ml, MGH 107 PFU/ml, CAR 109

PFU/ml.

To evaluate acid stability, a model was developed resembling the average pH values and retention

times present in the digestive tract of 22-days broilers. Phage suspensions in excipients were

consecutively transferred in three solutions of hydrochloric acid (HCl) at different pH values: 2 to 5

and 5 to 6. Calcium carbonate and magnesium hydroxide were effective at protecting the phages

from acid with the concentration of phages reduced by three orders of magnitude (1010 to 107

PFU/ml). Magnesium hydroxide the best excipient, while saccharose had no protective effect.

The pharmacokinetic assay was designed with the following conditions: a phage cocktail (with a

titer up to 1010 PFU/ml) was dispensed into the drinking water of 22-day old broilers (n=20). Four

treatments were applied: one negative control group with saline solution without phages (n=5);

another negative control group with the most protective excipient and without phages (n=5);

phages in saline solution with no protective excipient (n=5); and phages with the most protective

excipient (n=5). Blood samples were taken at 0, 2, 4, 8, 24 and 48 hours from the beginning of the

treatment. Fecal samples were taken at 0, 2, 4, 8, 24 and 48 hours after the beginning of the

treatment. Phage titers in these samples were measured by quantitative PCR using a single phage as

a marker for the cocktail. The purpose of this study was to improve our understanding of the

practicalities of phage treatment in a real scenario to advance phage therapy in the industry.




Characterization of lytic bacteriophages active against drug resistant clinical

isolates of Pseudomonas aeruginosa

Kirill Sergueev 1, Nathan Brown 2, Jenny He 1, Akhil Reddy 1, Mikeljon Nikolich 1, and Andrey

Filippov 1 *

1 Department of Bacteriophage Therapeutics, Bacterial Diseases Branch, Walter Reed Army

Institute of Research, 503 Robert Grant Ave., Silver Spring, MD, USA; 2 Department of Infection,

Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.


Pseudomonas aeruginosa is a frequent and important cause of multiple infections, with common

multidrug resistance and robust biofilm formation. Expanding antibiotic resistance limits

therapeutic options and necessitates the search for alternative antibacterials including lytic

bacteriophages (phages). Multiple publications have shown high efficiency of phage therapy

against experimental and veterinary P. aeruginosa infections. Since P. aeruginosa phages have

limited host ranges, the recommended approach is to use phage cocktails to cover the diversity of

clinical isolates. Rational formulation will require the use of phages that belong to different

families and bind to different receptors to prevent phage resistance.

The objective of this work was to isolate and characterize a diversity panel of potentially

therapeutic phages active against P. aeruginosa. Phages were isolated from sewage and

environmental waters, using the cultures of several clinical isolates of P. aeruginosa for

enrichment. The phages were first screened based on plaque morphology and host ranges

determined on a small number of strains, and the final screening was done using DNA isolation and

digestion with EcoRV. This allowed for the identification of 30 new different bacteriophages lytic

for P. aeruginosa. Based on full genome sequencing and electron microscopy, the phages belong to

five different phylogenetic groups. The genome sizes varied from 32 to 89 kb. Host range testing

on 52 multidrug resistant strains of P. aeruginosa mostly isolated in military hospitals revealed

lytic spectra ranging from 11% to 71%. Overall, 93% of strains were susceptible to one or more

phages from the panel. Three phages demonstrated a marked lytic activity against P. aeruginosa

PAO1 biofilms. Therefore, several of the 30 diverse lytic P. aeruginosa phages we isolated and

characterized are promising therapeutic candidates.




Bacteriophage engineering for therapeutics

Aurelija Grigonyte, Andrew Millard*, Alfonso Jaramillo

University of Warwick, UK


Bacteriophages are being increasingly explored as an alternative to antibiotics. The ability to

engineer bacteriophages will allow advantageous alterations of their phenotype, e.g., increased host

range, making them more pertinent as therapeutics.

In this study, we show how marker-based (trxA) as well as marker-less (CRISPR-Cas9) selection

can be used for engineering using bacteriophage T7 as a model system. Here we generated a

chimeric phage for which T7 was used as the main scaffold and appended with a tail fiber from

BBP-1 phage.

We have showed that the marker based method is more suitable for the tail fiber engineering. In

developing this system, we also observed co-infection between T7 and engineered phage, where

both wild type and engineered phage are required for a successful infection (figure below). The

idea of co-infection challenges the T7 exclusion principle previously delineated in the literature.




Hypersensitivity to phages is a rare phenomenon

Karolina Wojtyna*, Zuzanna Kaźmierczak, Joanna Majewska, Dorota Lecion, Anna Kłopot,

Weronika Kęska, Paulina Miernikiewicz, Barbara Owczarek, Marek Harhala, Andrzej Górski,

Krystyna Dąbrowska

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla

12, 53-114 Wroclaw, Poland


Phages can induce specific antibody production in mammals. According to the current state of

knowledge there are no reports documenting hypersensitivity to phages, suggesting that it is not

common. However, hypersensitivity to phages, if present, could have a serious impact not only on

phage therapy, but on all other medical applications of phages.

We investigated T4 as a model phage and 676Z, A3R, F8 - therapeutic phages from the Phage

Therapy Unit. First, we examined human sera to determine presence or absence of phage-specific

IgE in population. Sera were obtained from 50 healthy volunteers who had neither been subjected

to phage therapy nor involved in phage work. Second experiment was the evaluation of phage-

specific IgE level in sera of mice exposed to phages. Mice were challenged orally (4 x 109 pfu/ml

in drinking water continuously for 100 days) or intraperitoneally (1010 pfu, injections were repeated

after 20 and 50 days). Sera were collected at following time points: day 1, 10, 50 and 100 after first

immunization.

To analyze hypersensitivity-promoting conditions, we used oral mice allergy model. Mice were

injected subcutaneously with highly purified phage preparation in dose 5 x 109 pfu with an addition

of Al(OH)3 as an adjuvant promoting hypersensitivity reaction. The injection was repeated after 14

days. Seven days after the second sensitization mice were given phage preparations in drinking

water in dose 5 x 109 pfu/ml. IgE levels specific for tested phages in all experiments were evaluated

by ELISA.

We did not observe high level of phage-specific IgE in any of examined samples. In human

population only one sample (a person with other symptoms of general hypersensitivity problems)

had detectable level of phage-specific IgE. No increase of IgE production in mice exposed to

phages in normal conditions was observed. In oral allergy model only one group of animals (676Z)

responded with a low increase of specific IgE level. Other groups did not respond in IgE level,

even though the adjuvant was applied.

These results demonstrate that hypersensitivity to phages is a rare phenomenon. In our study, it

appeared in very specific, allergy-inducing conditions. This reaction also seemed to depend on

individual phage properties. However, the exact mechanism of this reaction still remains unknown.

Project supported by Wroclaw Centre of Biotechnology, programme The Leading National

Research Centre (KNOW) for years 2014-2018.




Real-time biodistribution imaging of intravenous and intraperitoneal

Pseudomonas aeruginosa bacteriophage using molecular labeling

Derek Holman1, Matthew P. Lungren2, Jonathan Hardy3, Chris Contag1,2, 3, Francis Blankenberg2*

1Department of Pediatrics, Division of Medical Genetics and Biochemistry,

Stanford University School of Medicine, Stanford, CA 94305, USA, 2Department of Radiology, Division of Pediatric Radiology and Nuclear

Medicine, Lucile Packard Children’s Hospital, Stanford, CA 94305, USA, 3Department of Microbiology and Immunology, E150 Clark Center MC 5427,

Stanford University School of Medicine, Stanford, CA 94305, USA


Bacteriophages are ubiquitous viruses which have adapted to infect and replicate within target

bacteria, their only known hosts, in a strain specific fashion with little to no cross infectivity. The

recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting

bacteriophages for the imaging and treatment of bacterial infection in humans.

In order to develop a method with which to image, in real-time, the biodistribution of phage

treatments via radiolabeling, we examined the effects of hydrazinonicotinamide (HYNIC)

conjugation and removal of bacterial DNA on the infectivity, biodistribution, and radionuclide

imaging of a Pbunavirus bacteriophage lytic for a clinically relevant strain of Pseudomonas

aeruginosa, a common gram negative bacterial pathogen often resistant to multiple antibiotics.

We found all but the briefest reaction of concentrated bacteriophage with HYNIC (≤ 3 minutes)

resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and

removal of bacterial DNA was needed to avoid high non-specific uptake of HYNIC-labeled

bacteriophage within the liver and spleen as well as prolonged circulation in the blood. We also

demonstrated the surprisingly wide soft tissue and organ biodistribution and rapid

pharmacokinetics of Tc99m-HYNIC-labeled bacteriophage in normal mice as well as its imaging

characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas

aeruginosa. In conclusion, the preservation of bacteriophage infectivity and removal of all bacterial

containments including DNA are critical methodologic considerations in the labeling of

bacteriophages for imaging and therapy.




Assessment of bacteriophage cocktails against MDR biofilms Anna C. Jacobs1*, James M. Regeimbal2, Luis A. Estrella3, Javier Quinones3, Matthew S. Henry3,

Nathan A. Franklin4, Samandra Demons1, Biswajit Biswas3, Joseph C. Wenke4, Theron C.

Hamilton3, Michael G. Stockelman2, Stuart D. Tyner1.

1Walter Reed Army Institute of Research, Silver Spring, MD; 2Naval Medical Research Center,

Silver Spring, MD; 3Naval Medical Research Center-Frederick, Fort Detrick, MD; 4U.S. Army

Institute of Surgical Research, San Antonio, TX.


Objectives: Some of the most challenging infections among war wounded are those caused by

biofilm-producing, multidrug resistant bacteria (MDR), which may require extended antibiotic

treatment and can result in treatment failure. Our research is focused on developing phage cocktails

to target complex MDR infections, including those associated with implants or orthopedic

hardware. We have developed a pipeline for the isolation, purification, and pre-clinical assessment

of phage cocktails against P. aeruginosa and S. aureus in preclinical trauma models. Our main

objective is to develop therapeutic phage cocktails that can be used in conjunction with antibiotics

to treat complex wound infections.

Methods: Phages were isolated from raw sewage water, and phage cocktails were compounded

using high throughput phage lysis assay. Phage combinations that resulted in synergistic bacterial

killing were further assessed in in vitro assays, including biofilm assays to determine if phages

could penetrate and destroy mature biofilms, and checkerboard assays to evaluate phages in

combination with antibiotics. Phage cocktails were also evaluated in in vivo mouse and rat wound

models. The measured outcomes of these animal models included weight loss post-surgery, clinical

signs of infection, bacterial burden as determined by bioluminescence signal, CFU, wound size,

and healing time.

Results: In vitro biofilm assays of P. aeruginosa or S. aureus showed that the phage cocktails

could reduce established biofilm by at least 50% based on crystal violet staining. In a proof-of-

concept mouse dorsal full-thickness cutaneous wound model, S. aureus-infected, phage-treated

mice had significantly smaller wounds than the control-treated mice (P ≤ 0.01) through day 10

post-infection, with a median area of 44 mm2 compared to 67 mm2. A preliminary study assessing a

S. aureus phage cocktail in a clinically-relevant rat femur defect model resulted in complete

clearance of bacteria from bone and hardware of several of the phage-treated rats, while all control-

treated rats had a bacterial burden of 106 CFU in bone and hardware.

Conclusions: Bacteriophages represent a novel therapeutic option for recalcitrant, MDR bacterial

infections. Our preliminary results suggest that phage cocktails can reduce biofilm and bacterial

burden in vitro and in in vivo animal models. Studies are ongoing to further evaluate both P.

aeruginosa and S. aureus alone and in the presence of antibiotics.




Novel Klebsiella bacteriophages to control the spread of

Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumonia

in hospital setting

Jongsoo Jeon*, Dongeun Yong, Kyungwon Lee

Department of Laboratory Medicine and Research Institute of Bacterial Resistance,

Yonsei University College of Medicine, Seoul, Korea


Objectives: In recent years, antimicrobial resistance has become a major medical threat

worldwide. Among them the rapid increase in Klebsiella pneumoniae carbapenemase (KPC)-

producing Klebsiella pneumoniae is one of the particular global issues challenging the health care

setting. In order to overcome the problems, bacteriophages have been newly reviewed as an

alternative strategy for the control of these pathogens.

Methods: Fifty-eight KPC-K. pneumoniae clinical strains were collected from a university hospital

in South Korea and these strains were used to isolate lytic phages from sewage water samples. Host

ranges of isolated phages were determined by the spot test and these phages were characterized by

transmission electron microscopy, one-step growth curve, adsorption rate, thermal/pH stability

analysis and host cell lysis test.

Results: In this study, we isolated and purified 4 K. pneumoniae phages (Bϕ-U2874, Bϕ-N54, Bϕ-

N98, Bϕ-N137), which cause lysis of KPC-K. pneumoniae strains, from sewage water at a hospital

in South Korea and these phages were characterized in detail. In morphological properties of

phages by TEM, all K. pneumoniae phages belong to the family Siphoviridae with an isometric

head and a long flexible tail. These phages exhibited a very broad spectrum against clinical KPC-K.

pneumoniae isolates used in this study, but they did not lyse other bacterial strains including Gram-

negative or positive strains. These phages showed high absorption rate > 75-99% within 5 min and

bust size of 24-74 PFU/cell. Most of them exhibited stability at 40-50°C and pH 4-10. All phages

showed strong host cell lytic activities at MOI=10.

Conclusions: Overall, several in vitro results revealed that 4 novel K. pneumoniae phages have a

potential as an antimicrobial agent to control the spread of KPC-K. pneumoniae strains in clinical

settings and can be considered as therapeutic agents for infectious diseases caused by KPC-K.

pneumoniae strains. Further studies will be performed on next-generation sequencing (NGS) of

selected phages and in vivo test for efficacy and safety of phages by using mouse infection model.




V. cholerae O1 phages: effective subtyping and potential therapeutic mean for

cholera outbreaks

T. Kokashvili1, N. Janelidze1, T. Koberidze1, K. Porchkhidze1, G.Tsertsvadze1, A. Huq2, and M.

Tediashvili1

1 George Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia; 2 Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA

The disease cholera, caused by highly variable waterborne bacterium V. cholerae O1, remains a

serious public health concern worldwide, especially in the developing countries with warm climate.

The global climate change increases the possibility of its emergence in the non-endemic regions.

Thus, development of effective means of detection, identification and subtyping of V. cholerae,

also for prevention and treatment of cholera outbreaks, is of high-priority. Specific bacteriophages

can be used for wider phenotypic diversification and tracking of V. cholera isolates in the

environment, and also to prevent and treat infections, caused by antibiotic resistant V. cholerae

strains.

The strain collections of the Eliava Institute, Georgia, and Maryland Pathogen Research Institute,

UMD, USA, were screened to determine the susceptibility to V. cholerae – specific phages,

isolated in Georgia. The clinical and environmental strains of V. cholerae O1 (382 strains in total)

and 16 strains of V. cholerae O139, collected in different geographical regions (Georgia, South

East Asia, South America, Haiti, West Africa, etc,) were used in the study along with the set of 14

Eliava phages specific to V. cholerae O1 and non- O1/non139.

These phages were characterized by virion morphology, serologic properties and DNA restriction

profiles. The phage lytic spectrum was determined by spot test technique followed by estimation of

the phage titer on selected bacterial strains. The strains of V. cholerae O1, obtained from patients,

appeared to be more susceptible to the Eliava phage set: in average 95,2% of strains were lysed by

at least one phage. Some differences in the phage susceptibility pattern was observed for particular

groups of V. cholerae O1 isolates: phages lysed all Haiti strains, 95% of Bangladeshi strains, and

91 % of isolates from different regions.

The Georgian environmental isolates of V. cholerae O1 showed less susceptibility (77 %) to V.

cholerae phages that can be explained by high genetic and phenotypic variability of environmental

V. cholerae strains. The susceptibility patterns to eight phages, selected for the phage-typing set,

allowed to divide clinical V. cholerae O1 isolates into a number of phage groups, with the number

of strains in each group depending on strain collection and geographical origin.

The results obtained have confirmed high distinctive features of specific bacteriophages for

subtyping of V. cholerae isolates of different origin as well as obvious potential of lytic phages for

future therapeutic applications in cholera outbreaks.




Phage Typing, Antibiotic Resistance and Genomic Rep-PCR Fingerprinting

of Clinical Salmonella Isolates from the Caucasus Region

Elene Kakabadze,1* Khatuna Makalatia,1 Maia Merabishvili,1,2 Nino Grdzelishvili,1 Nata

Bakuradze,1 Mkhtar Mkrtchyan,3 Marina Goderdzishvili,1 Jean-Paul Pirnay,2 Nina Chanishvili,1

Anahid Sedrykyan3 1George Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia; 2Queen

Astrid Military Hospital, Brussels, Belgium; 3Institute of Molecular Biology, Academy of

Sciences, Yerevan, Armenia


Fluoroquinolone-resistant Salmonella infections are emerging to cause considerable

morbidity and mortality worldwide. In reaction to the use of antibiotics in farming and veterinary

practices, MDR phenotypes have emerged. As Salmonella enterica serovars are widely diverse and

spread through the food chain, rapid increase in antibiotic resistance is also observed in clinical

strains. Therefore, rapid identification and treatment solutions are direly needed to treat MDR

infections. The use of lytic phage-based systems offers a triple benefit: rapid and cost effective

typing using therapeutic agents, which can potentially lyse the examined MDR strains.

In this study we have focused on isolates from patients with Salmonellosis, who have not

yet received any antibiotic therapy. In total, 71 non-typhoid Salmonella enterica clinical isolates

from the “Nork” Clinical Hospital of Infectious Diseases (Yerevan, Armenia) and 22 strains from

the Referral Laboratory at the Republican Clinical Hospital of Infectious Diseases (RCHID, Tbilisi,

Georgia) were selected.

Isolates were maintained on Salmonella-Shigella selective media. Serotyping by polyvalent

antisera for flagellar (H) and lipopolysaccharide (O) antigens, and by biochemical analysis, were

performed in the respective clinical settings. MALDI-TOF analysis was performed to confirm the

identification at the genus level. Semi-automated rep-PCR fingerprinting profiles were determined

using the DiversiLab® system and analyzed using Pearson’s correlation analysis. An UPGMA

dendrogram was constructed and strains showing a similarity value above 95% were marked as

linked (cluster). The thus obtained clusters were compared with Salmonella group B, C, D, E, F, G,

K, L, O, R reference libraries for serotype identification. Antibiotic resistance profiles (different

classes of antibiotics) were determined in accordance with the CLSI guidelines for standard disk

diffusion assays. Phage typing was performed using the line spot-test method. For this purpose, 5

multivalent (active against Salmonella and Escherichia coli), 3 phage mixtures and 7 Salmonella

specific phage lines were selected.

Species identification by phage typing confirmed the MALDI-TOF identifications. Forty-

one percent of the tested clinical isolates were shown to be MDR, while none of the strains showed

complete resistance to the tested phages. Despite of their resistance to different classes of

antibiotics, all isolates were susceptible to at least one of the phage lines (clones) used in our typing

system. According to rep-PCR fingerprinting, 93 examined isolates clustered into 24 different

cluster-serotypes. One isolate from RCHID showed 97.3% similarity with a Salmonella Dublin

reference strain, one cluster harbored 14 strains with >95% similarity to a Salmonella typhimurium

reference strain and another cluster consisted of 21 strains identified as Salmonella enteritidis. As

expected, no correlation was observed between strain genotypes (rep-PCR fingerprints) and phage

types. A robust lytic phage-based characterization system can be relevant as a tool for

epidemiological studies and as a rapid screening method for therapeutic or prophylactic phage

applications in food, veterinary or human clinical settings.




Drosophila melanogaster as a model to study host-phage infection, immunity

and interactions with the microbiome

Thomas Leech*, Amanda Bretman

University of Leeds, England


Bacteriophage therapy has long promised an enticing alternative to antibiotics in the treatment of

bacterial infections, but over 100 years since the discovery of bacteriophages, they are still yet to

realise their therapeutic potential. A number of aspects of bacteriophage biology have hindered

progress, amongst which the relative dearth of information available regarding phage-host

immunology and the unintended off-target effects of bacteriophage infection on host microbes, are

key.

In order to better understand the complex relationship between bacteriophage infection, the host

immune response and the microbiome, a suitable model is required. The fruit fly Drosophila

melanogaster is a useful and well established model of innate immunity and the microbiome due to

its genetic tractability, ease of housekeeping and the conserved nature of many key genes and

proteins, as well as it’s relatively simple resident bacterial community.

Here, we demonstrate the significant potential that D. melanogaster has in helping to elucidate

specific aspects of phage biology such as immune inactivation of therapeutic phages,

immunological sensitisation by phage infection and perturbation of the host microbiome structure

by potential therapeutic bacteriophages. D. melanogaster may become a powerful model in the

advancement of phage therapy.




Evaluation of phages cocktails for inhibition of V. parahaemolyticus in

aquaculture

Carlos Lomelí-Ortega; Sergio Martínez-Díaz

Microbiology Laboratory, IPN-CICIMAR, La Paz, B.C.S., 23096, México.


Vibrio parahaemolitycus is an important cause of disease, mortality, and economical losses in the

shrimp aquaculture industry, and in recent years has been associated to early mortality syndrome

(EMS), which is a new disease that has been detected at shrimp farms in Asia since 2010. New

therapies are necessary for control of this kind of diseases, and phage therapy is an alternative

prophylaxis treatment of bacterial infectious diseases that has gained interest. Phage therapy is the

use of naturally occurring viral predators of bacteria, and are recognized for their ability to reduce

the pathogen populations.

In this study, we evaluated individual effect of 12 phages against V. parahaemolyticus, and select

the best cocktail for biocontrol in artemia culture. A single application of each phage inhibited

growth of V. parahaemolyticus even 24h post application (p<0.05). Dual applications presented

interference between phages, with phages A3S, CVH4, and 4P the worst phages for combination

use.

On the other hand, phages VH5, SH, SP, PL14D, and VPMS1 were the best phages for dual

combination with themselves. A cocktail of VH5, SP, PL14D, and VPMS1 in equal proportion was

the best combination against V. parahaemolyticus, reducing growth in 73 % ± 0.9 during 24h post

application of a single dose. The cocktail was evaluated against four EMS positive strains and was

able to stop growth during 5h post application. A significant difference of 15 % was seen at the end

of the test (p<0.05). The next step will be the evaluation of activity in an in vivo model with

Artemia, to determinate the best conditions for application.




Humoral response to staphylococcal bacteriophages applied per os in

a murine model

Joanna Majewska, Zuzanna Kaźmierczak, Karolina Wojtyna, Dorota Lecion, Katarzyna Hodyra-

Stefaniak, Anna Kłopot, Barbara Owczarek, Marek Harhala, Paulina Miernikiewicz, Andrzej

Górski, Krystyna Dąbrowska

Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,

R. Weigla 12, 53-114 Wrocław, Poland


Objectives: In our previous studies, we showed that a model bacteriophage T4 applied orally over

a long period of time induced production of anti-phage antibodies in mice, both in the gut and in

the blood. Here we investigated a similar model in which two staphylococcal bacteriophages: A3R

and 676Z were applied per os.

Methods: C57BL/6J mice were given purified phage preparations in PBS, mixed with drinking

water (1:1) to a final concentration of 4×109 pfu/ml continuously for 100 days. Control group was

given drinking water with PBS. Phage treatment was followed by 130 days without the phages and

subsequent second continuous treatment with staphylococcal phages until the experiment was

terminated. Throughout the whole experiment blood and fecal samples were collected and

evaluated for the presence of viable phage particles and anti-phage antibodies (IgG, IgA, IgM).

Antibody levels were measured by ELISA immunoassay.

Results: Phages were detectable in the blood in small titers only during the first week of the

treatment. On the contrary, high phage titers (107-6×109 pfu/g) were simultaneously observed in

fecal samples, decreasing gradually to reach an approximate value of 102 and 103 pfu/g (for A3R

and 676Z, respectively) after 5 weeks and eventually decreasing to undetectable levels at the end of

the first phage treatment. Second treatment resulted in viable phage particles being again detectable

in feces. However, phage titers were significantly lower (5,7×102 for A3R; 2,1×103 for 676Z) than

in the initial phase of the experiment and decreased to undetectable levels within 25-38 days.

Decrease of phage titers in faeces correlated with an increase of IgA, but not IgG. Plasma IgA

levels started to increase 4-5 weeks after the initiation of phage treatment, reached its maximum

within 72-85 days and then gradually decreased after phage treatment was terminated. However,

second administration of phage preparations induced IgA production within 10 days. A marked

increase in plasma IgG was observed as soon as two weeks following the initiation of the

treatment. IgG levels then continued to rise until day 36 and – contrary to IgA – remained at high

concentration throughout the whole experiment. IgM peak was observed on day 15.

Conclusions: These data show that staphylococcal bacteriophages applied per os induced humoral

response in vivo, interestingly, with a lower contribution of primary response, but with a significant

level of response in IgG and IgA. Continuous oral treatment with phage preparations and the

induction of humoral responses resulted in a decreased phage passage through the murine

gastrointestinal tract. Increase in IgA levels correlates with a decrease of phage titers in feces.

Acknowledgements: This work was supported by the National Science Centre in Poland grant

UMO-2012/05/E/NZ6/03314 and by the Wroclaw Centre of Biotechnology, programme The

Leading National Research Centre (KNOW) for years 2014–2018.




Formulation and Encapsulation of Bacteriophages for Targeted Controlled

Release

Danish J. Malik1*, Goran T. Vladisavljević1, Martha R. J. Clokie2, Gurinder Vinner1, Salvatore

Cinquerrui1, Francesco Mancuso1

1Chemical Engineering Department, Loughborough University, Loughborough, UK; 2 Department

of Infection Immunity and Inflammation, University of Leicester, Leicester, UK.


Objectives: The aim of the work was to evaluate the use of novel microfluidic and membrane

based techniques for encapsulation of purified (using ultrafiltration and ion exchange)

bacteriophages in stimuli responsive polymers (microparticles) and liposomes (microparticles and

nanoparticles) for treating multi-drug resistant gastrointestinal infections.

Methods: Bacteriophage K (lytic, S. aureus, myovirus) and Felix O1 (lytic, S. enterica, myovirus)

were used in the study. Phages were amplified using a Sarorius Biostat® operated in batch mode.

Phages were purified using centrifugation, ultrafiltration, anion exchange chromatography and size

exclusion chromatography to remove host cell proteins and host cell DNA. Encapsulation of SYBR

green labelled phage in micro- and nanoparticles was shown using confocal microscopy. TEM

imaging of encapsulated phage in liposomes was carried out. Phage stability upon exposure

(exposure time 3hr at pH 2) to simulated gastric fluid and subsequent phage release kinetics was

studied using simulated intestinal fluid (phage titre measured using plaque assay). The effects of

formulation (e.g. polymer or lipid composition), phage titre and experimental setup conditions were

investigated on phage encapsulation, phage viability and storage stability. The phages were also

spray dried and freeze dried using excipients to improve long-term storage stability.

Results: We show that we can achieve significant concentration of highly purified phages (~1012

PFU/ml) using downstream processing operations by removing host cell proteins and host cell

DNA. We have proof-of-principle results demonstrating controlled encapsulation of phages in pH

responsive polymeric microparticles and liposomal nanoparticles using microfluidic and membrane

encapsulation techniques. Our approach allowed precise control over phage loading per particle

(i.e. control over phage dose). We show how scalable production of highly uniform micro- and

nanoparticles may be achieved using microfluidic and membrane emulsification with control over

particle size through manipulation of microfluidic hydrodynamic conditions, membrane shear and

fluid properties (e.g. viscosity). Through changes to the formulation and the particle size, we also

demonstrate control over the release dynamics (burst and sustained release formulations) of the

phages in response to a pH or enzyme trigger. We demonstrate that encapsulated phages may be

stored under refrigerated conditions following drying by addition of disaccharides such as trehalose

as excipients.

Conclusions: We have demonstrated that novel microfluidic and membrane based processes can

be successfully used to encapsulate phages in stimuli responsive micro- and nanoparticles. These

systems would allow delivery of phages to the gastrointestinal tract giving unprecedented control

over the phage dose and the release profile to treat multi-drug resistant infections.




Identification of the Type IV Pilus as the Receptor for Bacteriophage DLP1

Adherence to Stenotrophomonas maltophilia and Pseudomonas aeruginosa

Jaclyn G. McCutcheon, Danielle L. Peters and Jonathan J. Dennis*

University of Alberta, Edmonton, Alberta Canada


The opportunistic bacterial pathogen Pseudomonas aeruginosa is a major cause of nosocomial

infections, particularly in immunocompromised individuals, and is the second most prevalent

pathogen found in the lungs of cystic fibrosis patients. The inability to control P. aeruginosa

infections with antibiotics due to its intrinsic and adaptive multi-drug resistance and range of

virulence factors is cause for concern. A possible alternative treatment is phage therapy, the clinical

application of bacteriophages to eradicate target bacteria. While bacteriophages typically have a

very narrow host range, a novel phage DLP1 previously isolated in our lab is capable of infecting

across taxonomic orders, lysing strains of both P. aeruginosa and Stenotrophomonas maltophilia,

another highly antibiotic resistant bacterial pathogen. To explain this unique ability, we have

identified and are characterizing the primary receptor for DLP1 and the mechanism of attachment

to both host species.

Initial screening of a 2,242 member P. aeruginosa PA01 mutant library suggested that DLP1 uses

the type IV pilus, a virulence factor on the surface of the bacterium, as its primary receptor for

adherence. Results show that mutants with transposon insertions in genes encoding pilus structural

and regulatory components are resistant to DLP1 infection. Subsequent complementation of the

disrupted pilin subunit genes in PA01 restored DLP1 adherence and lysing ability. Preliminary data

from a cross-species complementation experiment using appropriate S. maltophilia D1585 pili

genes in a PA01 pili deficient background also restored phage binding and lysis, suggesting that a

similar pili structure serves as the receptor for DLP1 in the main S. maltophilia host strain, D1585.

Visualization using electron microscopy shows that DLP1 interacts with pilus structures on the

surface of PA01 and D1585. When the host bacteria is grown in media containing mucin, a major

component of mucus and a nutrient that facilitates colonization of the lungs by pathogenic bacteria,

the bacteria show increased twitching motility indicative of induced pilin expression. This increase

in pilin expression enhances the association of DLP1 to its host bacteria, visualized by transmission

electron microscopy.

Future research will examine mutagenesis of pilin associated genes in the main S. maltophilia host

strain, D1585, with the prediction that this will knockout DLP1’s ability to adhere to and lyse the

cells. This research will further characterize DLP1 as a candidate for phage therapy in the treatment

of highly antibiotic resistant bacteria from multiple genera.




Determination of safety and therapeutic potential of environmental waste

water lytic bacteriophages against multidrug resistant Staphylococcus aureus

(MDRSA) in BALB/c mice

Oduor Joseph M. Ochieng’1, 2, 3*, Onkoba Nyamongo 4, Maloba Fredrick 5, Arodi Washington

Ouma 6, Atunga Nyachieo 1

1Institute of Primate Research (IPR), Nairobi-Kenya, 2 KAVI-Institute of Clinical Research,

University of Nairobi-Kenya, 3Haartman Institute, University of Helsinki-Finland, 4Technical

University-Kenya, 5 School of Applied Pure Science, Kenyatta University, Nairobi-Kenya, 6 School

of Medicine, Kenyatta University, Nairobi- Kenya


The use of phage therapy as an alternative method of treating infections caused by multidrug

resistant bacteria is gaining ground as most bacteria are becoming resistance against the commonly

available antimicrobials.

Objective: The study sought to determine safety and therapeutic efficacy of environmentally

obtained Staphylococcus aureus lytic phages against multidrug resistant S. aureus (MDRSA) in

mice.

Methodology: Phages and MDRSA were isolated from sewage and waste water collected from

within Nairobi County. The isolated S. aureus bacteria were screened for resistance towards;

ceftazidime, oxacillin, vancomycin, netilmicin, gentamicin and erythromycin, trimethroprim-

sulfamethoxazole and cefuroxime. Thirty BALB/c mice were randomly assigned into three groups;

the MDRSA infection group (n=20), the phage-infection group (n=5) and non-infection group

(n=5). After 24 or 72h post-infection (p.i.) with MDRSA, the infected mice were either treated with

a single dose of clindamycin (8mg/kg/bwt) or 108 PFU/ml of S. aureus phage or a combination

treatment (clindamycin and S. aureus phage). Efficacy and safety of the treatments were

determined by monitoring animal physical health post-infection as well as gross pathology and

histopathology. Bacteremia was determined daily for 10 days and used to establish therapeutic

efficacy of the phage.

Results: Treatment with phage was efficacious (100%) compared to clindamycin (62.25%) at 24h

p.i and 87.5% at 72h p.i., and the combination therapy, 75% at 24h p.i. and 100% at 72hrs p.i.

Efficacy of the treatment regimens was dependent on the time of treatment post infection. The mice

infected with MDRSA and treated with phage had no bacteremia at day 7 post-treatment compared

to those treated with clindamycin and combination therapy (P < 0.001). There were no tissue

abscesses, inflammation in the brain, lungs and liver tissues of phage treated mice compared to

those treated with clindamycin and combination therapy.

Conclusion: Staphylococcus aureus phage obtained from sewage and waste water from within

Nairobi County was safe and possessed therapeutic efficacy against MDRSA bacterium.




Characterization of Pseudomonas aeruginosa phage-resistant clones obtained

by controlled phage infection

Tomasz Olszak1, Pawel Markwitz1, Grzegorz Gula1, Miguel Valvano2 and Zuzanna Drulis-

Kawa1*

1Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77, 51-148

Wrocław, Poland 2Centre of Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences,

Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK


Phage therapy is a promising strategy to combat bacterial infections, but it has also some

drawbacks. Besides difficulties in the standardization of phage preparations and regulatory issues

related to the use of phage products in humans, a major biological problem is the onset of phage

resistance upon phage treatment. The emergence of the phage-resistant mutants has been

recognized in phage therapy application. One of the simplest, effective, and widely accepted

practice of counteracting the effects of phage resistance is the use of phage cocktails containing

several phages with a similar host spectrum, but different attachment mechanism.

Most microbial surface structures recognized by phages are also important bacterial virulence

factors that strongly stimulate the immune system of infected humans. Therefore, changes in the

expression level or the structure modification of these receptors reduce bacterial virulence. In this

study, we examined the diversity of Pseudomonas aeruginosa clones obtained as a result of

controlled infection by single bacteriophages and cocktails composed of two or three

bacteriophages. Three well-characterized bacteriophages of the Caudovirales order were selected

for these experiments: phiKZ (fimbriae-dependent, giant Myoviridae) LUZ7 (fimbriae-dependent,

Podoviridae) and KTN6 (LPS-dependent, Myoviridae). Isolated bacterial mutants were tested for

twitching, swarming and swimming motility, the intensity of pyoverdine, pyocyanin and biofilm

production, the LPS patterns and the sensitivity to antibiotics, phages, complement system and

phagocytosis.

Our results indicate that the selection pressure caused by phage infection leads to modification or

even loss of important virulence factors (i.e. type IV fimbriae or LPS O-antigen). It was confirmed

that the virulence of isolated phage-resistant clones was reduced. In most cases, observed changes

remain stable, what suggests their mutational character. Nevertheless, bacterial resistance based on

the loss or modification of the particular receptor did not always result in cross-resistance to other

phages recognizing the same receptor.




Characterization of novel broad-spectrum Citrobacter freundii bacteriophage

Graça Pinto, Hugo Oliveira, Ana Oliveira and Joana Azeredo*

CEB – Centre of Biological Engineering, LIBRO – Laboratório de Investigação em Biofilmes

Rosário Oliveira, University of Minho, 4710-057 Braga, Portugal


Citrobacter spp. although frequently ignored, is an emergent nosocomial bacterium able to cause

various superficial and systemic life-threatening infections. Considered to be a hard-to-treat

bacterium, due to the acquisition of resistance to a large range of antibiotics, new measures of early

and efficient therapies are needed. Bacteriophages, the natural predators of bacteria, have elicited

interest due to their high specificity, low inherent toxicity, and ability to kill antibiotic-resistant

bacteria. To assess the potential for phage therapy, we have characterized and sequenced a novel

broad host range T4virus: the myovirus vB_CfrM_CfP1, that specifically infects Citrobacter

freundii (>85% of 21 strains tested). To demonstrate its value as an antimicrobial alternative, CfP1

was fully characterized, shown to be stable at a large range of temperature and pH values, capable

of killing multidrug-resistant clinical isolates (showing resistance up to 12 antibiotics), and with a

burst size of 45 PFU/cell. Genomically, CfP1 presents a dsDNA molecule of 180,219 bp, coding

273 CDSs and sharing 46 to 94% of nucleotide identity to other Citrobacter phages. The absence

of genes with known function encoding for virulence factors, its high killing activity of suspended

cultures and the elevated stability at a wide range of pH suggest that this phage possesses suitable

properties for therapy.




Approaches to bacteriophage therapeutics for Staphylococcus aureus

Melissa D. Ray*, Roger D. Plaut, E. Scott Stibitz

US Food and Drug Administration, Division of Bacterial, Parasitic, and Allergenic Products,

Center for Biologics Evaluation and Research, 10903 New Hampshire Ave., Silver Spring,

Maryland


Staphylococcus aureus can be a commensal organism as well as a serious pathogen capable of

causing skin abscesses, wound infections, endocarditis, osteomyelitis, pneumonia, and toxic shock

syndrome. The treatment of these infections has become difficult because of the emergence of

strains resistant to multiple antibiotics. Bacteriophage therapy has received more attention recently

as an alternative approach to combat multidrug-resistant bacterial infections.

Our work involves developing a platform to investigate the genetic aspects of, and utility of,

bacteriophage therapy aimed at decolonization of methicillin-resistance S. aureus (MRSA). The

utility of S. aureus bacteriophage K and the Eliava Fersisi bacteriophage cocktail have been

assessed in a mouse model of MRSA nasal and lung carriage. We used a bioluminescent S. aureus

NRS384 derivative, SAP231, to colonize mice. Staphylococcal bacteriophage K and the Eliava

Fersisi cocktail were used for decolonization treatment. Bacteriophage K is a virulent, polyvalent

phage that lyses both coagulase-positive and -negative staphylococci. Bacteriophage K and the

Eliava Fersisi bacteriophage cocktail were shown to comprehensively lyse a panel of twenty-two

MRSA strains. Preliminary results show a significant decrease in MRSA nasal and lung carriage

using the Eliava Fersisi cocktail.

One frequently raised issue is the potential ability of bacteria to develop resistance to phage. This

study also characterizes S. aureus mutants resistant to bacteriophage K in vitro. Using S. aureus

strain NRS384, nine genetically independent spontaneous bacteriophage K resistant mutants were

isolated. Whole genome sequencing identified single-nucleotide polymorphisms (SNPs) in each of

the nine strains. Interestingly, none of SNPs are predicted to be null mutations, but all resulted in

amino acid substitutions in the affected gene products. These mutations provide insight into

potential phage receptors, replicative pathways, and other cellular components that are crucial for

phage growth. The results of these genetic screens lead to a number of testable hypotheses that we

will pursue regarding interactions between phage K and host cells.

Although there have been numerous anecdotal reports describing instances of successful phage

therapy, there is still a need for controlled clinical trials to assess its effectiveness. Overall, this

work should allow us to address important questions regarding phage therapy in a preclinical

platform.




Accounting for transient or phenotypic host resistance in modeling infections

by phage cocktails in complex systems

Juliano Bertozzi-Silva1, Dominic Sauvageau1*

1Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada


Objectives: The successful implementation of phage cocktails in applications such as phage

therapy is often impeded by the limited information available for a given system. Mathematical

models can help alleviate these issues by providing predictions for population dynamics. However,

many current models forego the impact of important factors such as the presence of organisms

competing with the host, adsorption efficiency, and, with mounting evidence, transient or

phenotypic host resistance. The aim of this study was to develop and validate experimentally a

model taking these factors into account to provide accurate information on multi-phage infections

in complex systems.

Methods: The system studied consisted of two competing Lactobacillus plantarum phages (phages

B1 and B2), the host L. plantarum, and the yeast Saccharomyces cerevisiae (included as an non-

susceptible organism competing with the host for resources). A kinetic model was developed to

describe growth, interactions and resource allocation of each population present. The model

parameters were determined experimentally through one-step growth (lysis time, lysis rate

constant, latent period, burst size), adsorption (adsorption rate constant, adsorption efficiency), and

microbial growth experiments (maximum specific growth rate, substrate affinity constant, yield).

Transient or phenotypic host resistance was accounted for by the inclusion of two reversible states,

susceptible and non-susceptible hosts, in the bacterial population. The reaction rate constant and

equilibrium constant were determined through model fitting of population dynamics in single

phage infections. Various relationships between susceptible and non-susceptible host states were

tested.

Results: The model was validated using phages B1 and B2, L. plantarum and S. cerevisiae

populations growing concurrently in a bioreactor (closed system). Not only was the model able to

describe the evolution of populations accurately, but it was also predictive. Using the model, it was

possible to predict the behaviour of the various populations based on the initial multiplicity of

infections of each phage, and the initial cell loads of bacteria and yeast. Additionally, the model

provided inferred information which is otherwise not possible to obtain in real-time, such as the

ratio of susceptible and non-susceptible host bacteria, and the individual titers of competing phages

B1 and B2.

Conclusions: The model developed, taking into account adsorption efficiency and transient or

phenotypic host resistance, provided valuable information on population dynamics, even for multi-

phage systems. The model can be applied to a variety of applications and settings where multiple

organisms and phages are present, from phage therapy and biocontrol to ecological studies.




Characterization of four virulent bacteriophages isolated from sewage in

Waukesha Wisconsin and their use to combat Pseudomonas aeruginosa

infections

Christine Schneider*, Eric Graham, and Kimani Njoya, William C. Hutchins3

1Department of Life Sciences, Carroll University, Waukesha WI 53186; 2Penn State College of

Medicine, Hershey, PA; 3University of Wisconsin Milwaukee, Milwaukee WI


The global increase in antibiotic resistant bacteria threatens to undermine our ability to perform

many life-saving medical procedures. Pseudomonas aeruginosa is a gram-negative opportunistic

pathogen that is a significant cause of nosocomial infections as well as the most common cause of

lung infections in cystic fibrosis patients. The CDC has categorized P. aeruginosa as a serious

threat due to prevalence of multidrug resistant strains and the clinical significance of this pathogen.

Clearly, alternative strategies to combat P. aeruginosa infections are needed.

Although the interest in using virulent phages to combat bacterial infections dropped out of favor in

the United States after the development of antibiotics, there is now renewed interest in using phage

therapy to combat antibiotic resistant bacteria. Bacteriophage specific to P. aeruginosa would be of

clinical interest as an alternative to antibiotics for the treatment of infections with multidrug

resistant strains or to control biofilm formation in hospital settings to reduce the risk of nosocomial

infections. Toward that aim, we isolated four virulent phages from sewage and here we describe the

characterization of these novel bacteriophages.

To identify novel phage therapy candidates to combat P. aeruginosa infections, we attempted to

isolate bacteriophages from raw, filtered sewage using an enrichment protocol with a cystic fibrosis

clinical mucoid strain of P. aeruginosa (FRD1) and a spontaneous non-mucoid mutant (FRD2). All

four of the virulent phages described here were isolated using FRD2. Unfortunately, we were

unable to isolate phages using the mucoid strain FRD1. Host range analysis revealed that individual

phages lysed 30-40% of the seventeen P. aeruginosa strains examined with more lab strains being

lysed than clinical isolates. Interestingly, the efficiency of plating values (EOP) were typically

higher than 1 for most of the clinical isolates indicating the phage had a higher plaque forming

efficiency on the clinical strain relative to FRD2. In addition, one isolate (BB2) was able to lyse

FRD1, although with reduced efficiency relative to FRD2 (EOP=0.29). Each phage isolate was

able to inhibit the planktonic growth of FRD2 completely for at least 16 hrs, with BB2 completely

blocking growth for 24 hrs. Whole genome sequencing and EM results indicate these phages are

Myoviridae most similar to the ubiquitous and well-conserved family of PB-1 like phages.




Phages targeting Streptococci of the oral cavity

Muireann Kate Smith1, Lorraine Draper1, Cillian Hayes1, Olivia Connolly1, Marion Dalmasso1†,

R. Paul Ross1 and Colin Hill1

1APC Microbiome Institute, University College Cork, Cork, Ireland, †Present address: Normandie

Univ, UNICAEN, ABTE, 14000 Caen, France


Streptococci are important pathogens and commensals of the mouth, skin, intestine, and upper

respiratory tract. Infections caused by these bacteria can vary in severity from mild

throat infections and those associated with tooth decay, to those that are systemic and life-

threatening. In many cases such infections are derived from pathogenic species that are the

causative agents of a number of invasive and non-invasive infections and diseases such as

Streptococcal pharyngitis, Scarlett fever, pneumonia, necrotising fasciitis and toxic shock

syndrome. In addition to known pathogens, commensal Streptococci are known to become

opportunistic pathogens should they gain access to the bloodstream from the oral cavity. Examples

of such commensals include: Streptococcus mutans, the leading cause of dental caries that affects

2.4 billion people worldwide; and Streptococcus anginosus that has been implicated in

subcutaneous abscesses, bacteraemia and infections of the upper respiratory tract.

Discovery of new and effective antimicrobial agent(s) in the current battle against antibiotic

resistance is of global concern. Thus, bacteriophages (phages) have been identified as promising

antibacterial agents that could be used to prevent or treat Streptococcal infections. In this study,

four different phages targeting clinically relevant Streptococci were isolated and characterised.

Their respective hosts were identified to be S. mutans, S. anginosus, S. pyogenes and S. infantis.

Following characterisation, two of these phages were determined to be lytic and effectively

diminished S. mutans and S. pyogenes in both planktonic and biofilm forms.

This bactericidal effect was further amplified by combining phages with the food grade bacteroicin

nisin. While both antimicrobials were found to be effective alone, an enhanced bactericidal effect

was witnessed when they were combined. Not only are these antimicrobials highly effective

against Streptococci, they offer a safe and natural means to protect against and treat bacterial

disease. Thus, making the combination of phages and nisin an attractive, practical and attainable

contribution to addressing Streptococcal infections in human health.




Preventing Phage Resistance Through Tail Fiber Mutagensis

Kevin Yehl, Sébastien Lemire, Andrew C. Yang, Hiroki Ando, and Timothy K. Lu*

Massachusetts Institute of Technology, Cambridge, MA 02139, USA


The rapid emergence of antibiotic-resistant infections is prompting increased interest in alternative

antimicrobials. Acquisition of resistance against a new antimicrobial is a large issue in their

development. We created a high-throughput method to engineer antimicrobial bacteriophages that

achieve long-term suppression of bacterial growth and prevent resistance appearance. Analogous to

antibody specificity engineering, we direct mutations to small host range determining domains of

the phage’s tail fiber using protein structure as a guide to synthesize large (>107 different members)

“phagebody” libraries. This approach generates high functional diversity while minimizing

disruptions to the overall protein structure. We demonstrate that phagebodies isolated from such

libraries surpass natural phages at suppressing bacterial resistance appearance. We anticipate that

this approach will facilitate the creation of next-generation antimicrobials that defeat resistance,

and that it can be extended to other viral scaffolds for a broad range of applications.

Author Index (Bold text indicates that the author is attending this meeting)

Aarestrup, Frank ................................. 122

Abas, F. ................................................ 93

Abdulkarim, M. S. ................................ 93

Abedon, Stephen ............................... 107

Adamia, Rezo ....................................... 60

Adhya, Sankar ...................................... 17

Aertsen, Abram .................................... 71

Ahern, Stephen ................................... 111

Ahmed, Rafath ..................................... 28

Akella, Srividya.................................... 33

Akter, Mahmuda .............................. 108

Alem, Farhang ...................................... 99

Alemnah, Yonas ................................... 49

Ali, Monsur .......................................... 30

Allison, Cody ..................................... 129

Altheide, Samantha ............................ 129

Alzbutas, Gediminas .......................... 121

Anany, Hany ....................................... 30

Ando, Hiroki ................................ 34, 184

Arevalo, Alejandra ............................... 29

Astudillo, Ariel ................................... 158

Attai, Hedieh ....................................... 27

Atterbury. Robert .................... 154, 159

Azeredo, Joana ................................... 42

Bai, Jaewoo ................................... 83, 86

Baig, Abiyad ............................. 154, 159

Bailey, Barbara ..................................... 53

Baker, James ........................................ 17

Baker, Kristi ......................................... 53

Bakuradze, Nata ................................. 171

Balamurugan, Sampathkumar .............. 28

Balarjishvili, N ................................... 133

Baquero, Diana ..................................... 29

Baran, Nava .......................................... 16

Baraniak, Anna ................................... 144

Barbu, Magda ..................................... 33

Barendregt, Arjan ............................... 139

Barnabas, Shaun ................................. 128

Barr, Jeremy ....................................... 53

Barrow, Paul ............................... 154, 159

Barth, Zachary ................................. 138

Batumbo, Doudou ................................. 32

Bednarek, Agnieszka .......................... 144

Bekker, Linda-Gail ............................. 128

Benesik, Martin ................................... 146

Berisio, Rita ........................................ 130

Bertozzi-Silva, Juliano ........................ 181

Bhandare, Sudhakar ................... 84, 159

Bhowmick, Tushar ........................ 25, 85

Birkholz, Nils ...................................... 140

Biswas, Biswajit ..................... 49, 62, 168

Blankenberg, Francis .......................... 167

Blasdel, Bob ........................... 60, 64, 126

Blinkova, Olga .................................... 124

Bliskovsky, Valery ................................ 17

Blumberg, Richard ................................ 53

Bolocan, Andrei ................................. 155

Bolton, D ............................................. 134

Bompangue, Didier ............................. 32

Bondy-Denomy, Joseph ...................... 43

Bonnain, Chelsea ............................... 110

Bordwell, Brianna ............................... 129

Borges, Adair ........................................ 43

Borne, Yannick ................................... 125

Breitbart, Mya ..................... 20, 110, 132

Bretman, Amanda ............................... 172

Breyne, Koen ........................................ 91

Briers, Yves................................. 126, 130

Brister, J. Rodney ............................. 124

Britton, Warwick................. 156, 157, 158

Brøndsted, Lone .................... 24, 88, 111

Brouns, Stan ........................................ 44

Brown, Nathan .............................. 95, 164

Brown, Pamela ...................................... 27

Brum, Jennifer .................................... 15

Bryan, Dan ........................................... 60

Buceta, Javier ...................................... 150

Buck, K.N ........................................... 110

Bunk, Boyke ....................................... 137

Butcher, James .................................... 147

Buttimer, Colin .................................. 125

Cadieux, Brigitte ................................... 84

Cambillau, Christian ............................ 69

Campbell, Danielle ........................... 149

Campion, Ciorsdan ............................. 155

Cano, Patricia ....................................... 77

Capolupo, Angela ............................... 139

Caprara, S. .......................................... 110

Cardona-Félix, César.......................... 101

Carlson, Michael .................................. 16

Carlson, Paul ........................................ 54

Caro-Quintero, A.................................. 90

Carrigy, Nicholas ....................... 32, 156

Carstens, Alexander ............................. 46

Casey, Aidan ...................................... 125

Casey, Pat ..................................... 50, 160

Cenens, William ................................... 71

Chan, Benjamin .................................... 32

Chan, Hak-Kim .......... 75, 156, 157, 158

Chang, Rachel ............................. 75, 156

Chanishvili, Nina ............................... 171

Chen, Ke ............................................... 75

Chen, Mandy ...................................... 129

Chitwood, Adrienne ........................... 129

Christensen, Tokala ............................ 129

Chun, Jihwan ...................................... 86

Cinquerrui, Salvatore ......................... 175

Citorik, Robert ................................... 97

Clavijo, Viviana .................................. 29

Clement, Frédéric ................................. 48

Clokie, Martha ....... 32, 52, 95, 112, 175

Coffey, Aidan ............................. 125, 134

Colavecchio, Anna ............................... 84

Colom, Joan .............................. 154, 159

Connerton, Ian ...................... 32, 147, 162

Connerton, Phillippa .......................... 162

Connolly, Olivia ................................. 183

Contag, Chris ..................................... 167

Coulson, Carollyne ............................. 129

Criss, Nichole ..................................... 129

Cucic, Stevan........................................ 30

Culley, Alexander .............................. 115

Cuypers, Maxime ................................. 65

Dabee, Smritee ................................... 128

Dąbrowska, Krystyna 74, 145, 166, 174

Dąbrowski, Kamil .............................. 144

Dalmasso, Marion ................. 50, 160, 183

Danis-Wlodarczyk, Katarzyna ........ 126

Das, Mayukh .................................. 25, 85

Dauley, Melinda .................................. 129

Davis, Christopher .............................. 129

de Crécy-Lagard, Valérie ...................... 46

de la Croix, Nick ................................... 71

De Smet, Jeroen .................................... 64

De Vos, Daniel ...................................... 32

Debarbieux, Laurent ..................... 78, 116

Dedon, Peter .......................................... 46

Degnan, Patrick ................................... 149

Del Rosario, Yvette ............................... 33

Demons, Samandra ....................... 49, 168

Dennis, Jonathan ......................... 151, 176

Devonshire, Daniel ............................. 129

Dewi, Fitriya ......................................... 32

Di Santo, James ..................................... 78

Dies, Marta .......................................... 150

Dirks, Clarissa ..................................... 129

Dishaw, Larry ....................................... 20

Doan, Matthew ...................................... 62

Dobbs, Kyle ........................................ 129

Donado, Pilar ........................................ 29

Donovan, David ................................... 98

Dopitova, Radka ................................. 146

Doskar, Jiri .......................................... 146

Draper, Lorraine ................. 50, 160, 183

Drulis-Kawa, Zuzanna ..... 126, 130, 178

Durham, Jacob .................................... 129

Dutilh, Bas ........................................... 18

Edner, N.M............................................ 44

Egilmez, Halil ..................................... 112

Eldougdoug, Noha ................................ 30

ElRakaiby, Marwa ............................ 161

El-Shibiny, Ayman ...................... 32, 162

England, Whitney ............................... 118

Espy, Michael ..................................... 129

Estrella, Luis ................................. 62, 168

Etobayeva, Irina .................................. 99

Evans, Charlotte .................................... 95

Evans, Gary ......................................... 141

Fagerlund, Robert ............................... 139

Fane, Bentley ...................................... 132

Farfan-Esquivel, Juan ..................... 163

Farlow, J ............................................. 133

Fellouse, Fred.................................... 100

Fernández, Lucía ............................ 87, 89

Ficoseco, María ................................. 123

Fieseler, Lars ...................................... 125

Figlar-Barnes, Jarred .......................... 129

Filip, Carlos .......................................... 30

Filippov, Andrey ........................ 133, 164

Fineran, Peter ............. 21, 139, 140, 141

Finlay, Warren.................................... 156

Fish, Randy ......................................... 60

Flint, Annika ...................................... 147

Forde, Amanda ................................... 155

Fortuna, Wojciech ................................ 57

Franklin, Nathan ................................. 168

Fredrick, Maloba ................................ 177

Froissart, Remy .................................. 128

Funderburk, Parke .............................. 129

Furrer, Evan........................................ 129

Gage, Brianna ..................................... 129

Gallagher, Tara ................................... 118

Galtier, Matthieu ................................ 116

Galyov, Ed ......................................... 112

Gambino, Michela ........................ 88, 111

Gamieldien, Hoyam ........................... 128

Gammage, A. ....................................... 77

Ganichev, D.A. ..................................... 59

García, Pilar.................................. 87, 89

Gasiūnas, Giedrius ............................. 121

Gawor, Jan.......................................... 144

Gencay, Y. Emre .......................... 24, 88

Gilberts, Daniel .................................. 129

Gill, Jason ................................... 47, 131

Glassbrook, Andrew ........................... 129

Głowacka, Aleksandra ....................... 144

Gniadkowski, Marek .......................... 144

Goderdzishvili, Marina ...................... 171

Gogokhia, Lasha .................................. 32

Goldin, Sveta ........................................ 16

Golomidova, Alla ................................. 70

Gonzalez, Carlos ............................ 25, 85

González-Menéndez, Eva .................... 87

Goodridge, Lawrence ........................... 84

Górski, Andrzej ..... 57, 144, 145, 166, 174

Graham, Eric ....................................... 182

Graham, Zachary................................... 20

Grdzelishvili, Nino .............................. 171

Green, Jenny ....................................... 129

Griebel, Philip ....................................... 77

Griffiths, Mansel ................................... 30

Grigonyte, Aurelija ..................... 26, 165

Gromadka, Robert ............................... 144

Grosten, Kyle ...................................... 129

Guerin, Emma ........................... 113, 155

Guerrero-Ferreira, Ricardo ................... 70

Guild, Brandon ...................................... 28

Gula, Grzegorz .................................... 178

Gunathilake, Damitha ...................... 127

Gutiérrez, Diana ............................ 87, 89

Ha, Eunsu........................................... 142

Hakami, Ramin ..................................... 99

Halmillawewa, Anupama .................... 127

Hamilton, Theron .................... 49, 62, 168

Hansen, Lars ......................................... 46

Happel, Anna-Ursula ........................ 128

Happonen, Lotta .................................. 135

Hardy, Jonathan .................................. 167

Harhala, Marek ................. 145, 166, 174

Harrison, Melissa ................................ 156

Harrison, Sophie ............................... 129

Hatcher, Eneida ................................... 124

Hatfull, Graham ............................ 46, 156

Hawsawi, Hajar ..................................... 30

Hayes, Cillian ...................................... 183

Hayes, Connie ....................................... 77

Hayes, Sidney....................................... 77

He, Jenny............................................. 164

Hebert, Elvira ...................................... 123

Heck, Albert ........................................ 139

Hejatko, Jan......................................... 146

Hejnowicz, Monika ............................. 144

Hendrix, Hanne ............................. 64, 125

Henry, Marine ....................................... 78

Henry, Matthew ...................... 49, 62, 168

Hernández, R ....................................... 90

Hill, Colin ..... 50, 113, 125, 155, 160, 183

Hillebrand, Roman ................................ 46

Hinton, Debbie .............................. 65, 66

Hobbs, Zack ........................................ 91

Hodyra-Stefaniak, Katarzyna ..... 145, 174

Holguin, Angela ................................ 100

Holman, Derek ................................. 167

Holmfeldt, Karin ........................ 117, 121

Honaker, Ryan...................................... 91

Hooper, Annelise................................ 129

Hoyle, Naomi ...................................... 12

Hudson, Corey ..................................... 38

Huq, A ................................................ 170

Hurwitz, Bonnie ................................. 19

Hutchins, William .............................. 182

Hutinet, Geoffrey................................ 46

Hyman, Paul ............................... 81, 120

Hynes, Michael .................................. 127

Indráková, Adéla ................................ 153

Irwin, Casey ....................................... 129

Iskandriati, Diah ................................... 32

Israelsson, Stina ................................. 117

Iyer, L. .................................................. 66

Jackson, Simon ........................... 140, 141

Jacobs, Anna ............................... 49, 168

Jaiani, E ................................................ 92

Janda, Lubomir ................................... 146

Janelidze, N ................................. 92, 170

Jang, Ho Bin ....................................... 126

Jaramillo, Alfonso .............................. 165

Jaspan, Heather .................................. 128

Jaumdally, Shameem.......................... 128

Jauniškis, Vykintas ............................. 121

Jeon, Jongsoo .................................... 169

Jiménez, H ............................................ 90

Johnson, Allison ................................... 99

Jumba, Mirriam .................................... 94

Jun, Jin Woo ....................................... 135

Jurtz, Vanessa ..................................... 122

Kadija, Ermir ................................... 114

Kaestner, Coleman ............................. 129

Kaikabo, Adamu .......................... 32, 93

Kakabadze, Elene ............................. 171

Kallus, Katherine................................ 129

Kamwiziku, Guyguy ............................ 32

Kang'ethe, Erastus ................................ 32

Kariuki, Samuel .............................. 32, 94

Kaźmierczak, Zuzanna ........ 145, 166, 174

Kazwala, Rudovick ............................... 32

Kearns, Daniel..................................... 152

Keen, Eric ............................................ 17

Kemeraitė, Jolita ................................. 121

Kęska, Weronika ................................. 166

Khukhunashvili, T................................. 92

Khvichia, G. .......................................... 12

Kieper, Sebastian ................................ 139

Kilcher, Samuel ................................... 39

Kincaid, Randall ................................. 63

Kinkhabwala, Anika ............................. 91

Kirby, Bronwyn .................................... 22

Kirzner, Shay ........................................ 16

Klaus, James ......................................... 17

Kłopot, Anna ....................... 145, 166, 174

Klumpp, Jochen .................................... 39

Klykov, Oleg ....................................... 139

Knaus, Nolan....................................... 129

Knipling, Leslie..................................... 65

Knirel, Yuriy ......................................... 70

Koberidze, ........................................... 170

Kok, Jan .............................................. 102

Kokashvili, T....................................... 170

Koledin, Tamara ................................. 129

Konkol, Melissa .................................. 152

Kot, Witold ........................................... 46

Kozlova, Yu .................................... 55, 59

Kraus, Randolph ................................. 129

Krause, Kurt ........................................ 139

Krishnakumar, Raga ............................. 38

Kuhl, Sarah ........................................... 60

Kuipers, Oscar..................................... 102

Kutateladze, M .............................. 60, 133

Kutter, Betty .... 60, 68, 75, 123, 157, 158

Kvachadze, L ...................................... 133

Labrie, Simon ...................................... 115

LaFave, Matt ......................................... 33

Lakshman, N ......................................... 77

LaLonde, Tessa ................................... 129

Langevin, S. .......................................... 60

Latka, Agnieszka ............................... 130

Lau, Britney .......................................... 38

Lavigne, Rob ...... 40, 48, 64, 71, 96, 125,

126, 154

Lazarou, Michael.................................. 53

Le, Tram ............................................... 85

Leavitt, Justin ................................... 131

Lecion, Dorota.................... 145, 166, 174

Ledbetter, Stacia ................................. 129

Lee, Kyungwon .................................. 169

Lee, Yan-Jiun ..................................... 147

Leech, Thomas .................................. 172

LeGault, Kristen ............................... 143

Leigh, Brittany ................................... 20

Leiman, Petr ......................................... 70

Lemire, Sébastien ....................... 34, 184

Lepique, Ana ...................................... 105

Letarov, Andrey ................................... 70

Letarova, Maria.................................. 23

Letkiewicz, Sławomir .......................... 57

Leung, Chung ....................................... 78

Leung, Sharon .................... 156, 157, 158

Leung, Vince ........................................ 30

Lévesque, Alice ................................. 115

Li, Jian .................................................. 75

Liberti, Assunta .................................... 20

Lim, Loong-Tak ................................... 28

Lindell, Debbie ................................... 16

Linden, Sara ................................. 99, 103

Lindhard, Barbara .............................. 122

Liu, Y. .................................................. 44

Łobocka, Małgorzata ....................... 144

Lockwood, Madesyn .......................... 129

Loessner, Martin .................................. 39

Lomelí-Ortega, Carlos ............. 101, 173

Lopez, Anthony .................................. 129

Łoś, Marcin ......................................... 79

Lourenço, Marta .............................. 116

Lu, Rebecca .......................................... 91

Lu, Timothy ........................... 34, 97, 184

Ludwig, Kody .................................... 129

Luna, Sharmin .................................... 108

Lund, Ole ........................................... 122

Lundin, Daniel.................................... 117

Lungren, Matthew .............................. 167

Luque, Antoni ...................................... 53

Maes, M ................................................ 96

Maestri, Alice ........................................ 32

Magass, Nacia ..................................... 129

Mageeney, Catherine ........................ 150

Mahmud, Shahin ................................. 108

Mahony, Jennifer .................................. 69

Maidanik, Ilia ........................................ 16

Maina, Alice ................................... 32, 94

Majewska, Joanna............. 145, 166, 174

Majkowska-Skrobek, Grazyna ............ 130

Makalatia, Khatuna ............................. 171

Makumi, Angela ...................... 32, 69, 71

Malagon, Francisco ............................... 17

Malik, Danish .............................. 95, 175

Malki, Kema ...................................... 132

Mancuso, Francesco ............................ 175

Marcelli, Barbara .............................. 102

Marchon, Bruno .................................... 91

Markwitz, Pawel ................................. 178

Martin, Mark ....................................... 132

Martínez, Beatriz............................. 87, 89

Martínez-Díaz, Sergio ................. 101, 173

Martiny, Jennifer ................................. 118

Mašlaňová, Ivana ................................ 153

Mateczun, Alfred .................................. 62

Mattinen, Laura ................................... 135

Maura, Damien ................................... 116

Maxwell, Howard ............................... 139

Mazurkiewicz-Kania, Marta ............... 145

McAuliffe, Olivia ........................ 125, 134

McCann, Angela ................... 50, 113, 160

McCliment, Trevor ............................. 129

McCutcheon, Jaclyn ......................... 176

McDonnell, Siobhan ........................... 155

McKitterick, Amelia ........................... 80

McNamara, Bryanne ........................... 129

Melkova, Katerina............................... 146

Melo, Luis ............................................. 42

Merabishvili, Maia ........................ 48, 171

Meyer, Evelyne ..................................... 91

Międzybrodzki, Ryszard .................... 57

Miernikiewicz, Paulina ....... 145, 166, 174

Millard, Andrew ...... 26, 52, 95, 136, 165

Miller, Stefan ....................................... 35

Mkrtchyan, Mkhtar ............................ 171

Moineau, Sylvain ................... 46, 76, 115

Mokres, Lucia ...................................... 91

Moodley, Arshnee .............................. 154

Morales, Sandra ........... 58, 75, 157, 158

Morello, Eric ........................................ 78

Morozov, Andrew .............................. 112

Morozova, V.V. ............................. 55, 59

Mosier, Philip ....................................... 99

Mosterd, Cas ....................................... 76

Motoi, Maria ........................................ 61

Muessner, Christina ............................. 39

Mukhopadhyay, Suchetana ................ 152

Munoz, Robert ................................... 129

Mwaura, Francis ................................... 94

Myagmarjav, Bat-Erdene ................... 152

Nadareishvili, L. ................................... 12

Nagel, Tobi .......................................... 32

Nakavuma, Jesca ................................ 32

Nale, Janet ........................................... 32

Napper, Scott ........................................ 77

Napuli, Alberto ................................... 129

Nasinyama, George ............................ 32

Nawaz, Ayesha ................................... 135

Nazarov, Sergey ................................... 70

Needham, Micah ................................ 129

Neguț, Alina ........................................ 61

Neitzel, James .................................... 129

Nelson, Daniel ............... 36, 99, 103, 104

Nelson, Scott ....................................... 67

Neve, H .............................................. 134

Neve, Horst ........................................ 125

Nguyen, Sophie .................................... 53

Nielsen, Morten .................................. 122

Nikolich, Mikeljon .................... 133, 164

Nilsson, Emelie ......................... 117, 121

Nizharadze, D. ...................................... 12

Njoya, Kimani .................................... 182

Noben, Jean-Paul........................ 125, 126

Nobrega, F.L. ....................................... 44

Nocis, Melyssa ................................... 129

Novacek, Jiri ...................................... 146

Nowak, Sylwia ................................... 145

Nowotny, Taylor ................................ 129

Ntaongo, Julien ..................................... 32

Nyachieo, Atunga .............................. 177

Nyamongo, Onkoba ............................ 177

O’Mahony, Jim ................................... 125

O’Sullivan, Lisa ................................. 134

Oakley, Brian ........................................ 98

Oany, Arafat ........................................ 108

Ochieng, Joseph................................. 177

Odishelidze, N....................................... 12

Ogden, Luke ........................................ 129

Oliver, Andrew .................................. 118

Oliveria, Hugo..................................... 125

Olszak, Tomasz ................................. 178

Omran, Maha ...................................... 161

Ostapchuck, Yuri ................................ 124

Ouma, Arodi ....................................... 177

Owczarek, Barbara .............. 145, 166, 174

Padman, Benjamin ................................ 53

Pajunen, Maria .................................. 135

Pamungkas, Joko................................... 32

Pantůček, Roman ...................... 146, 153

Parker, David ...................................... 129

Parks, Olivia ........................................ 129

Parsley, Todd ....................................... 37

Passarell, María ................................... 123

Passmore, Jo-Ann ............................... 128

Patterson, Adrian................................. 141

Pearce, F. Grant................................... 139

Pekarsky, Irena ...................................... 16

Pernisova, Marketa ............................. 146

Perry, Benjamin .................................. 127

Peters, Danielle .......................... 151, 176

Peterson, Todd ...................................... 33

Petrova, Zaritza ................................... 156

Phillips, Kaden .................................... 129

Phillips, Kenya ...................................... 27

Pinhassi, Jarone ................................... 117

Pipia, L. ................................................. 12

Pires, Priscila......................................... 42

Pirnay, Jean-Paul .................. 11, 32, 171

Piya, Denish ......................................... 47

Plastow, Rian ...................................... 129

Plaut, Roger ....................................... 180

Plevka, Pavel ....................................... 153

Pollet, S ................................................ 96

Pope, Robert ......................................... 62

Pope, Welkin ...................................... 156

Porchkhidze, K ............................. 92, 170

Portilla, Silvia ....................................... 89

Prince, Jeffrey ...................................... 17

Prokhorov, Nikolai ............................. 70

Pruidze, N. ............................................ 12

Prüssing, Tessa ..................................... 88

Przybilski, Rita ................................... 141

Putonti, Catherine ............................ 119

Quaraishi, Habib ................................ 129

Quinones, Javier ........................... 62, 168

Radford, Devon .................................. 28

Rajamanickam, Karthic ........................ 77

Ramsey, Jolene ................................. 152

Raqib, Rubhana .................................. 108

Rauch, Benjamin .................................. 43

Ray, Melissa ...................................... 180

Raya, Raul .......................................... 123

Reddy, Akhil .............................. 133, 164

Redgwell, Tamsin ................. 26, 52, 136

Regeimbal, James ................... 49, 62, 168

Restrepo-Córdoba, Marcela ............... 127

Reyes, Alejandro .................... 13, 29, 90

Riccio, Cristian ..................................... 70

Richardson, Toby ................................. 33

Richter, Manuela .................................. 91

Rihtman, Branko .................................. 26

Rimbey, Jeanette .................................. 27

Ritchie, Jenny ..................................... 159

Roach, Dwayne ................................... 78

Roberts, Justin .................................... 129

Robertson, Rosanna ............................. 65

Rodríguez, Ana .............................. 87, 89

Rogóż, Paweł ....................................... 57

Rohde, Christine ................................. 137

Rohde, Manfred.................................. 137

Rohwer, Forest ..................................... 53

Roman, Jessica ..................................... 49

Rombouts, S. ........................................ 96

Ross, Alexa ........................................ 120

Ross, R. Paul 50, 113, 125, 155, 160, 183

Rossiter, Shayna ................................. 129

Roundtree, Breck ................................ 129

Rouse, Michael .................................... 49

Rowley, D. Treva .................................. 98

Russell, William .................................... 47

Ryan, Feargal ................................ 50, 160

Ryu, Sangryeol ........................ 83, 86, 142

Sacher, Jessica ................................... 147

Salmond, George................................. 141

Samir, Reham ...................................... 161

Săndulescu, Oana .................................. 61

Saunders, Candace .............................. 129

Sauvageau, Dominic ................... 156, 181

Sazinas, Pavelas .................................... 26

Scanlan, Dave ..................................... 136

Scheltema, Richard ............................. 139

Schlosser, Kyle ..................................... 53

Schmidt, Imke ..................................... 137

Schneider, Christine ......................... 182

Schoeniger, Joseph ................................ 38

Schooley, Robert ................................... 62

Schouler, Catherine ............................. 154

Schubert, Alyxandria .......................... 54

Scoville, Marissa ................................. 129

Sedrykyan, Anahid .............................. 171

Seed, Kimberley ............ 41, 80, 138, 143

Seil, Alexea ......................................... 129

Sergueev, Kirill ........................... 133, 164

Shanahan, Fergus ................................ 155

Shang, Xiaoran .................................. 104

Shapiro, Jason ..................................... 119

Shearer, Jonathan .................................. 49

Shohadai, Petal .................................... 129

Shoporov, Andrey ............................... 113

Šiborová, Marta................................... 153

Sillankorva, Sanna ................................ 42

Šimoliūnas, Eugenijus......................... 121

Simons, Mark ........................................ 49

Singhm,Devika...................................... 78

Skurnik, Mikael........................... 56, 135

Smith, George ....................................... 27

Smith, Keelan ...................................... 129

Smith, Muireann ............................... 183

Songailienė, Inga................................. 121

Sørensen, Martine ................................. 24

Speicher, David ................................... 51

Spröer, Cathrin ................................... 137

Squeglia, Flavia .................................. 130

Staals, Raymond ................... 21, 139, 141

Staes, Ines ............................................ 71

Staffe, Cæcilie .................................... 111

Stanbro, Josh ........................................ 49

Staub, Sean ......................................... 129

Stibitz, Scott ................................ 14, 180

Stintzi, Alain ...................................... 147

Stockelman, Michael .................... 49, 168

Stothard, Paul ..................................... 151

Strange, Philip ...................................... 28

Streinu-Cercel, Adrian ......................... 61

Streinu-Cercel, Anca ............................ 61

Studer, Patrick ...................................... 39

Sulaiman, Lanre ............................... 105

Šulčius, Sigitas .................................. 121

Swift, Stephen ...................................... 98

Szymanski, Christine ......................... 147

Taha, Omar ......................................... 162

Talukder, Kaisar ................................. 108

Taylor, Corinda .......................... 140, 141

Tediashvili, M .............................. 92, 170

Tedoradze, I.......................................... 12

Temple-Rosebrook, Louise .................. 99

Thanki, Anisha ..................................... 95

Thompson, Charles ............................ 129

Tikunova, N.V. ............................. 55, 59

Tolstoy, Igor ....................................... 124

Trindade, Marla .................................... 22

Tsersvadze, G ....................................... 92

Tsertsvadze, G .................................... 170

Turner, Paul .......................................... 32

Tyner, Stuart ................................. 49, 168

Valvano, Miguel ................................. 178

Van Belleghem, Jonas ........................ 48

van Charante, F .................................... 96

van Rijswick, Irma ............................... 69

van Sinderen, Douwe ........................... 69

Van Vaerenbergh, J .............................. 96

van Zyl, Lonnie .................................... 22

Vananzo, Steven ................................. 129

Vaneechoutte, Mario ............................ 48

Vara, Leonardo ..................................... 47

Vehring, Reinhard ......................... 32, 156

Velayudhan, Vimalkumar ............. 50, 160

Venneman, S ......................................... 96

Vidal, Amber....................................... 129

Villa, Luisa .......................................... 105

Villamizar, Santiago ......................... 106

Vincent, Antony .................................. 115

Vincent, Warwick ............................... 115

Vinner, Gurinder ........................... 95, 175

Vives, Martha ........................ 29, 106, 163

Vladisavljević, Goran ......................... 175

Vlassov, V.V. ........................................ 59

Volckaert, A .......................................... 96

von Henkelman, Jenny ........................ 129

Vrbovská, Veronika ............................ 153

Waddell, M. Brett ................................. 65

Wagemans, Jeroen .............................. 96

Wagner, Julian ...................................... 38

Wandro, Stephen ................................. 118

Wang, Jiping ......................................... 75

Ware, Vassie ....................................... 150

Waśko, Patryk ................................... 148

Waters, Jerel .......................................... 98

Watkins, Siobhan .............................. 119

Watson, Bridget................................... 21

Watters, Chaselynn ............................... 49

Weber-Dąbrowska, Beata ............. 57, 144

Weigele, Peter ..................................... 147

Weihe, Claudia .................................... 118

Weitz, Joshua ........................................ 78

Wenke, Joseph .................................... 168

West, Jennifer ...................................... 66

Weston, Thomas ................................... 53

Westra, Edze ........................................ 45

Wheat, Gordon ...................................... 60

White, Stephen ...................................... 65

Whiteson, Katrine ............................... 118

Whitford, Crystal ................................ 129

Wilkinson, Max................................... 139

Williams, Chandler ............................. 129

Williams, Kelly .................................... 38

Wittmann, Johannes ......................... 137

Wojtyna, Karolina ............... 145, 166, 174

Wolfe, David ........................................ 62

Wolput, Sanne ...................................... 71

Woolston, Joelle .................................. 31

Woźnica, Wioleta ............................... 144

Yakovets, E.A ...................................... 55

Yang, Andrew .............................. 34, 184

Ye, Wei .............................................. 100

Yeasmin, Mahmuda ........................... 108

Yehl, Kevin ................................. 34, 184

Yong, Dongeun ................................... 169

Yost, Christopher ............................... 127

Young, Ry ...................................... 47, 85

Zablocki, Olivier ................................. 22

Zaczek, Maciej ...................................... 91

Zdorovenko, Evelina ............................. 70

Zeman, Michal................................... 153

Zeng, Lanying ...................................... 72

Zermeño-Cervantes, Lina ................... 101

Zhang, Junjie ....................................... 73

Zhvania, P. ............................................ 12

Zidek, Lukas ....................................... 146

Zielonka, Katarzyna ............................ 145

Zschach, Henrike .............................. 122

Save the dates! ISVM Viruses of Microbes, July 9th – 13th 2018 We are really pleased to invite you to the 2018 Viruses of Microbes meeting: Biodiversity and

future applications that will take place in Wrocław, Poland.

The Wrocław meeting will be the 5th flagship meeting for the International Society of Viruses of

Microbes, following conferences organized in Paris 2010, Brussels 2012, Zurich 2014, and

Liverpool 2016, and bringing together research communities studying viruses infecting bacteria,

archaea, algae, fungi and protozoa. The conference will cover a broad range of topics, from

evolutionary and ecological aspects, through viral molecular structures, to practical applications in

industry and medicine. A special focus will be given to microbial virus encoded enzymes and

phage therapy, as these represent the main Polish experience in the field. Sessions will include:

Viral entry and exit in the variety of microbial hosts

Transcription and replication in viruses of microbes

Host-virus interactions: structure and function, microbial resistance to viral infection

Microbial virus encoded enzymes: fundamentals and application

Biotechnology and molecular engineering of microbial viruses

Phage therapy (I) fundamentals, (II) application, and discussion panel: direction to the future

Diversity and evolution

Ecology and systems biology

Application to market: food production, agriculture, environmental, diagnostics

All of our invited speakers are international experts, many of whom are new to speaking at a VoM

meeting so will bring fresh and innovative perspectives.

Dating back to the 10th century, Wrocław is a beautiful

city and a thriving multicultural centre attracting visitors

from all over the world. Its extensive heritage combines

almost all religions and cultures of Europe! Wrocław is

Poland’s fourth largest city, situated in the south-

western part of the country, on the banks of the Oder

river and its four confluents. With over 100 bridges,

Wrocław is often called the Venice of the North or the

City of Bridges. Besides its fascinating architecture and

being the cultural and academic centre of Lower Silesia, Wrocław is also famous for its dwarfs –

over 300 bronze figurines are scattered and hidden around the city. The city offers many cultural

attractions: 8 theatres, 14 museums, Opera House and National Forum of Music. The conference

venue – a spectacular, modern Conference Centre at the University of Wrocław – is located right in

the heart of the city, in front of the main building of the University and within walking distance of

the vibrant and colorful Market Square (Rynek) and picturesque Ostrów Tumski with a XIII

century gothic cathedral. The opening ceremony and choir concert will be held in the magnificent

Aula Leopoldinum, a representative baroque auditorium of the University of Wrocław. Social

events will also include welcome party in Oratorium Marianum, a get-together barbeque at the

Botanic Garden, and a special evening in the unique africarium of the Wrocław ZOO.

We look forward to seeing you in Wrocław! Please contact Prof. Zuzanna Drulis-Kawa

([email protected]) or Dr Krystyna Dąbrowska ([email protected]) if

interested in sponsorships or with any questions, comments or suggestions. Website with further

information and registration form will be available soon.

22nd biennial evergreen international phage...

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