ISSN 1479-2699The magazine of the Society for Applied Microbiology ■ December 2011 ■ Vol 12 No 4

WOUNDINFECTION

■ The evolving challenge of war wounds ■ Is the use of honey for the treatment of biofilms a sticky subject?

■ Bateriophages’ function in wound healing ■ New SfAM website ■ Biofocus: EU legislation ■ StatNote 27

■ Science Communication Award winner ■ Environmental Microbiology Lecture 2011 report ■ PECS: writing up

■ Summer Conference 2011 report ■ Winter Meeting 2012 ■ SfAM events in 2012 — save the dates!INSID

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03

contentsthe magazine of the Society for Applied Microbiology

December 2011 ■ Vol 12 No 4 ■ ISSN 1479-2699

26

members

04 Editorial: Lucy Harper talks about wound infection

07 New Members: we welcome our new members

08 President’s column: Martin Adams talks about peer review

09 CEO’s column: latest developments in the Society

10 Membership matters

41 In the loop: writing up

42 Public Engagement Grant: report

44 Sponsored Lecture Grant: report

45 Students into Work Grant reports

47 President’s Fund reports

publications

13 Journal watch

news

11 Biofocus: legislation from the European Union

12 Science Communication Project report

features

26 The evolving challenge of war wounds

30 The use of honey for the treatment of biofilms

33 Bacteriophages’ function in wound healing

37 StatNote 27: identification of bacterial strains

meetings

15 SfAM events in 2012: save the dates!

16 Summer Conference 2011 report

22 Communications Award 2011 winner report

23 Environmental Microbiology Lecture 2011 report

24 Winter Meeting 2012 programme and booking form

commercial

50 Advertisements and news from our Corporate members

Microbiologist is published quarterly by the Society forApplied Microbiology. ISSN 1479-2699. Registered in theUK as a charity and Company limited by guarantee.Registered in England and Wales: 6462427. RegisteredCharity: 1123044.

© Society for Applied Microbiology 2007-2011. Materialpublished in Microbiologist may not be reproduced,stored in a retrieval system, or transmitted in any formwithout the prior permission of the Society.

Editor: Lucy Harper. lucy@sfam.org.uk

Contributions: These are always welcome and should beaddressed to the Editor at: lucy@sfam.org.uk

Advertising: Lucy Harper. Tel: +44 (0)1234 326709.email: lucy@sfam.org.uk

Design and print: Pollard Creativity Limited Tel: +44 (0)1933 664700.email: micro@pollardcreativity.co.uk

Cover: © Pollard Creativity Limited

Society for Applied Microbiology, Bedford Heights,Brickhill Drive, Bedford MK41 7PH, UK.Tel: +44 (0)1234 326661. Fax: +44 (0)1234 326678.

www.sfam.org.uk

information

24Winter Meeting 2012

30Honey for thetreatment of biofilms

04

Lucy Harper

members

Microbiologist ispublished quarterly bythe Society for AppliedMicrobiology, aregistered charity. ISSN 1479-2699.

Copy Dates:

Vol 13 No.1 March 2012Friday 23 Dec 2011

Vol 13 No.2 June 2012Friday 23 March 2012

Vol 13 No.3 Sept 2012Friday 22 June 2012

Vol 13 No.4 Dec 2012Friday 28 Sept 2012

Disclaimer: The Societyassumes no responsibilityfor the opinionsexpressed bycontributors. The viewsexpressed by Societyofficers and staff do notnecessarily represent theofficial position of theSociety. Readers shouldnote that scientificmaterial is not refereedand represents only theviews of the authors.The claims of advertiserscannot be guaranteed.

Subscriptions:A subscription toMicrobiologist isincluded in the annualSfAM membership fee.For further informationabout the many benefitsof membership pleasesee page 6.

Advertising:Information aboutadvertising inMicrobiologist and howto submit advertise mentscan be found on theSociety website.

Website: our website(www.sfam.org.uk) isa timely source of up-to-date information on allSociety matters andmaintains acomprehensive archiveof articles and reports ona variety ofmicrobiological topics.

When did you last fall over andcut yourself? For me it was asa child, and I’m happy to say

it’s been a long time since I encounteredan open wound. We’re all familiar withthe amazing, healing ability of thehuman body, but if wounds becomeinfected, the formation of biofilms canmean this process is interrupted. Somepeople encounter wounds as a matter ofcourse as part of their daily routine andon page 26 we hear about thecomplications of treating wounds ofconflict. As the authors say, this isn’tnew: “The fight against thedevelopment of infection in battlewounds is not a new problem.Sumerian carvings describe the useof beer and hot water to wash woundswhich were then dressed with aplaster of herbs or fruits. The ancientEgyptians used honey and sometimesanimal faeces to dress wounds and

Hippocrates describes the washing of woundswith wine and the drainage of pus to reduceinflammation.”

Hmmmm… I’m not sure about its efficacy,but I like the idea of treating wounds withwine…

But on a more serious note, the mention ofhoney leads me nicely on to our second featureon page 30 which describes the use of honey inthe treatment of biofilm infections. Again, this isnot a new phenomenon: “Honey is an ancient

wound remedy that has been re-introduced into modern medicalpractice and a range of licensed products that contain honey areavailable on prescription in Australasia, Europe and North America.They range from sterile medical grade honey in tubes, honeyincorporated into ointments or impregnated on to dressings.”

In the final feature article, we look at a pet love of mine —bacteriophages and their use in the treatment of wound infections. Turn topage 33: “Bacteriophages are ubiquitous and more abundant than anyother nucleic acid-based entities. They display incredible variationsin how they adhere to their target host cell, breach the outer defences,replicate, and eventually lyse their host to infect new cells. Added tothis complexity are vastly different strategies for phage infectionspossessed by lytic versus temperate bacteriophages. However, throughpersistence the therapeutic use of bacteriophages is emerging.”

In keeping with the approaching festive season, this issue is also one ofcelebration. We celebrate the winner of this year’s Communication Award,Professor Joanna Verran on page 22 and, as with each issue of themagazine, we celebrate the work that’s funded by many SfAM grants,including the Sponsored Lecture Grant and the Public Engagement Grant.

All the information you’ll need about our grants is available on the newSfAM website http://www.sfam.org.uk/en/grants--awards/index.cfm andwe’ve made it easier for you to apply for grants, by providing a facility todo this online. You can read all about the new SfAM website on page 10and remember, the site will continue to develop and evolve with the needsof our Members, Non-Member visitors and the development of newtechnologies. So get in touch with your ideas.

Finally, at this time of celebration, I would like to wish all readers afantastic festive season and a wonderful 2012.

editorialLucy Harper talks about wound infection— the main theme of this issue ofMicrobiologist

We are always lookingfor enthusiastic writerswho wish to contributearticles to the magazineon their chosenmicrobiological subject.

For further informationplease email the editor,Lucy Harper at:lucy@sfam.org.uk

contribute

05

COMMITTEE MEMBERS

HON PRESIDENT: Professor Martin Adams, School of Biomedical & MolecularSciences, University of Surrey, Guildford, Surrey GU2 7XHemail: m.adams@surrey.ac.uk

HON GENERAL SECRETARY: Dr Mark Fielder, School of Life Sciences, KingstonUniversity, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EEemail: m.fielder@kingston.ac.uk

HON MEETINGS SECRETARY: Dr Andrew Sails, Health Protection Agency,Newcastle Laboratory, Institute of Pathology, Newcastle General Hospital, WestgateRoad, Newcastle NE4 6BEemail: andrew.sails@hpa.org.uk

HON TREASURER: Mr Steve Davies, Microbiology Department, Northern GeneralHospital, Herries Road, Sheffield S7 5AUemail: steve.davies@sth.nhs.uk

ORDINARY COMMITTEE MEMBERS UNTIL JULY 2012

Mr Mark Reed, Pro-Lab Diagnostics, 7 Westwood Court, NestonCheshire CH64 3UJemail: mreed@pro-lab.com

Dr Sally J Cutler, School of Health and Biosciences, University of East London,Stratford Campus, Romford Road, London E15 4LZemail: s.cutler@uel.ac.uk

Dr Samantha Law, NCIMB, Ferguson Building, Crabstone Estate, Bucksburn,Aberdeen AB21 9YAemail: s.law@ncimb.com

Dr Alison Kelly, School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EEemail: a.kelly@kingston.ac.uk

ORDINARY COMMITTEE MEMBERS UNTIL JULY 2013

Dr Louise Fielding, Food Research and Consultancy Unit, Cardiff School of HealthSciences, University of Wales Institute Cardiff, Llandaff Campus, Western Avenue,Cardiff CF5 2YBemail: lfielding@uwic.ac.uk

Dr Irene Grant, Institute of Agri-Food and Land Use, School of Biological Sciences,Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BLemail: i.grant@qub.ac.uk

Dr Katie Laird, De Montfort University, The Leicester School of Pharmacy, Faculty ofHealth & Life Science, Hawthorn Building, Leicester, LE1 9BHemail: klaird@dmu.ac.uk

ORDINARY COMMITTEE MEMBERS UNTIL JULY 2014

Professor Christine Dodd, Division of Food Sciences, School of Biosciences, Universityof Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RDemail: christine.dodd@nottingham.ac.uk

Dr Clare Taylor, School of Life, Sport & Social Sciences, Edinburgh Napier University,Sighthill Campus, Sighthill Court, Edinburgh, EH11 4BNemail: cl.taylor@napier.ac.uk

CHIEF EXECUTIVE OFFICER: Philip Wheatemail: pfwheat@sfam.org.uktel: +44 (0)1234 326661

COMMUNICATIONS MANAGER: Lucy Harperemail: lucy@sfam.org.uktel: +44 (0)1234 326709

COMMUNICATIONS OFFICER: Clare Doggettemail: clare@sfam.org.uktel: +44 (0)1234 327679

MEMBERSHIP & FINANCE CO-ORDINATOR:Julie Wrightemail: julie@sfam.org.uktel: +44 (0)1234 326846

EVENTS ORGANIZER: Sally Cryeremail: sally@sfam.org.uktel: +44 (0)1933 382191

ADMINISTRATOR: Julie Buchananemail: julieb@sfam.org.uktel: +44(0)1234 326661

executive committee

society office staff

contact point

Society for Applied MicrobiologyBedford Heights, Brickhill Drive, Bedford MK41 7PH, UK.

tel: +44 (0)1234 326661fax: +44 (0)1234 326678email: communications@sfam.org.ukwww.sfam.org.uk

FEATURES EDITOR: Claire Cassaremail: c.cassar@vla.defra.gsi.gov.uk

FEATURES EDITOR: Louise Fieldingemail: lfielding@uwic.ac.uk

FEATURES EDITOR: Clare Tayloremail: cl.taylor@napier.ac.uk

REGULAR CONTENT EDITOR: Alison Kellyemail: a.kelly@kingston.ac.uk

GRANTS EDITOR: Louise Hill-Kingemail: louise@hill-king.com

publications subcommittee

06

membershipbenefits

■ Full Ordinary Membership gives access to ourmany grants and awards, online access to theJournal of Applied Microbiology, Letters in AppliedMicrobiology, Environmental Microbiology,Environmental Microbiology Reports and MicrobialBiotechnology, copies of Microbiologist, preferentialregistration rates at Society meetings and access tothe members’ areas of the website.

■ Full Student Membership confers the samebenefits as Full Membership at a specially reducedrate for full time students not in receipt of a taxablesalary.

■ Associate Membership is only open to thosewith an interest in applied microbiology without itbeing a prime aspect of their job. For example,school teachers and those taking a career break; onmaternity leave, or working temporarily in otherareas. It does not provide access to any journals orSociety grants and awards.

■ Honorary Membership of the Society is byelection only and this honour is conferred onpersons of distinction in the field of appliedmicrobiology. Honorary Members have access to ouronline journals.

■ Retired Membership is available to FullMembers once they have retired from theiremployment. Retired Members are entitled to all thebenefits of Full Membership except grants andaccess to the Society’s journals.

■ Corporate Membership is open to allcompanies with an interest in microbiology.Corporate Members benefits include:● Quarter page advertisement in each issue of Microbiologist (which can be upgraded to a larger size at discounted rates).

● The opportunity to publish press releases, company news, etc., in each issue of Microbiologist.

● FREE banner advert on the Society website with a direct link to your company site.

● Up to three members of company staff attending Society meetings at members’ rate (this means a 50% discount on non member registration rate).

JOIN US!You can apply for membership on, or offline. Toapply offline, please contact the Membership &Finance Co-ordinator, Julie Wright on +44 (0)1234326846, or email julie@sfam.org.uk. Alternatively,write to her at:

The Society for Applied Microbiology, BedfordHeights, Brickhill Drive, Bedford MK41 7PH, UK.

options

www.sfam.org.uk

The Society for Applied Microbiology is the voice of appliedmicrobiology within the UK and was founded in 1931. Society membersplay a leading role in shaping the future of applied microbiology, and enjoymany benefits, including:

■ The opportunity to apply for one of our many grants or funds.■ Eligibility to win any of our awards or nominate a candidate for the

SfAM Communications Award.■ Access to our five peer–reviewed Journals: Journal of Applied

Microbiology, Letters in Applied Microbiology, Environmental Microbiology, Environmental Microbiology Reports and Microbial Biotechnology.

■ Free access to the entire collection of digitized back files for JAM and LAM dating back to 1938.

■ A topical quarterly magazine, Microbiologist.■ Substantially reduced rates for attendance at SfAM meetings and

conferences.■ Networking with worldwide professionals in over 80 countries.■ Access to private members’ area of the SfAM website.■ Monthly email bulletins with the latest news from SfAM.■ Invitation to the annual Environmental Microbiology lecture.■ Fostering cross disciplinary research.■ A 25% discount on the extensive Wiley–Blackwell collection of titles. Detailed information about all these benefits and more can be found on

the Society website at: www.sfam.org.uk.

GRANTS & AWARDS: Many grants, awards and prizes are available tomembers including the W H Pierce Memorial Prize and prizes for studentoral presentations and posters at the Summer Conference. In addition tothese substantial awards, the Society has funds to assist members in theircareers as microbiologists. These include the President’s Fund, ConferenceStudentships, Sponsored Lecture Grants and the popular Students intoWork Scheme.

Full details of all the Society’s grants and awards can be found on thewebsite together with application forms.

JOURNALS: The Society publishes two monthly journals: Journal ofApplied Microbiology and Letters in Applied Microbiology. We alsoproduce this quarterly colour magazine, Microbiologist, which containsfeatures, topical news stories and full details of our meetings. The Societyis also a partner with Wiley–Blackwell in the monthly journals:Environmental Microbiology, Environmental Microbiology Reportsand Microbial Biotechnology.

All Full and Student Members receive free access to the online versionsof the Society’s journals, and can also submit papers to our journals via anonline submission service.

MEETINGS: We hold three annual meetings; the Winter Meeting is aone-day meeting with parallel sessions on topical subjects. The SpringMeeting is a one–day meeting tailored for personnel in clinicalmicrobiology. The Summer Conference is held every July and comprises amain symposium, a poster session, the AGM and a lively socialprogramme. All members are invited to our prestigious annual lecture heldto commemorate the success of our Environmental Microbiology journal.We also hold joint ventures with other organizations on topics of mutualinterest.

WEBSITE: The website is the best source of detailed information on theSociety and its many activities. It has fully interactive membership areaswhere you can find archive issues of Microbiologist, exclusive SfAMdocumentation and much more.

members

07

NEW MEMBERS

We would like to warmly welcome thefollowing new members and hope thatyou will participate fully in the activitiesof the Society.

Australia

R. Cavicchioli; D. Mahoney; T. Kostic; A. Loy; M. Wagner

Belgium

V. Castiaux

Canada

J. Foght; L. Y. Stein

Chile

R. T. Espeso

Denmark

M. Kuhl

Germany

M. Friedrich; G. Molinari; V. Muller; B. Tummler

India

N. Desai; D. Kaushik; R. Manickam; H. Panwar; S. Sharma

Ireland

N. Borchert; K. Burgess; M. Friel; A. Grainger; N. Kennedy; M. Lenahan; O. Lynch; M. Martins; E. M. McCabe; M. McCusker; K. McDonald; H. Meredith; D. O’Leary

Italy

F. Nazzaro

Jordan

M. Mustafa

Kenya

M. M. Malenge

Netherlands

B. Lugtenberg

Nigeria

E. O. Adeleke; T. C. Adias; R. Anyanwu; F. V. Daniels; F. Esumeh; N. N. Iwuala; A. I. Obasi; V. A. Oriaku; B. A. Osopale

Spain

A. Jofre; B. Nogales; J. Ramos; F. Rojo

Sweden

U. M. Romling

UK

M. Adnan; Y. Ahad; H. R. Ahmad; S. Akhtar; R. Akthar; S. Ali; A. Ali; F. Alkhaleefah; G. Allardyce; E. Allegra; W. Allison; A. Angel; W. Armour; J. P. Ashton; A. Ashton; N. Astbury; S. M. Athi Narayanan; N. A. Baharin Md Daud; A. Bendall; J. Betts; L. Bishop; B. Blane; S. Bradley; J. Braid; D. Butler; C. Cass; M. Charnick; G. Cole; J. Collins; A. Collins; I. Concepcion; S. Cowper; D. Dadnam; W. Davies; K. Dyson; A. Edwards; A. Fisher; M. Frost; H. Garner; D. Green; N. J. Grover; B. Hadef; M. Hallinan; A. Hammond; K. R. Hardie; R. E. Harris; J. Hawkins; K. Ho; V. Horta de Passo; Z. Hoskins; A. Hunt; R. Ingham; R. Jackson; S. A. James; K. Jenkins;A. Johnson; S. Keane; S. Khalaj; C. Loo; N. Mallon; B. Manku; J. A. Marr; M. D. M. Martin; T. McGenity; A. McIntyre; K. Metson; N. Mistry; A. Mohamudally; R. Monson; J. C. Murrell; S. Nicholson; A. O. Olaifa; I. T. Olonade; C. A. Pallister; B. Pandya; S. Park; K. Patel; H. J. Plant; J. Portman; J. Potrykus; R. Praptiwi; S. Raghvani; B. Reynolds; S. Rubinchik; P. Scanlan; V. Sewell; D. Sharp; N. Stone; B. Suarez Martinez-Falero; R. Swann;C. Swift; D. Taylor; N. Thiru; D. S. L. Thomas; F. Urbas; M. Veses Garcia; C. Waitt; K. Ward; J Wardle; J. M. Warrillier-Grant; S. Weaver

USA

B. Baros; D. Buckley; D. Cherney; R. Chmielewski; T. Crippen; M. Dworkin; R. Gunsalus; J. Handelsman; K. Harper; M. G. Klotz; R. Knight; M. N. Kramer; R. Li; A. Olson; A. Pearson; T. Poole; E. Rose; D. Schaffner; R. Schoenborn; C. Stoltenow; J. Stratton; J. Vornhagen; T. Wood; J. Zehr

Corporate

Biolog Inc

Microbiologics

Membership changes

08

members

It can be irksome when someone fails torecognize your genius, as may happen whenanonymous referees reject a paper you have

submitted. I have known people (not myself, ofcourse) driven almostapoplectic on suchoccasions, chewing (inpre-electronic submissiondays) the corners of thereturned manuscript,cursing the referees fortheir myopia and wishingto see the Editor slowlyrotating over a bed ofred-hot coals as aninterim measure whilethey decide on a moreappropriate fate for them.

In cooler, morerational moments though,we all recognize that the

current system has enormous strengths thatoutweigh these occasional setbacks. If you havefailed to convince the referees of the novelty andworth of your submission then perhaps there is aneed for redrafting to bring out the key pointsmore clearly, or possibly a need for moresupportive evidence. If, despite such efforts, yourpaper still does not find a home, then there isalways the consolation that other great mindshave gone unrecognized in their lifetime — aflower…born to blush unseen.

Publication supported by the system of peerreview remains central to maintaining some formof quality control to smooth the path of scientificprogress. Without it, huge resources would beunnecessarily wasted following up erroneousclaims and trying to repeat the unrepeatable. Theproduction of scientific journals entails anenormous amount of work on the part of manyindividuals and we are all heavily indebted toEditors, members of Editorial Boards andindividual reviewers for this frequentlyunrecognized and unrewarded activity. They give

of their time, usually outside their normal full-time employment, to do work that is oftenunheralded and that does not usually providemuch grist for the career enhancement andpromotion mill.

SfAM gains an enormous amount from itsassociation with a number of excellent journals –our own Journal of Applied Microbiology,which goes back to 1938, and, from 1985,Letters in Applied Microbiology, as well asthose that we publish in partnership with Wiley-Blackwell — Environmental Microbiology,Microbial Biotechnology and EnvironmentalMicrobiology Reports. For many microbiologiststhey are the first choice for publication ofresearch papers and reviews in their respectivefields, they are internationally acclaimed and playa significant role in the high internationalstanding of the Society.

There is also a much more tangible way inwhich the Society and all SfAM members benefitfrom our association with these journals: thesubstantial income they generate. Without thosefunds the Society would be unable to subsidizeour three annual meetings, our regional meetingsand the whole panoply of grants to anything likethe same extent. The Students into Work Grant,the New Lecturer’s Research Grant and theInnovative Project awards, to name but three ofthe many available to members, are enormouslysuccessful in promoting and developing appliedmicrobiology. Demand has increased dramaticallyin recent years but the amounts allocated aregenerous so there is still a good chance ofsuccess. They serve an increasingly importantrole as sources of funding that are simply notavailable from many other grant awarding bodiesor are increasingly scarce in these financiallystraitened times.

To close my first President’s column, I’d liketo thank my predecessor in the Presidency,Professor Geoff Hanlon, for all his excellent workover the last three years and for leaving theSociety in such thriving good health. I thoroughlyenjoyed working with him and we are allextremely grateful for his work and the way hehelped maintain the Society’s reputation for itsfriendly and welcoming character. It has alwaysbeen an aspiration of the Society back to its earlydays as the Society of AgriculturalBacteriologists to engender “…an atmosphereof informality and cordiality…” and I hopeand intend to continue that admirable tradition.Finally, may I wish all our Members the very bestfor the coming season and a very successful andhappy New Year.

president’scolumnSfAM’s new President, Professor MartinAdams talks about peer review, scientificjournals and the benefits of membership

Martin AdamsPresident of the Society

09

For many people this time of year is a timefor celebration and reflection over the last12 months. Indeed, this year the Society has

reason to celebrate once again. It is pleasing toreport that as I am writing this column (lateSeptember) membership numbers are at least100 higher compared to the same time in 2010.A further reason to celebrate is that for the firsttime ever, applications for our two most populargrants (President’s Fund and Students intoWork) were oversubscribed in 2011. If you arethinking of applying for either of these grants in2012 I would strongly recommend you applyearly.

Our scientific meetings were very popular thisyear. In particular, the Summer Conference inDublin was extremely successful with recordnumbers of attendees, including many StudentMembers who had successfully applied for astudentship to attend. It is also pleasing to reportthat a large number of abstracts were received.Next year’s Summer Conference (Edinburgh, 2 to5 July, 2012) promises to be popular, so if youare eligible to apply for a studentship pleaseensure you apply early or indeed if you are a FullOrdinary Member, early registration isrecommended.

The Society can look forward with confidence.As I have stated many times membership offerstremendous value for money. We have introduceda further initiative which makes membershipsubscription even better value. Full OrdinaryMembers who pay a subscription fee should haverecently received a renewal notice and for thefirst time we are offering a facility whereby if amember pays for two years’ membership theywill get a third year’s membership free.Therefore, anybody taking up this offer will notneed to renew their membership until 2015. Itwill also mean that in effect membership costsjust over £34 per year (if paid by credit card) forthose three years. If you would like furtherdetails please contact either Julie Wright(julie@sfam.org.uk) or Julie Buchanan(julieb@sfam.org.uk).

Another brand new initiative for the comingyear is the introduction of a new category of

membership — eStudent Member. This categoryof membership is designed for undergraduateswho have an interest in microbiology. It is freeof charge and applications are welcome fromanywhere in the world. A PowerPoint slide isavailable (julieb@sfam.org.uk) should you wishto promote this category of membership in yourown institution. Full details, including fullbenefits and terms and conditions can be foundby visiting www.sfam.org.uk. Initial response tothis initiative has been very encouraging.

Another new initiative for 2012 is that for thefirst time the next Spring Meeting (18 April2012) will be a joint meeting with the Institute ofBiomedical Science (IBMS). The meeting formspart of the centenary celebrations of the IBMS.In the morning the meeting will cover“Historical Perspectives of Microbiology” andthen in the afternoon there will be concurrentsessions covering “Sepsis and Implants” and“Virology — typing and its applications”. Afull programme will be available online in 2012.

As I’ve referred you tothe website a couple oftimes in this column, itseems appropriate tomention the new SfAMwebsite which went liveon 7 October 2011. Ihope you have enjoyedusing the new site and ifyou’ve not yet visited Iwould encourage you togo to www.sfam.org.ukand have a browse. And do get in touch with usat communications@sfam.org.uk with any ideasor suggestions you have for the site. To readmore about the new website, see page 10.

All that remains is for me to wish you all ahappy festive period and a prosperous New Year.

Philip WheatChief Executive Officer

ceo’s columnPhilip Wheat reports on the latest

developments within the Society

Statistical Analysis in Microbiology: StatNotesBy Richard A Armstrong and Anthony C Hilton. Published by Wiley-Blackwell/SfAM, 2010

StatNotes has been designed specifically for microbiolo gists who are involvedin experimental research and need to draw accurate conclusions from theirfindings. It features 28 StatNotes that together enable you to understand thebasic principles of statistics, choose the correct statistical methods to analyseyour experi mental data, and work with a variety of commercially availablestatistical software packages. Written specifically for microbiologists,StatNotes enable you to choose which statistical methods should be appliedto analyse and draw correct conclusions from your experimental data.

members

As you will know by now, the new SfAMwebsite went live on 7 October 2011! You willhave received an email reminding you of your

username andpassword which you’llneed to access theMember’s area of thesite. Do feel free tobrowse the site and letus know what youthink. We hope youlike the new site whichhas so much more to

offer both Member and Non-Member visitors. Inresponse to the Member’s Questionnaire wedistributed in 2009, and taking on board whatMembers and Non-Member stakeholders havetold us via a specific questionnaire and

SfAM Honorary General Secretary MarkFielder has beenconferred with thetitle Professor ofMedicalMicrobiology byKingston University.Congratulations toMark on this verywell deservedachievement.

membershipmatters

Congratulations

10

S. Peter Borriello has been appointed as ChiefExecutive of theVeterinary MedicinesDirectorate. All atSfAM would like tocongratulate him onhis newappointment.

interviews about the website, we’ve added newfunctionality to the site and put in place a newnavigation system which means you’ll find iteasier to find what you’re looking for. There arenews listings, events booking and links to oursocial media pages on Facebook and LinkedIn.You can also keep up with the latest microbiologynews on our Twitter feed, which appears on thehome page.

Members will find a new improved ‘Member’sarea’ which includes new online grant applicationand a new forum for our Special Interest Groups.

But it doesn’t stop there. We will continue todevelop the site with the latest information andusing the latest technological developments. Socontact us (communications@sfam.org.uk) withyour ideas and tell us what else you’d like to seeon the SfAM website. Happy browsing!

New SfAM website

11

Dr Mark Downs, PhD, FSBChief Executive, Society of Biology

bioFocusMark Downs reports: legislation from the European Union

The Society of Biology is a single unified voice for biology:

■ advising Government and influencing policy.

■ advancing education and professional development.

■ supporting our members.

■ engaging and encouraging public interest in the life sciences.

For further information visit:

www.societyofbiology.org

Like it or not, all biologists need to be aware of legislation.It creates the framework for the operation oforganizations, places responsibility individually or

corporately and creates our ethical framework. Regulation issomething we all want less of, but can’t do without. Despite allthe rhetoric from successive Governments, legislation is agrowth area. Everyone knows it is important and can have adramatic impact on the way we work and run our lives, yet nosingle person has the capacity to follow it all and “do the dayjob”. This is where professional bodies come in — identifyingpriority areas, summarizing the key issues, consulting expertsand representing members’ interests wherever possible. At theSociety of Biology we are trying to do this for both ourindividual and organizational members focussing on the cross-cutting and generic issues such as science funding, education,training, skills and ethical frameworks.

Dependent upon how you define new legislation, 70 to 90%now originates from the European Union. The most commontype of legislation is a Directive. These are proposals putforward by the EU’s civil servants (the Commission) and thendebated and amended by the Member States (the Council) andthe European Parliament. The Member State negotiations takeplace behind closed doors and (for the UK) are led by homedepartment officials and their colleagues from the “Embassy”to the EU (UKREP). In parallel through one or morecommittees, and then typically two readings in Parliament,MEPs shape their own text through public debate. At thispoint there are two sets of different text for the same purpose!After typically months of negotiation, publicly and privately,the end is then almost in sight, well sort of: agreementbetween Member States and the Parliament is often still adistant dream. To resolve disputes a bizarre process known asconciliation is invoked whereby the EU Parliament andMember States delegations (led by the rotating Presidency)argue it out privately on a time limited basis until a consensusis reached, often through the night. If there is none, thelegislation fails to become a Directive. This might often be the

best outcome but given all the work everyone has put in thereis a danger that it will seem more attractive to have badregulation than none at all.

All clear? Probably not! It is far from a transparent process.Once a Directive is published Member States usually have 24months to implement (“transpose”) it into their domestic law.Directives set minimum requirements seeking to harmonizelaw across the EU driving down costs for business andincreasing common standards for EU citizens.

My experience from working within the system is that it ispretty much basic horse trading. Forget evidence basedpolicymaking — importantthough it often is. This ispolitical in every sense.There are red-lines betweenministers, betweendepartments, differingMember State views,domestic and EU lobbygroups and the need to getagreement from an almostnon accountable EuropeanParliament.

OK, so I’m being harsh.But how many reading this article can name their MEP letalone comment on what stance they have taken on keypolitical issues? Do they support good science or understandthe breadth, impact and value of biology? The truth is they arelargely anonymous and when that happens accountability isless obvious. The EU focus for science is often on the hugeFramework research programmes. But, the wider regulationagenda must never be ignored.

Influencing the outcome of the EU decision-making processis not straightforward. But it can be done. Large, broadspectrum groups with a clear, well-argued and balancedmessage are difficult to ignore domestically or at an EU level.But, there needs to be recognition of the differing issues andinterests across the EU, and timing of lobbying has to beright. For example, months of hard fought changes to drafttext can be lost or changed in an instant by last minutelobbying of the EU equivalent of a party whip.

In my view, the overall legislative burden is not set tochange, and this Government’s and Parliament’s appetite forconsultation on both the legislative and policy agenda seem atan all-time high both for Westminster and the devolvedadministrations. Since the formation of the new coalitionGovernment the Society has dealt with dozens of consultationshaving also considered responding to many more!

We strive to represent the views of biologists and yourexpert knowledge, and your opinions are vital ingredients inthis. Please remember to have your say and get involved withthe policy agenda through SfAM colleagues or directly. Forweekly updates on general science policy issues subscribe toour free Science Policy Newsletter (email:policy@societyofbiology.org) and visit the website’s policypages to see some of our work.

news

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Forging networksfor collaboration

BritishScienceFestival

Lucy HarperCommunications Manager

news

gathered in elegant surroundings of the GreatVictoria Hotel in Bradford. We all heard a fewintroductory words from Society of Biology ChiefExecutive Mark Downs, followed by the SBsMedia and Events Executive Jenna Stevens-Smithwho described how the project formed, how itdeveloped and her vision for the future. Next weheard from Jen Middleton who welcomed allStempra members — especially those based inthe North of England and had the nice job ofasking people to eat canapés and drink wine. I’msure a lot of successful networking went on thatnight — the best way of launching a report whichfosters the formation of networks andcollaboration.

To download the report and listen to a podcastof my interview with Jenna Stevens-Smith aboutthe report, visithttp://www.sfam.org.uk/en/news/index.cfm/sbscicommreport/ and click on the link.

The Society of Biology and Stempra held anevent at the British Science Festival thisyear. The event was to launch a report

written by Jenna Stevens-Smith of the Society ofBiology (SB) which represents the culmination ofover six months work in fostering collaborationbetween members of staff at the MemberOrganizations (MOs) of the SB.

The Science Communication Project involvedrelevant staff members of MOs, as well asexternal representatives from advisory bodiessuch as: the Department for Business Innovationand Skills (BIS), Nature, and the Science MediaCentre. Each meeting focussed on a differentarea of science communication, and membersshared ideas and best practice as well as usefulhints and resources in:

■ Interacting with the media.■ Communicating science policy.■ Public engagement.■ The Internet as a means of communication.

On Tuesday 13 September the great and thegood in the world of Science Communication

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publications

involvement of the electron acceptor in substrateactivation. J. Zedelius, R. Rabus, O. Grundmann, I.Werner, D. Brodkorb, F. Schreiber, P. Ehrenreich, A.Behrends, H. Wilkes, M. Kube, R. Reinhardt and F.Widdel, Vol. 3, No. 1.

Powering microbes with electricity: direct electrontransfer from electrodes to microbes. D. Lovley,Vol. 3, No. 1.

Major accoladesfor MicrobialBiotechnology

Journal now indexedin PubMed, Scopusand ThomsonReuters-ISI, andawarded an SJRIndicator!

Microbial Biotechnology, published jointly byWiley-Blackwell and SfAM, has been accepted forinclusion in widely read databases PubMed,Scopus and Thomson Reuters (ISI). It has alsoreceived a SCImago Journal Rank (SJR) Indicator of0.234 in recognition of the excellent quality of itspapers.

Microbial Biotechnology is a sister of the premiermicrobiology journals Environmental Microbiology,Molecular Microbiology and Cellular Microbiology.It publishes papers of original research reportingsignificant advances in any aspect of microbialapplications.

“More than 90% of microbial diversity still remainsto be discovered. It is this new biodiversity that willbecome the treasure chest of new and improvedbiotechnological developments and applications inthe sectors of chemicals, pharmaceuticals, energy,mining, materials, agriculture, food, andenvironmental protection,” said Editor-in-ChiefProfessor Ken Timmis. “This journal harnessesoriginal research reporting advances belonging tothe upper 25% in the field in any aspect ofmicrobial applications.”

The journal invites potential contributors to bepart of the growing success of MicrobialBiotechnology, submit their article online and takeadvantage of the author benefits offered. Theseinclude:

■ Broad, inclusive scope representing all current and emerging topics in applied microbiology.

■ Regular Special Issues specifically targeting topical themes.

■ Editorial team who are leaders in the variousfields of microbial biotechnology.

The following articles were the mostdownloaded articles from the Society forApplied Microbiology’s journals betweenJanuary and September 2011.

Journal of Applied Microbiology

Antimicrobial agents from plants: antibacterialactivity of plant volatile oils. H. J. D. Dorman andS. G. Deans, Vol. 88, No. 2.

Antimicrobial activity of essential oils and otherplant extracts. K. A. Hammer, C. F. Carson and T.V. Riley, Vol. 86, No. 6.

A study of theminimum inhibitoryconcentration andmode of action oforegano essential oil,thymol and carvacrol.R. J. W. Lambert, P. N.

Skandamis, P. J. Coote and G.-J.E. Nychas, Vol. 91,No. 3.

Letters in Applied Microbiology

Extraction methods and bioautography forevaluation of medicinal plant antimicrobial activity.A. Nostro, M.P. Germanò, V. D’Angelo, A. Marinoand M.A. Cannatelli, Vol. 30, No. 5.

Antibacterial activity of selected plant essential oilsagainst Escherichia coli O157:H7. S.A. Burt andR.D. Reinders, Vol. 36, No. 3.

Antibacterial activity of plant extracts onphytopathogenic Xanthomonas campestrispathovars. S. Satish, K. A. Raveesha and G. R.Janardhana, Vol. 28, No. 2.

Environmental Microbiology

Referees’ quotes — 2010. Vol. 12, No. 12.

Fresh fruit and vegetables as vehicles for thetransmission of human pathogens. C. N. Berger, S.V. Sodha, R. K. Shaw, P. M. Griffin, D. Pink, P.Hand and G. Frankel, Vol. 12, No. 9.

Global patterns in the biogeography of bacterialtaxa. D. R. Nemergut, E. K. Costello M. Hamady,C. Lozupone, L. Jiang, S. K. Schmidt, N. Fierer, A.R. Townsend, C. C. Cleveland, L. Stanish and R.Knight, Vol. 13, No. 1.

Environmental Microbiology Reports

Environmental reservoirs of Vibrio cholerae andtheir role in cholera. L. Vezzulli, C. Pruzzo, A. Huqand R. R. Colwell, Vol. 2, No. 1.

Alkane degradation under anoxic conditions by anitrate-reducing bacterium with possible

journalWatchNews about the Society’s journals

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publications

Felicity HowlettWiley-Blackwell

■ Online submission and review process, leading to faster decision times.

■ Early View — article by article publishing.

■ Exceptional exposure to researchers and institutions worldwide.

■ Indexing in Thomson Reuters (ISI), PubMed and Scopus.

Recently published Special Issuesinclude:

Extremophiles, Lactic Acid Bacteria, Streptomyces,Marine omics, Biocatalysis, Microbiology of energybiotechnology, Life of microbes that interact withplants, and Bioremediation.

Forthcoming Special Issues willcover:

Microbial vaccines and immunomodulators,Microbial resource management, and Biodefence.

The most cited papers in the journalto date are:

Metabolic engineering to enhance bacterialhydrogen production. T. Maeda, V. Sanchez-Torresand T. K. Wood, Vol. 1, No. 1.

Identification of furfural as a key toxin inlignocellulosic hydrolysates and evolution of atolerant yeast strain. D. Heer and U. Sauer, Vol. 1, No. 6.

T-DNA insertion, plasmid rescue and integrationanalysis in the model mycorrhizal fungus Laccariabicolour. M. Kemppainen, S. Duplessis and A. G.Pardo, Vol. 1, No. 3.

The most read papers in 2010 were:

Bioactive compounds from marine bacteria andfungi. A. Debbab, A. H. Aly, W.H. Lin and P.Proksch, Vol. 3, No. 5.

Prokaryotic whole-transcriptome analysis: deepsequencing and tiling arrays. R. J. Siezen, G.Wilson and T. Todt, Vol. 3, No. 2.

Microbial fuel cells. L. P. Wackett, Vol. 3, No. 2.

Microbial Biotechnology is available to institutionsand individuals via a paid subscription fee. Torecommend the journal to your librarian today,please visit http://bit.ly/recommendmbt andcomplete the short online form.

For further information on the journal, please visitwww.microbialbiotech.com.

MicrobiologyOpen: a special Societyfor Applied Microbiology Memberoffer

“I am pleased and honoured to introduceMicrobiologyOpen, anew journal fromWiley-Blackwell.”MicrobiologyOpen is apeer-reviewed journaldelivering rapiddecisions and fastpublication of microbialscience, a field which isundergoing a profoundand exciting evolutionin this post-genomicera. The journal’s

Editor-in-Chief, Pierre Cornelis, explains theconcept:

“Why then a new journal? The term ‘open’ hastwo meanings: first, MicrobiologyOpen is an openaccess journal, but, more importantly, it alsomeans that it is open to all aspects ofmicrobiology.”

The journal gives priority to reports of qualityresearch, pure or applied, that further ourunderstanding of microbial interactions andmicrobial processes.

“MicrobiologyOpen launches specifically to servethe broad microbiology community…[it] is aresponse to the growth of research beingundertaken and the data now available. It is clearthat the community will benefit from the increasedflexibility provided by the online only, open accessformat.”

All articles published by MicrobiologyOpen arefully open access: immediately freely available toread, download and share. To cover the cost ofpublishing MicrobiologyOpen charges apublication fee. SfAM Members benefit from a10% discount on the article publication charge forMicrobiologyOpen, when they submit a paperdirectly to the journal. The usual price forpublishing in the new open access journal is$2,175 / £1,400 / €1,650. As a SfAM member,you pay just $1,957.50 / £1,260 / €1,485. Totake advantage of this special offer, simply enterthe Society Member Account Code, which can beobtained from the Society office by contactingeither julie@sfam.org.uk orjulieb@sfam.org.uk, when presented withpayment options when submitting to the journalwww.microbiologyopen.com.

Ellie KeyWiley-Blackwell

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Wednesday 18 April 2012

The Stratford Q Hotel, Stratford-upon-Avon, UK

2 - 5 July 2012

The George Hotel, Edinburgh, UK

For further information on these events please visit sfam.org.uk or contact Sally Cryer■ Email: sally@sfam.org.uk ■ Telephone +44 (0)1933 382191

Wednesday 11 January 2012

The Royal Society, London, UK

■ Including the Denver Russell Memorial Lecture

● Microbiological safety of imported food

● Microorganisms and climate change

Winter Meeting

Spring Meeting6th broadening microbiology horizonsin biomedical science meeting

■ Including the Procter and Gamble Applied Healthcare Microbiology Award Lecture

Summer Conference● Microbial resistance to antibiotics andbiocides ● Natural and experimentaladaptation in bacteria ● Bioremediation

■ Including the Lewis B Perry Memorial Lecture: Globalization of antimicrobial resistance. Didier Pittet, University Hospital in Geneva

SfAM events in 2012 — save the dates!

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Summer Conference 2011 reportFood MicrobiologyMonday 4 - Thursday 7 July 2011 Clontarf Castle, Dublin, Ireland

Message from the Honorary Meetings Secretary

Firstly, I would like to thank all of you who attended thisyear’s Summer Conference at the Clontarf Castle Hotel,Dublin and I hope you enjoyed it as much as I did. This

Summer Conference was one of the most successful we haveorganized in many years. Although there is a comprehensivereport describing the meeting in this issue of Microbiologist Idid want to make a few more general comments.

This summer we welcomed over 230 delegates in totalrepresenting 33 countries spanning all five continents whichreflects how “international” the Society has become. Wealso had to close our registration to delegates several weeksbefore the meeting due to us reaching the capacity of thelecture rooms. This is an action I can’t recall ever happeningat a SfAM Summer Conference before. We also awarded 29studentships to enable our Student Members to attend andpresent their research work and we would encouragestudents to apply for studentships again in the future. Thenumber of abstracts submitted for poster presentation waswell over 100 plus; four student oral presentations were

selected to compete for the “Best Student OralPresentation”.

A lot of work goes into organizing the SummerConference and I would like to thank all of the SfAM staffand Committee for all of the hard work they put in to makeit such a success.

In summer 2012 the Summer Conference moves to TheGeorge Hotel in the centre of the beautiful city of Edinburghwhere we will have another excellent program addressingMicrobial resistance, Natural and experimental adaptation inbacteria and Bioremediation.

We hope to see you all there and remember to book earlyto avoid disappointment!

Andrew SailsSfAM Honorary Meetings Secretary

Clontarf Castle Hotel in Dublin provided a very grandsetting for four fascinating days of lectures on ‘Foodmicrobiology’. On the Monday a number of delegates

attended the ‘risk assessment workshop’ with Deon Mahoney,FAO, Bangladesh and John Bassett, Unilever, UK. This gavedelegates an insight into risk assessment in the food industryand the level of detail required. After lunch a teamwork

exercise got everyone thinking about the detail andappropriate information needed in a risk assessment. Whatseemed like a ‘good’ risk assessment to begin with, by the endof the afternoon, and after much critical discussion, turnedout to be in fact a really ‘bad’ risk assessment. The key pointsfrom this exercise were that risk assessments in the foodindustry are complicated and have to be conducted

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Chris Low from the Scottish Agricultural College startedthe session on ‘Pathogen updates’ with an informative updateon verotoxigenic E. coli. Verocytotoxigenic E. coli (VTEC)were first recognized in 1977 becoming recognized assignificant pathogens during the 1980s. Most often associatedwith serotype O157:H7 (although not exclusively), thisorganism was elevated up the political agenda followingseveral significant outbreaks in Scotland and England.Understanding the pathogenesis of these strains is pivotal tosuccessful implementation of control measures. Infection isusually by direct or indirect contact with ruminants, thoughwaterborne and foodborne sources are also well documented.Despite this, most infection is sporadic. In cattle infection iswithout clinical consequence, with the organism typicallycolonizing the distal rectum being shed for up to three weeks.The H antigen (H7) is important for attachment and somesuccess has been seen in reducing colonization among orallydosed cattle, but is not licensed for UK use. The toxin isencoded by bacteriophage with VT2 being correlated withgreatest disease severity. Among cattle it is believed thatcarriage of the bacteriophage endows a selective advantage,but in man this correlates with pathogenic potential. Morerecently another VT positive novel strain has emerged inGermany associated with 810 cases and 27 deaths. This is a

different serogroup, 104, and shows greatest homology withan enteroaggregative E. coli isolated from Africa. Beansprouts have been incriminated as the likely source ofinfection in Germany. The timing of this outbreak with thispresentation ensured that this talk was truly a “hot topic”without a spare seat left in the lecture theatre.

This was followed by Simon Park, University of Surrey,talking on Campylobacter. Being the leading cause offoodborne infection their reduction is of paramountimportance. Despite their ability to result in such an enormityof human infection, the organism remains restively “fragile”with little in the way of resistance, or ability to surviveextreme environmental stresses. Of particular interest aretheir strict demands for a microaerophilic atmosphere. Thiscould offer a means of controlling Campylobacter. Similarly,5% ethanol is inhibitory for Campylobacter. Potentially thiscould be combined with oxygenation as a means of reducingCampylobacter. What the clinical consequences are of usingan oxygenated diet remains to be explored?

Marion Koopmans from RIVM, in the Netherlands went onto describe many foodborne viruses, including the headlinestealing, norovirus, followed by hepatitis A and hepatitis Eviruses. Despite the highest levels of infection, norovirus hasthe least clinical impact, with much higher mortality beingassociated with the hepatitis A virus. In order to recognizeoutbreaks of infection and gain insights into theirepidemiology, a global network, “noronet” has beenestablished. Through such a resource, levels of foodborneversus person-to-person infection can be monitored togetherwith new or emerging genotypes.

When looking at levels of virus in food-handlingestablishments, it was no surprise that during an outbreak ofinfection; the toilet was highly contaminated, with 61% ofsamples being positive. This dropped to 3.1% in the absenceof outbreaks, with the kitchen only yielding 1.6% of positivesamples.

Correlations have been made between genotypes andvirulence, with new waves of infection emerging through

throughout the food chain (from farm to table).In the evening all delegates had the chance to tour the

Guinness Storehouse and see what makes the perfect pint ofGuinness before the Lewis B Perry Memorial Lecture. AlanReilly (Food Safety Authority of Ireland) gave a fascinatingtalk about ‘food safety in a global environment’ and howcommunication is key. He also praised social media, such asTwitter, emphasizing how it influences the communicationbetween scientists, journalists and the general public.

After the lecture a Guinness style buffet was prepared fordelegates, along with a complimentary pint of Guinness.

Samantha Price PECS Events Officer

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antigenic variants every couple of years. Epidemiologicalanalysis can be hampered by the ability of these viruses toremain infective after frozen storage of foods.

Hepatitis A virus, in contrast to norovirus above, showssignificantly more geographical and food-related clustering.Current oyster die-offs occurring near France are beingassociated with an influx of oysters from other producers suchas China, Japan and Korea. Whether this will result in changedpatterns of infection is under scrutiny.

Taking us back to bacterial pathogens, John Threlfall wenton to describe how Salmonella could be considered as multi-flexible pathogens. We were then enlightened by the diversityand often unpredictable changes being witnessed inSalmonella spp. causing human infections. Not only have weseen the acquisition of increasing levels of chromosomalencoded resistance, typically associated with chromosomalislands, but also plasmid encoded resistance traits. Outbreaksof infection have occurred with different phage typesassociated with diverse food vehicles ranging from porksausages, eggs (poultry and duck), bean sprouts, to reptilefood such as “pinkies” (suckling mice mass-produced forreptile consumption). Furthermore, an upsurge in infectionswith monophasic variants of Salm. enterica withchromosomally-encoded resistance to ampicillin,streptomycin, sulphonamides and tetracyclines (ASSuT) havebeen seen in Europe.

Furthermore, changing patterns of food-production mightgive rise to changing opportunities for infection, such asorganic versus conventional farming. Reduction ofSalmonella requires a multifactorial approach includingprudent use of antimicrobials; stringent biosecurity; and goodfarm hygiene levels.

Initially known as “sausage poisoning”, Mike Peck went onto describe the history of the neuroparalytic intoxicationknown as botulism from foodborne; infant botulism; to woundbotulism. Although production of potent botulinum toxinunifies Clostridium botulinum species, the microorganismsproducing this toxin are microbiologically heterogeneousbelonging to four distinct groups. Of these, Cl. botulinumGroup I producing a proteolytic toxin and the non-proteolyticCl. botulinum toxin Group II toxins are responsible for mostepisodes of botulism. More recently, other species names havebeen attributed to botulinum toxin producing strains.

The toxin itself is astoundingly toxic with just 3g beingenough to kill all humans in the UK, and 400g enough to killmankind worldwide! Despite this, therapeutic applications ofthe toxin are now becoming commonplace. Though clinicalcases of botulism are rarely encountered, the implicationsboth for the individual and the economy are huge. Survivorsof infection may require years to regain mobility. For food-producers, recovery is often impossible, as seen with thedemise of hazelnut yoghurt producers in the UK. The“botulinum cook” of 121°C for 3 minutes or for minimallyheated chilled foods, around 90°C for 10 minutes are essentialto prevent this notorious toxaemia.

Sally Cutler SfAM Main Committee

The afternoon’s session on ‘Pathogen updates’ commencedwith Seamus Fanning from University College Dublin giving anoverview of Cronobacter spp. Ireland produces 15% of theglobal supply of infant nutritionals including powdered

formula and due to the nature of Cronobacter spp. being ableto withstand dry environments this organism is a healthconcern within these products, with 42% of cases ofmeningitis caused by Cronobacter spp. being fatal. The FDAmethod for identification was updated in 2009 with theoriginal protocol being linked to PCR for more accurateidentification. The control of Cronobacter spp. in themanufacturing environment is vital and thus quality controlprocesses have been put in place including PFGE, to identifypersistent strains and mapping transmission to define theecology of the organism in the production environment andthus be able to produce an evaluated risk model. The secondtalk was given by Niall Logan from Glasgow Caledonian onBacillus spp. and its relatives. Niall began by giving us thehistory of an arctic venture in the 19th century which led toBacillus subtilis being isolated in the arctic in 1936. Bacillusspp. cause opportunistic infections in wounds, abscesses, theocular system to name a few; however it is also prominent infood, with B. cereus, B. licheniformis, B. subtilis and B.pumilus often being isolated. Bacillus spp. can cause diseasein two forms; diarrhoeal where the vegetative organismsporulates and produce enterotoxins and emetic illness wheresymptoms are induced by heat stable toxins. Food relatedoutbreaks are often associated with milk, ice cream,sandwiches and reheated rice from Chinese restaurants. Thebiggest worry though is that Bacillus spp. are often used inprobiotics!

The ‘Epidemiology of foodborne disease’ session waskicked off by Hilde Kruse from the WHO, Italy, discussing thecurrent challenges to microbial food safety, highlighting theincreasing cases of emerging diseases in the last 60 to 70years with 335 new infectious diseases being identified and 95of these being associated with food, this is thought to be dueto changes in agriculture and food processes and also climatechange. To combat this, a new food safety initiative was put inplace in 2007 called the Foodborne Disease BurdenEpidemiology Reference Group (FERG). To date some of theachievements of this task force include, assessing the true

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estimate of mortality of children in Africa and South East Asiafrom foodborne diseases and determining the actual numberof the population that have peanut allergies (1 to 2%).

Katie Laird SfAM Main Committee

Following a tea break and an opportunity to visit the tradeshow, Marion Wooldridge from the AHVLA talked about‘Climate change and the challenge of new pathogens’. Mariongave evidence of how climate change has impacted vectorsresulting in changes in pathogen prevalence, incursion andpersistence. Marion also highlighted that such pathogens areemerging in new areas as a result of changes in host rangesand susceptibility as well as through mutations. One exampleof the impact of climate change on vectorborne viruses is themutation of a vial envelope in Aedes aegypti (yellow fevermosquito) to the Aedes albopictus responsible for the CHIKVoutbreak.

This talk was followed by Alec Kyriakydes, the Head ofQuality, Safety & Supplier Performance at Sainsbury’s whospoke about the ‘Retailer’s perspective on food safety’. In histalk he discussed the changing trends in food choices andfactors affecting shift in infection and disease, this includedagricultural and industrial factors as well as the impact ofsourcing and supply. A decline in research base, funding andfood scientists, coupled with the emergence of new hazardswere discussed as was the importance and influence of socialmedia in food microbiology.

Last thing on Tuesday was the student session, a popularand key highlight for Student Members at the SfAM SummerConference. The focus of these sessions was careerdevelopment for PhD students and early career scientists. Thisyear’s theme was titled ‘Enhance your employability’. Duringthis session SfAM Communications Officer, Clare Doggett,talked about interview skills and transferable skills which willincrease employment potential after which SfAMCommunications Manager and Editor of Microbiologist, LucyHarper, talked about communication skills and the importance

of communicating clearly and correctly as “poorcommunication causes problems”. This talk was followed byProfessor Mark Fielder who, as always, provided anentertaining but informative talk on presentation skills.Finally, Sorcha Mulcahy, Career Development Adviser atUniversity College Dublin, spoke about getting the most fromyour CV.

The student session served as a reminder to theparticipating PECS members on important but occasionallyneglected aspects of ‘student development’ however, asexpected it was a light-hearted affair with great feedback.

Emmanuel Adukwu PECS Events Officer

The first talk on Wednesday morning concluded Session 2.Hilde Kruse, WHO, Italy, spoke about ‘The threat of antibioticresistance in the food chain for human health’. Antibioticresistance doesn’t respect phylogenetic, geographical orecological borders and any use of them can select forresistance. Two types of hazards are recognized: directhazards from ingestion or handling of food containingantibiotic resistant bacteria and indirect hazards associatedwith transfer of antibiotic resistance genes.

After giving examples of specific organisms with well-documented resistance to antibiotics, Hilde described theWHO European strategy for tackling antibiotic resistance froma food safety perspective. She cited seven action areas andseven key messages for countries. A copy of a WHOpublication on this topic was included in the delegate packs. Itwas stressed that an intersectoral, multi-faceted response isrequired at the national and international level.

Microbiological risk assessment was the theme for Session3 which was chaired by Louise Fielding. The session wasopened by Sarah Cahill, Food and Agriculture Organization ofthe United Nations, Italy, discussing ‘Recent global riskassessments and impact on Codex standard setting’. Shebegan by providing some background information aboutbodies such as Codex Alimentarius, the World TradeOrganization and the Joint FAO/WHO Expert Meetings onMicrobiological Risk Assessment (JEMRA).

The use of risk assessments and scientific advice in thesetting and adoption of standards has improved over the lastfive years. Sarah used the example of Cronobacter spp. inpowdered infant formula to show the improvements from thetime of alerts in 2003 through to adoption of microbiologicalcriteria for follow-up formula by Codex in 2009.

A website www.mramodels.org which allows real-timemodelling has been successful in making people less afraid ofrisk assessments. Sarah described a risk management toolwhich has been developed for control of Campylobacter andSalmonella spp. in chicken meat. It is undergoing pilottesting currently and should be available by the end of theyear as a support tool for risk managers. This new approach isless prescriptive and allows more flexibility than previousapproaches and so encourages risk assessment at the nationallevel.

The final presentation of the morning was given by MaartenNauta, DTU, Denmark, on ‘Recent developments inCampylobacter risk assessment’. Such risk assessmentsprovide estimates of absolute risk, relative risk and addedvalue and are needed for risk-based food safety target setting.MedVetNet Workpackage 24, which entailed a comparison of

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Campylobacter quantitative microbiological risk assessments(QMRAs) from four EU countries, was summarized. Despitemany differences between the models used, similarconclusions were drawn. For example, high concentrations ofbacteria pose the largest risk and logistic slaughter has littleeffect.

After commenting on the recently issued EFSA ScientificOpinion on Campylobacter control, Maarten devoted the restof his talk to the use of risk assessment in definingmicrobiological criteria as an instrument to control foodsafety. He remarked on the difficulty of modelling theconsumer phase and presented the results of a comparison ofeight such models for Campylobacter. Finally, furthersimplification of QMRA modelling was described as anongoing challenge.

Louise Hill-King Grants Editor

The afternoon session was opened by Pirkko Tuominen(Food Safety Authority, Finland) who discussed ‘SalmonellaRisk Assessment in Finland’. Pirkko spoke about the history ofSalmonella control in Finland and how the risk has beenassessed for over 10 years. She explained how the nationalSalmonella control programme (SCP) is in place to ensurethat Salmonella prevalence shall not exceed 1% at any stageof beef, pork, broiler, turkey or egg production. If, duringsampling, Salmonella is detected within the SCP, follow-upaction must be taken. Pirkko also explained how productionchain quantitative microbiological risk assessments (QMRAs)have also been exploited e.g. cost-benefit analysis, samplingprocedure comparison and planning of food safetymanagement metrics. She explained how the QMRAs had beenconverted from independent model chains into one combinedBayesian network. The aim of the Bayesian approach is toallow risk managers to make revisions on an annual basis,follow the Salmonella situation, respond quickly to changesand guide food operators and inspectors efficiently. Pirkkoconcluded that the SCP had been estimated cost effective andalthough Salmonella cases in Finland had remained the same,human cases acquired from Finland as well as from foodsunder SCP had decreased over the last 10 years.

Phil Voysey (Campden BRI, UK) continued on the topic ofmicrobial risk assessment by talking about ‘Listeria risk inbutter’. Phil described how butter is not normally regarded ashazardous; however there have been a number of issuesrelating to listeriosis which has promoted their research intowhy L. monocytogenes should pose a risk in butter. Initially,the work concentrated on the requirements for growth of L.monocytogenes, the parameters of butter and butter-containing spreads (e.g. salt levels, temperature, water dropletsize, pH) and how these conditions allowed the growth of L.monocytogenes. The second phase of this project focused onthe characteristics of the butters and strains of L.monocytogenes that were associated with listeriosis outbreaksand the methods of manufacture, formulation and handling ofbutters. Phil went on to tell us the final phase of the practicalwork which involved testing of hypotheses gathered from thefirst two phases of the project. Once the methods ofinoculating butter with L. monocytogenes had beeninvestigated the effect of water droplet size, salt concentrationand tempering and temperature on the behaviour of L.monocytogenes was assessed. It was concluded that the

crucial factors were salt levels and water droplet size. Philhighlighted that these factors should be taken intoconsideration by butter manufacturers and how goodmanufacturing technique will prevent problems from thegrowth of L. monocytogenes.

Samantha Law SfAM Main Committee

After a tea and coffee break we all settled back to watchthe student presentations.

First up was Phillip Humphryes. Phillip gave anintroduction to the world of Leptospira vaccine proteomes,with a thorough discussion of the infection process andsparing none of the gruesome details of vaccine testing onhamsters. Phillip expressed his desire to find an alternative tohamster testing, which was shared by the audience. Next heexplained the various investigations he had undertaken inanalysing a variety of currently available vaccines. Theseincluded assays of the components, comparing the proteincontent and composition and separating various fractions tobetter understand commercial products.

Following on from Phillip, Samantha Price gave apresentation focused on an investigation into themycobactericidal and bactericidal properties of a novelcatalyst, which enhances the effects of hydrogen peroxide.She discussed the uses and mechanisms of hydrogen peroxideand the properties of the catalyst. There were a plethora ofgraphs, which showed the catalyst’s activity against aerobicBacillus spp., Mycobacterium spp. and Staphylococcusaureus. Interestingly, Samantha has found the leachate fromthe catalyst also has a residual effect on the organismsexposed to it. Further work will include re-testing in thepresence of soiling. Samantha concludes that this catalystcould certainly be useful in industries such as healthcare,where infection control is a constant battle.

Next Patience C. Obinna-Echem presented work whichinvestigated the bacterial and yeast communities which maybe responsible for fermentation of starchy-meal and which can

21

make it unsafe. There was a thorough background discussingthe importance of the fermented starchy-meal which is astaple food in Africa. Current production does not involve astarter culture and so the fermentation process is spontaneousand relatively inefficient. The main part of this presentationfocused on culture techniques and identification of specieswhich may be responsible for fermentation and spoilage. Anumber of lactic acid bacteria were commonly found.

Finally Steffi Bough gave a presentation on her workinvestigating the association between multi-drug efflux pumpsand Salmonella biofilms. She gave a comprehensivebackground of the various themes of the work includingSalmonella biofilm formation, the impacts of biofilms andefflux pump physiology. There were stunning visuals in theform of scanning electron microscope pictures of biofilms.She went on to discuss the contribution of differentcomponents of the pumps to biofilm production. Finally thetalk covered the relationships between expression ofregulators for biofilm production and the structure andlongevity of biofilms.

Next we had two lectures from recipients of SfAM awardsand grants.

Firstly Clare Taylor of Edinburgh Napier University, UK, arecipient of the SfAM New Lecturer Research Grant gave apresentation on ‘Microbes and metal: Metal transport duringbacterial infection’. Clare discussed her progress investigatingthe role of different gene regions thought to be involved in thetransport of cobalt, iron and other metals. Her work hasshown some interesting results, highlighting potential targetsfor novel anti-virulence therapies.

Finally the W H Pierce Prize Lecture was given this year byBrian Jones, University of Brighton, and was on exploring thehuman gut mobile metagenome.

Brian’s talk focused on investigations into the mobilegenetic elements (MGE) and bacteriophages found within thegut microbiota. This has been called the mobile metagenomeand can be likened to a virtual organ. The exciting possibilitiesfor scientific and pharmaceutical value are as yet unexplored.

The Conference continued well on into the evening with theAGM followed by the Summer Conference dinner, which washeld at Jameson’s Distillery. Delegates got a tour of the olddistillery followed by a delicious dinner and some Irishentertainment including Irish dancing and a live band.

Joanna Tarrant PECS Events Officer

The Thursday morning session, ‘Novel technologies tocontrol safety and stability’, began with an overview of Noveltechnologies from Bala Balasubramaniam (Ohio StateUniversity, USA). Bala began by outlining how alternative foodpreservation techniques, that can preserve heat sensitivefunctional components of food whilst still extending shelf lifeand being microbiologically safe, are becoming moreimportant as consumer interest in healthy eating increases. Hethen went on to detail how these novel techniques could begrouped into two categories, advanced thermal and non-thermal food processing. Advanced thermal techniquesmentioned included ohmic, microwave, and radio frequencyheating which have the benefit of being very fast but thedisadvantage that slow cooling of the food can affect itsquality. Non-thermal approaches discussed included highpressure, pulsed electric fields, irradiation and ultrasound, ingeneral these approaches preserved the quality of the food butwere not without their own disadvantages. He concluded bydiscussing how the increasing resistance of bacteria wouldplay a key role in the future of this field.

Stefan Toepfl (DIL German Institute of Food Technologies,Germany) continued the session by speaking on ‘PulsedElectric Fields’. Stefan began by giving a brief overview of thetechnique and its history, detailing how its application in thefood industry has been investigated. He continued bydescribing ongoing efforts to scale this technique up to beused in an industrial setting and explained how this could beoptimized.

Following this, Frank Devlieghere (Ghent University,Belgium) discussed ‘Use of packaging for food preservation’.To start he examined how the current trend in foodmanufacture is to reduce the number of preservatives addedto food whilst retaining its shelf life and microbial safety. Hethen went on to discuss how maintaining certain gaseousatmospheres within food packaging could be used to reducebacterial numbers and prevent spoilage. Frank concluded bymentioning how the optimal compromise between preservingthe quality of food whilst maintaining shelf life would be touse a combination of mild inactivation of the bacteriaalongside packaging in a modified atmosphere.

The last presentation of the session, and the conferencewas given by Alejandro M. Amezquit (Unilever, UK) on‘Pressure assisted thermal sterilization’. Alejandro describedhow the use of high pressure could raise a temperature of 80to 90OC to levels high enough to initiate sterilization, in amanner similar to classic thermal processes, with theadvantage that once the pressure is removed the temperatureof the food returns to normal levels. Alejandro went on todiscuss his efforts in validating this method usingClostridium botulinum before concluding that further workwas required.

Phillip Humphryes PECS

22

meetings

Professor Joanna Verran, was this year’s winner of theSociety for Applied Microbiology (SfAM)Communications Award in recognition of her vital work

conveying the principles of applied microbiology to thegeneral public. She has used a range of different activities, inparticular reading and discussing works of popular fiction tomake the subject easily understood by non-scientists.

She has been innovative in her approach tomicrobiology education and public engagementand is the founder of the “Bad Bugs BookClub” the first of which was held during the

SfAM Summer Conference in Manchester,2009. Her down-to-earth attitude andfriendly disposition make her an engaging

speaker. Jo’s warmth and accessibilitycombine with a sharp wit to impress and

communicate sometimes complex informationeffectively to a lay audience. This wasevident at the Summer Conference dinner,in Dublin, where she was presented heraward. She gave us a rundown of three

experiences of science communication, all delivered with skill,fun and a great deal of humour.

When asked about her achievement, Professor Verran said:“I am absolutely thrilled to have received this award. Ilove microbiology, and have always tried to encouragesimilar passion and interest amongst lots of different

audiences, so it is great to have received this recognitionfor my work. In my opinion, applied microbiology is asubject of importance and fascination to everyone, andSfAM does a great job keeping it in the public eye (orear?!) as ‘the voice of applied microbiology’.”

Jo is a regular contributor to TV news and featuresprograms including BBC’s The One Show, and she has beenquoted in many newspaper articles. She has worked with SfAMand other organizations on countless public engagementevents at science festivals across the UK, including SfAM’sown “Outbreak: engaging the public in infectious disease”for the British Science Festival. She’s also worked atManchester Science Festival, Cheltenham Science Festival, andmost recently she has talked about her public engagementwork at the Federation of European Microbiology SocietiesCongress in Geneva, Switzerland.

According to our President, Professor Martin Adams: “Jo’swide knowledge and infectious enthusiasm have made heran outstanding public communicator and ambassador forapplied microbiology. This award is well deservedrecognition of her innovative work in this area. Long mayit continue.”

Science CommunicationAward winner announced

Lucy HarperCommunications Manager

The SfAMCommunications Awardaims to recognizeindividuals who havecommunicated theirwork/applied microbiologyto the general public.

The overall aim of thisaward is to raise the profileof applied microbiologyand SfAM. The award willbe for £1000 andnominations must be fromFull Ordinary or StudentMembers with a deadlinein April each year.

More information aboutthis award can be foundon the SfAM website at:http://www.sfam.org.uk/en/grants--awards/sfam-communications-award.cfm together withan application form inAdobe PDF format.

about this award

Professor Joanna Verran (right) with SfAMCommunications Manager, Lucy Harper

23

This year’s Environmental Microbiology Lecture onceagain took place at the Royal Society of Medicine inLondon a fitting venue for this year’s talk, ‘Microbes

inside’.The evening started with tea and coffee and a chance to

catch up with members of the Society and supporters of SfAMbefore the talk began. Professor Willem de Vos, Professor atHelsinki and Wageningen Universities was this year’s veryworthy speaker on the topic of the intestinal microbiome.

Willem began by giving us a potted history of themicrobiology of the intestines starting with Leeuwenhoek’sanimalcules (little animals), some of the earliest references tomicrobes. Leeuwenhoek discovered animalcules afterbecoming ill to a diarrhoeal disease. Willem continued bygiving us all a little perspective explaining that not only didintestinal microbes outnumber our own cells by one or moreorders of magnitude, but that one microbial metagenome (thegenomic material present in one host’s microbiome) is 150times larger than the human genome! It was enough to makeus feel very insignificant.

Willem then moved on to discuss the different approachesthat are being used to understand the intestinal microbiota.Willem summarized some of the most recent papers coveringmany aspects of the intestinal microbiota includingcomparisons of the pili of lactobacilli (the bacteria commonlyused in probiotics) and the pathogenic vancomycin-resistantEnterococcus faecium. The pili in these bacteria are verysimilar and allow both probiotic and pathogenic strains tobind to intestinal mucus. Recent research found that theLactobacillus strain can outcompete the pathogenic bacteriaas an effective infection control method.

Willem went on to discuss the complexity of the gutmicrobiota. The majority of microbes within the intestineshave not been cultured and the majority of studies are nowbased on an “omics” approach. Willem explained that whilstthere are common networks of microbes, everyone’s

Environmental MicrobiologyLecture 2011

Clare DoggettCommunications Officer

microbiota is individual. Many influences alter or determinethe microbes in our gut, including blood group and conditionssuch as irritable bowel syndrome (IBS). Willem highlightedone particular study which demonstrated that people with IBShave similar microbiota which is distinctly different to that ofhealthy individuals.

Willem then focussed on one particular organismAkkermansia, which belongs to the verrucomicrobia phylum,the only intestinal bacteria to do so. This small organismdegrades the intestinal mucus and is present in most people’sintestinal microbiome. Interestingly, Willem pointed out,Akkermansia is abundant in individuals with a healthyappendix, but is only present at very low levels in individualswith appendicitis. This trend is also true of healthy individualscompared with those who have irritable bowel disease (IBD).Willem raised the possibility that Akkermansia could,therefore, act as a biomarker for a healthy intestine.

Willem went on to discuss health implications of theintestinal microbiota including the effect of extremely lowcalorie diets on the composition of the microbiota and thefascinating results of studies on faecal transplantation inClostridium difficile infection and people with insulinresistance. Willem ended his talk by reminding us “we feedour microbes, they talk to us, we benefit… we just have tounderstand and exploit this”.

Professor de Vos was presented with a commemorativeplaque by SfAM President Martin Adams and Chief Editor ofEnvironmental Microbiology Ken Timmis. The eveningfinished with a wine reception and a chance to discuss thefascinating talk.

If you would like to watch the lecture it is now available at:www.yada-yada.co.uk/Blackwell/SFAM2011/SFAM2011.html

Professor Willem de Vos(centre) receiving acommemorative plaquefrom SfAM President MartinAdams (left) and ChiefEditor of EnvironmentalMicrobiology Ken Timmis(right)

24

meetings

Wednesday 11 January 2012

The Royal Society, London, UK

■ Including the Denver Russell Memorial Lecture

● Microbiological safety of imported food

● Microorganisms and climate change

Winter Meeting

The programme for this meeting was correct at the time of going to press

10.00 – 10.30 Tea, coffee and registration

Chair: Martin Adams

10.30 – 11.15 The Denver Russell Memorial Lecture:To be confirmed

11.15 – 11.50 Monitoring the microbiological safety of imported foods Caroline Willis, HPA, Southampton, UK

11.50 – 12.25 www.spatialepidemiology.net — tools for mapping infectious disease epidemiology To be confirmed

12.25 – 13.30 Lunch

Session A Microbiological safety of imported food

Chair: Andy Sails

13.30 – 14.05 From the banal to the bizarre — microbiological hazards and imported foodsSue Jones, HPA, Southampton, UK

14.05 – 14.40 Salad days — foodborne outbreaks due to imported fruit and vegetables: hazards, vehicles & sources Christine Little, HPA, Colindale, UK

14.40 – 15.00 Tea and coffee

15.00 – 15.35 Salmonella and imported eggs and poultry Sarah O’Brien, University of Liverpool, UK

15.35 – 16.10 Safety of imported foods — a commercial perspective Kaarin Goodburn, Chilled Food Association, UK

Session B Microorganisms and climate change

Chair: Mark Fielder

13.30 – 14.05 Microbes as climate engineers Dave Reay, University of Edinburgh, UK

14.05 – 14.40 Climate change and communicable disease: what are the risks?Andrew Nichols, University of Plymouth, UK

14.40 – 15.00 Tea and coffee

15.00 – 15.35 Assessing the impact of climate change on vector-borne viruses in the EU through the elicitation of expert opinionPaul Gale, AHVLA, UK

15.35 – 16.10 Antibiotics and climate changeMarion Wooldridge, AHVLA, UK

16.10 – 16.45 Shifting trends in pathogen dynamics ona changing planet Paul Hoskisson, University of Strathclyde, UK

16.45 Close

Programme

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26

features

The evolvingchallenge ofwar wounds

Surgeon LieutenantAndrew Matheson,

Major Emma Hutley andGroup Captain Andrew

Green detail the historicaldevelopments in managing

infection in combat woundsand discuss the current

issues faced by British forcestreating wounds sustained inAfghanistan, elaborating on

some of the more unusualinfections seen and themanagement strategies

developed to deal with them

The very nature of conflict is to inflict wounds which, ifthey do not kill directly, have the potential to becomeinfected, causing further injury and death. The approach

to the challenge of preventing and treating combat woundinfections has changed throughout the centuries, however thechallenge continues to evolve and so must the management.

BackgroundThe main focus of current British military operations is

Afghanistan where there are about 9,500 troops. Current work

is with the Afghan Security Forces to maintain the hard wongains from the Taliban over the last 10 years and to train theAfghan Army and Police as they start to take control of theirnational security. As of June 2011, 331 British personnel havebeen killed due to hostile action in Afghanistan. In 2010 alone,806 members of the Afghan National Army and 1,250members of the Afghan National Police lost their lives.However, this loss of life is only part of the picture; severeinjuries and loss of limb(s) are a daily occurrence. In 2010,518 UK personnel were wounded in action

27

(http://www.mod.uk/DefenceInternet/FactSheets/OperationsFactsheets.htm2011) and the survival of tripleamputees is not unusual.

The UK medical services in Afghanistan consist of teams ofmedics and doctors, highly trained in battlefield traumaresuscitation, providing pre-hospital care in the ForwardOperating Bases (FOBs) and on every patrol. These teams aresupported by a rapidly deployable Medical EmergencyResponse Team (MERT) from the field hospital which includesspecialists in anaesthetics or emergency medicine. The MERT

flies as close to the point of wounding as possible to collectthe casualty who is stabilized and treated en route back to thehospital. The deployed hospital at Camp Bastion providesimmediate life and limb saving treatment for any injuredpersonnel, be they coalition forces, insurgents, Taliban orlocal civilians. UK personnel are aeromedically evacuated backto the UK, usually within 24 hours, on a Critical Care in theAir Support Team (CCAST) aircraft which provides an‘intensive care’ level of care. On return to the UK, all patientsgo to the role four hospital at University HospitalsBirmingham where they receive definitive care; usuallyinvolving multiple reviews of wounds, further debridementsand intensive care support. Rehabilitation is conducted at thedefence medical rehabilitation centre Headley Court.

The history of wound infectionsThe fight against the development of infection in battle

wounds is not a new problem. Sumerian carvings describe theuse of beer and hot water to wash wounds which were thendressed with a plaster of herbs or fruits. The ancientEgyptians used honey and sometimes animal faeces to dresswounds (Molan, 2006) and Hippocrates describes the washingof wounds with wine and the drainage of pus to reduceinflammation. The Ancient Greeks felt there were differenttypes of pus and the encouragement of benign pus was centralto therapy (Pikoulis et al., 2004), a practice continued byGalen.

The late Middle Ages brought a new focus on cautery ofwounds with hot irons or oil to prevent infection. In the 16thcentury a French surgeon, Ambroise Paré, introduced wounddebridement. The Napoleonic Wars meant French surgeonsremained at the forefront with amputation becoming thestandard of care. Dominique-Jean Larrey, Surgeon-in-Chief ofthe Napoleonic armies and favourite of the emperor, couldperform hip and shoulder joint amputations in 15 seconds and11 seconds respectively with a 75% success rate in preventinginfection (Pruitt, 2006).

The first descriptions of war wound bacteriology camefrom Fleming during The Great War. Initially, after injury,wounds were infected with anaerobes, streptococci and otherfaecal pathogens. By day seven, streptococci werepredominant and after 20 days a mixture of staphylococci andstreptococci existed. In particular, Fleming noticedClostridium welchii (now Cl. perfringens) in 81% ofwounds from 1 to 9 days after injury, but only 34% of woundsfrom 8 to 20 days (Fleming, 1915). World War 2 saw theintroduction of antibiotics with topical sulfanilamide powder,and then penicillin. These, along with further surgicaldevelopments such as delayed primary closure, quickerevacuation to improved hospital facilities and improvements ininfection control, reduced mortality from most wounds inWorld War 2. An exception was gas gangrene in the MiddleEast where MacLennan noted an increase in mortality of 25%compared to World War 1. He felt the underlying cause wasless extensive wound debridement than performed in Flandersin 1917/18. He concluded that antimicrobials could notreplace surgery as the primary method of wound treatment; aprinciple which is still important today (MacLennan, 1943).

In Korea, wound infections again declined, with improvedevacuation times following the introduction of helicopterambulances. The side effects of administration of antibioticprophylaxis following injury were also documented in Korea.

28

features

Surgeons in a receiving hospital in Tokyo observed a highincidence of infection with organisms resistant to penicillinand streptomycin in soldiers given those drugs followingwounding. Resistance to penicillin was demonstrated in 48 of58 cases and to streptomycin in 49 of 58 cases (Wannamaker& Pulaski, 1958). Prophylactic antibiotics leading tocolonization with resistant organisms remains a concern today.

Vietnam highlighted the importance of good woundmanagement and several studies of wound bacteriologyshowed an initial mix of Gram-positive and Gram-negativeorganisms, with an increasing proportion of Gram-negative(Enterobacter spp., Pseudomonas spp. and Proteus spp.)from day five onwards.

The harsh terrain of the Falklands conflict, combined withthe nature of the fighting and limited evacuation assets, led tolong delays before initial debridement could be performed.Where prophylactic antibiotics were not administered andsurgery was delayed by over three hours the infection rate was33% (Jackson, 2007).

The challenges of AfghanistanMost front-line troops work in the green zone, a stretch of

fertile ground along the Helmand River Valley. The ForwardOperating Bases (FOBs) and Patrol Bases are often basedwithin the towns and villages of this area and much of thedaily work is in the surrounding fields and wadis. This notonly makes evacuation of injured troops challenging, butmeans wounds are heavily contaminated with mud and foliage.

Seriously injured casualties arriving in the deployedhospital undergo Damage Control Resuscitation (DCR), whichaims to reverse the lethal triad of coagulopathy, acidosis andhypothermia resulting from their trauma. Surgery is focussedon arrest of haemorrhage, wound debridement, temporarystabilization of fractures and removal of contamination(clothing, dirt, foliage and potentially tissue from othercasualties). Initial wound management aims to “remove allforeign and non-viable material from the wound and socreate the right conditions for wound healing orreconstruction without infection” (Guthrie et al., 2011). Thechallenge is correctly determining how much tissue to debridein severely contaminated wounds; too much debridementremoves viable tissue which is essential for reconstruction at alater date while too little leaves dead tissue and foreignmaterial as a focus for infection. Furthermore, severelyinjured casualties should spend the minimum possible amountof time in theatre to prevent further acidosis, hypothermia andbleeding due to the ensuing coagulopathy. This meansdebridement as radical or thorough as the surgeon would like,may not be possible. Additionally, patients areimmunosuppressed due to the massive blood transfusionsrequired to keep them alive at a time that their wounds arestill contaminated. The combination of all these factors meansthat casualties have a high risk of wound infection. Patientswith these severe injuries (Injury Severity Scores of greaterthan 60) are now described as ‘unexpected survivors’ andsome have gone on to develop unusual late wound infectionswith invasive fungi including zygomycetes.

The challenges facing the deployed laboratory staff are alsodifferent to those faced in the NHS. Military laboratory staffmust multitask as microbiologists, biochemists,haematologists and transfusionists. The ability to automate islimited by the environment, need for mobility and logistic

challenges, so staff must be able to deal with a variety ofisolates with the traditional tools of microscopy, culture andsimple biochemical tests. Additionally, the deployed laboratoryis usually diverted to providing the huge numbers of units ofpacked red cells and platelets required whenever a severetrauma case arrives.

Microbiology of Afghan woundsThe most common isolate seen in infected wounds is

Staphylococcus aureus, followed by Pseudomonasaeruginosa, Enterobacter cloacae and Bacillus spp. Thisdata is from the Wound Infection Surveillance Program, whichrecords data on all wounds in UK soldiers evacuated toBirmingham. The diagnosis of infection in these wounds isbased on the CDC 1992 criteria that are also used in thenational Surgical Site Surveillance Program (Horan et al.,1992).

An interesting organism seen in some patients injuredwhilst patrolling in the green zone is Aeromonas hydrophila.Infection following injury in similar conditions, immersed inwater or wet muddy fields, has been previously reported.Zygomycetes and Aeromonas spp. were isolated from injuriessustained in waterlogged environments during the Asiantsunami (Hiransuthikul et al., 2005; Andresen et al., 2005).

Multiple resistant Acinetobacter baumannii-calcoaceticuscomplex (ABC) was a significant issue during operations inIraq. Much debate occurred as to the source of the organismand the clinical sequelae. The UK and US experiences andoutcomes varied markedly. From a UK perspective, nosignificant infections occurred in UK service personnel despitemany patients being colonized. This may be explained by themore narrow spectrum antibiotic policy used by the UKmedical chain and the significant attention given to infectioncontrol.

29

As mentioned earlier, a number of the ‘unexpectedsurvivor’ group of patients from Afghanistan have developedzygomycete infections. This is thought to be linked to the typeand severity of injury in these individuals (Evriviades et al.,2011). Zygomycetes such as Rhizopus spp., Apophysomycesspp., Mucor spp., Saksenaea spp. and Absidia spp. havecaused infection. The classical zygomycosis is invasiverhinocerebellar disease in poorly controlled diabetic patientswho develop sinusitis following inhalation of fungal spores.The infection then spreads into adjacent tissues causingthrombosis and local necrosis. Metabolic acidosis andimmunosuppression following cytotoxic or radiation treatmentare also recognized as risk factors for zygomycosis.

Zygomycete spores are common in the environment, andinoculation of spores can result in invasive infection followingsevere trauma with heavy soil contamination. Invasive fungalinfection with zygomycetes has been reported in traffic andfarming accidents (Vitrat-Hincky et al., 2009), following thetsunami in 2004 and more recently following a tornado inMissouri in 2011(http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6029a5.htm). Our ‘unexpected survivors’ are at particularrisk of invasive fungal wound infection due to the extensivesoil contamination and microvascular effects from blastwounds, metabolic acidosis from muscle death andimmunosuppression following large blood transfusions. Theseinfections have been reduced by pre-emptive use of antifungalagents in patients fulfilling a specific set of risk criteria(personal communication). Burn wounds are predisposed toinfection due to the loss of the protective layer of the skin,and zygomycosis of burn wounds has been reported inAfghanistan (Hospenthal et al., 2011).

Diagnosis of fungal disease is challenging in the deployedenvironment. Ideally, histological examination of tissues isrequired to demonstrate tissue invasion and infection, andculture of the fungi is needed for identification andsusceptibility profiles (Cuenca-Estrella et al., 2011). Tissueremoved during debridement of wounds is stained withfluorescent calcofluor white stain to look for fungal hyphae,and it may be possible to identify the organism from themorphology (Evriviades et al., 2011).

ConclusionThe challenge of treating war wounds is constantly

changing, depending on the nature of the conflict. InAfghanistan, improvements in pre-hospital care meanpreviously unsurvivable blast injuries are pushing the limit ofour medical and surgical ability. The optimum management ofthese ‘unexpected survivors’ is still unknown, with alterationsto surgical technique and the use of novel pre-emptiveantimicrobials under consideration in the face of delayedwound debridement. The Wound Infection SurveillanceProgram which has been running for three years will hopefullyallow us to learn more and adapt further to these new combatwound infection challenges.

■ Andresen, D., Donaldson, A., Choo, L., Knox, A., Klaassen, M., Ursic,C., Vonthethoff, L., Krilis, S. and Konecny, P. (2005). Multifocalcutaneous mucormycosis complicating polymicrobial wound infections ina tsunami survivor from Sri Lanka. Lancet, Vol. 365 (9462), pp876-878.

■ Cuenca-Estrella, M., Bassetti, M., Lass-Flörl, C., Racil, Z., Richardson,M. and Rogers, T. (2011). Detection and investigation of invasive moulddisease. J. Antimicrob. Chemother, Vol. 66 (Suppl 1), i15-i24.

■ Evriviades, D., Jeffery, S., Cubison, T., Lawton, G., Gill, M. andMortiboy, D. (2011). Shaping the military wound: issues surrounding thereconstruction of injured servicemen at the Royal Centre for DefenceMedicine. Philos. Trans. R. Soc. Lond. B. Biol. Sci., Vol. 366 (1562),pp219-230.

■ Fleming, A. (1915). On the bacteriology of septic wounds. Lancet, Vol.2, pp638-643.

■ Guthrie, H.C., Clasper, J.C., Kay, A.R. and Parker, P. J. (2011). Initialextremity war wound debridement: a multidisciplinary consensus. J. R.Army Med. Corps., Vol. 157 (2), pp170-175.

■ Hiransuthikul, N., Tantisiriwat, W., Lertutsahakul, K., Vibhagool, A. andBoonma, P. (2005). Skin and soft-tissue infections among tsunamisurvivors in southern Thailand. Clin. Infect. Dis., Vol. 41, (10), e93-6.

■ Horan, T. C., Gaynes, R. P., Martone, W. J., Jarvis, W. R. and Emori, T.G. (1992). CDC definitions of nosocomial surgical site infections, 1992:A modification of CDC definitions of surgical wound infection. Infect.Control. Hosp. Epidemiol., Vol. 13, pp606-608.

■ Hospenthal, D.R., Chung, K.K., Lairet, K. Thompson, E. H., Guarro, J.,Renz, E. M. and Sutton, D. A. (2011). Saksenaea erythrospora infectionfollowing combat trauma. J. Clin. Microbiol., [Epub ahead of print].http://jcm.asm.org/cgi/content/abstract/JCM.05095-11v1.

■ Jackson, D. S. (2007). Soldiers injured during the Falklands campaign1982. Sepsis in soft tissue limb wounds. J. R. Army Med. Corps, Vol. 153 (Suppl. 1), S55–S56.

■ MacLennan, J.D. (1943). Anaerobic infections of war wounds in theMiddle East. Lancet, Vol. 24 (6256), pp94-99.

■ Molan, P.C. (2006). The evidence supporting the use of honey as awound dressing. Int. J. Low. Extrem. Wounds., Vol. 5, pp40-54.

■ Pikoulis, E.A., Petropoulos, J.C., Tsigris, C, Pikoulis, N., Leppäniemi, A.K., Pavlakis, E., Gavrielatou, E., Burris, D., Bastounis, E. and Rich, N. M.(2004). Trauma management in ancient Greece: value of surgicalprinciples through the years. World J. Surg., Vol. 28, pp425-430.

■ Pruitt, B.A. Jr. (2006). Combat casualty care and surgical progress.Ann. Surg., Vol. 243, pp715-729.

■ Vitrat-Hincky, V., Lebeau, B., Bozonnet, E., Falcon, D., Pradel, P., Faure,O., Aubert, A., Piolat, C., Grillot, R. and Pelloux, H. (2009). Severefilamentous fungal infections after widespread tissue damage due totraumatic injury: six cases and review of the literature. Scan. J. Infect.Dis., Vol. 41, pp491-500.

■ Wannamaker, G.T. and Pulaski, E.J. (1958). Pyogenic neurosurgicalinfections in Korean battle casualties. J. Neurosurg., Vol. 15, pp512-518.

■ Fatal fungal soft-tissue infections after a tornado — Joplin, Missouri,2011. MMWR. Vol. 60, No. 29.www.cdc.gov/mmwr/preview/mmwrhtml/mm6029a5.htm July 2011.www.mod.uk/DefenceInternet/FactSheets/OperationsFactsheets.htm July2011.

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Surgeon Lieutenant Andrew Matheson1, Major EmmaHutley2 and Group Captain Andrew Green3

Royal Centre for Defence Medicine

1 2 3

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Is the use ofhoney for thetreatment ofbiofilms astickysubject?

Traditionally most bacteria were thought to exist in natureas independent, free-living or planktonic cells. Therecognition that most species exist in biofilms has

revolutionized our perception.Biofilms are three dimensional structures firmly attached to

surfaces. They are comprised of either single or multiplespecies organized into complex communities and encased in amatrix of mutually secreted slimy extracellular polymericsubstances (EPS). Although many different signals can elicitbiofilm formation, the way in which they develop is wellestablished (Figure 1). Initially, cells are attracted to aninterface where reversible attachment of planktonic cells ismediated by surface appendages (such as pili and flagella) orpolysaccharide and protein cell surface adhesins. Growth anddivision leads to the formation of a localized microcolony. Thecontinual emission and detection of signalling moleculesallows individual cells within the microcolony to estimate thenumber of similar cells within close proximity by chemicalcommunication or quorum sensing. When a critical number isexceeded, differential gene expression initiates biofilmformation, which results in irreversible attachment, loss ofmotility, increased virulence and EPS secretion. Gradually, theimmature biofilm matures into complex three dimensional ormushroom-like structures with fluid filled channels for theexchange of nutrients and waste products. Cells in establishedbiofilms exhibit slow growth rates and their susceptibility toantimicrobial agents is at least 500 times less than that ofplanktonic cells (Stewart & Costerton, 2001). Fragments ofmature biofilm can become detached by shear forces anddispersal of planktonic cells from the mature biofilm isinfluenced by nutritional triggers.

Biofilms in human diseaseBiofilms are widely distributed throughout the environment

and can give rise to either beneficial or detrimental effects.Their role in the corrosion of submerged metal structures, orocclusion of pipelines is well described. Biofilms that becomeestablished within the human mouth, gut or vagina mayprovide protection against infection. However, biofilms havebeen implicated in 65% of human infections (Potera, 1999)and that estimate is probably too low. Whereas acute

Figure 1. Biofilm imformation

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infections that respond to antibiotics are considered not toinvolve biofilms, persistent infections such as cystic fibrosis,osteomyelitis, periodontal disease, prostatitis, sinusitis andinfections associated with indwelling medical devices havebeen associated with biofilms. Recently, chronicity in woundswas linked to the presence of biofilms (James et al., 2008).Difficulties in treating biofilm infections stem not only fromtheir recalcitrance to antibiotics and antiseptics, but also totheir ability to suppress the normal host immune responses(Bjarnsholt et al., 2008). Innovative treatment strategies areurgently required.

Control of biofilms in woundsAlthough routine methods of diagnosing biofilms in human

tissues are not yet available, the financial and social burden ofchronic wound infections on healthcare resources hasprompted searches for effective antibiofilm agents. Theproblem of how to manage biofilms in wounds is currentlyreceiving much attention. Two approaches have beenidentified: biofilm prevention and biofilm disruption. The firstclinical study to employ strategies designed to disrupt biofilmsin ischaemic leg ulcers utilized sharp debridement and topicalapplication of lactoferrin and xylitol. This approach was calledbiofilm-based wound care (BBWC) (Wolcott & Rhoads, 2008).Removal of tissue from the wound bed aimed to disrupt thebiofilm and reduce its biomass. Lactoferrin was used toprevent biofilm formation by sequestering iron andstimulating bacteria to adopt a specialized form of motility,which precluded the formation of biofilms (Singh et al., 2002)and xylitol was used to block the adherence of Gram-positivebacteria to epithelial cell surfaces and tissue surfaces(Tapiainen et al., 2004). Combination therapies seem to bemore effective than single interventions.

Other possible treatments for biofilms in wounds includemaggots, garlic (Rasmussen et al., 2005), silver (Bjarnsholt etal., 2007) and honey. Honey is an ancient wound remedy thathas been re-introduced into modern medical practice and arange of licensed products that contain honey are available onprescription in Australasia, Europe and North America. Theyrange from sterile medical grade honey in tubes, honeyincorporated into ointments or impregnated on to dressings.One non-sticky, flexible sheet has also been developed forirregularly shaped wounds. The floral origin of the honeysutilized are not always declared on the labels of theseproducts, but include manuka (Leptospermum scoparium),buckwheat, chestnut or they can be multifloral.

Inhibition of bacteria by honeyHoney is a broad spectrum antimicrobial agent that has

been shown to inhibit a wide range of microbial species invitro; more recently honey has been reported to inhibitantibiotic-resistant and multi-drug resistant wound pathogenssuch as MRSA, vancomycin resistant enterococci (VRE),Acintetobacter and Pseudomonas aeruginosa (Cooper,Molan & Harding, 2002; George & Cutting, 2007; Kwakman etal., 2008; Blair et al., 2009). The chemistry of honey iscomplex and varies with floral origin, species of bee,harvesting and storage conditions. Antibacterial activity ofundiluted honey is derived from its high sugar content, lowwater content and low pH. In diluted honeys, antibacterialactivity may be attributed to the generation of hydrogenperoxide (Bang, Buntting & Molan, 2003), the presence of

methylglyoxal (Mavric et al., 2008; Adams et al., 2008) orbee defensin (Kwakman et al., 2010). Not all honeys possessall of these antimicrobial components (Kwakman et al.,2011). Manuka honey from New Zealand has been shown toprevent cell division in planktonic cultures of MRSA (Jenkins,Burton & Cooper, 2011) and to cause cell surface changes andlysis in Ps. aeruginosa (Henriques et al., 2010).

Inhibition of biofilms by honeyLaboratory investigations into the effect of honey on

biofilms indicate that planktonic cells are more susceptible tohoney than biofilms. Also, lower concentrations of honey arerequired to prevent biofilm formation than those required todisrupt established biofilms (Merckoll et al., 2009; Cooper,Jenkins & Rowlands, 2011). Inhibition of Ps. aeruginosabiofilms was found to be dependent on exposure time andhoney concentration (Okhiria et al., 2009).

A study to determine the effectiveness of honey ininhibiting biofilms of Ps. aeruginosa and Staphylococcusaureus grown in the Calgary biofilm device showed that Sidrhoney from the Yemen and manuka honey from New Zealandwere more effective than commonly used antibiotics. Sincethese test organisms have been implicated in chronicrhinosinusitis, the authors suggested that nasal lavage withsolutions of honey could have a role to play in treatingpatients with recurrent and persistent infections (Alandejani etal., 2009). Typically, manuka honey contains higher levels ofmethylglyoxal (MGO) than other honeys (Mavric et al., 2008;Adams et al., 2008). It was recently demonstrated that honeyscontaining at least 0.53 mg/ml MGO had biofilm-cidal activityagainst Staph. aureus. Furthermore the antibiofilm activity ofa non-MGO honey supplemented with MGO was observed tomimic that of manuka honey, but MGO did not account for allof the activity of manuka honey (Jervis-Bardy et al., 2011). Itis possible that further antibacterial components in manukahoney may yet be discovered.

It has been demonstrated that honey prevents biofilmformation in Ps. aeruginosa because molecules of fructose(which is the most abundant sugar in honey) competitivelyblock the PA-IIL lectin that mediates adhesion of thebacterium to fucosylated receptors on the membranes ofpotential host cells (Figure 2) (Lerrer et al., 2007). Failure ofPs. aeruginosa to bind to target host cells precludes infectionand biofilm formation.

Figure 2. Blocking of lectin PA-IIL in Pseudomonas aeruginosaby fructose molecules in honey (after Lerrer et al., 2007)

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Rose CooperProfessor of MicrobiologyUniversity of Wales Institute Cardiff

■ Adams, C.J., Boult, C.H., Deadman, B.J., Farr, J.M., Grainger, M.N., Manley-Harris,M., and Snow M.J. (2008). Isolation by HPLC and characterisation of the bioactivefraction of New Zealand manuka honey (Leptospermum scoparium) honey.Carbohydr. Res. Vol. 343, pp651-659.

■ Alandejani, T., Marsan, J., Ferris, W., Slinger, R., and Chan, F. (2009). Effectivenessof honey on Staphylococcus aureus and Pseudomonas aeruginosa biofilms.Otolarynology-Head and Neck Surgery, Vol. 139(1), pp107-111.

■ Bang L.M., Buntting C., and Molan P. (2003). The effect of dilution on the rate ofhydrogen peroxide production in honey and its implications for wound healing. J.Altern. Complement Med. Vol. 9(2), pp267-273.

■ Bjarnsholt, T., Kirketerp-Møller, K., Kristiansen, S., Phipps, R., Nielsen, A.K., Jensen,PØ., Høiby, N., and Givskov, M. (2007). Silver against Pseudomonas biofilms. APMIS,Vol. 175, pp921-928.

■ Bjarnsholt, T., Kirketerp-Møller, K., Jensen, P.Ø., Madsen, K.G., Phipps, R., Krogfelt,K., Høiby, N., and Givskov, M. (2008). Why chronic wounds fail to heal: a newhypothesis. Wound Repair Regen., Vol. 16(1), pp2-10.

■ Blair, S.E., Cokcetin, N.N., Harry, E.J., and Carter, D.A. (2009). The unusualantibacterial activity of medical-grade Leptospermum honey: antibacterial spectrum,resistance and transcriptome analysis. Eur. J. Clin. Microbiol. Infect. Dis., Vol. 28(10),pp1199-1208.

■ Cooper, R. A., Molan, P. C., and Harding, K. G. (2002). The sensitivity to honey ofGram-positive cocci of clinical significance isolated from wounds. J. Appl. Micro.,Vol. 93, pp857-863.

■ Cooper, R., Jenkins, L., and Rowlands, R. (2011). Inhibition of biofilms through theuse of manuka honey. Wounds UK, Vol. 7(1), pp24-32.

■ George, N.M., and Cutting, K.F. (2007). Antibacterial honey (Medihoney™): invitro activity against clinical isolates of MRSA, VRE, and other multiresistant Gram-negative organisms including Pseudomonas aeruginosa. Wounds, Vol. 19(9), pp231-236.

■ Henriques A.F., Jenkins R.E., Burton N.F., and Cooper R.A. (2010). The effect ofmanuka honey on the structure of Pseudomonas aeruginosa. Eur. J. Clin. Microbiol.Infect. Dis., Vol. 30, pp167-171.

■ James, G.A., Swogger, E., Wolcott, R., Pulcini, E., Secor, P., Sestrich, J., Costerton,J.W., and Stewart, P.S. (2008). Biofilms in chronic wounds. Wound Repair Regen.,Vol. 16, pp 37-44.

■ Jenkins, R., Burton, N., and Cooper, R. (2011) J. Antimicrob. Chemother., Epub7th September 2011 doi: 10.1003/jac/dkr340.

■ Jervis-Bardy, J., Foreman, A., Bray, S., Tan, L., and Wormald, P-J. (2011).Methylglyoxal-infused honey mimics the anti-Staphylococcus aureus biofilm activityof manuka honey: potential implication in chronic rhinosinusitis. Laryng., Vol. 121,pp1104-1107.

■ Kwakman, P.H.S., Van den Akker, J.P.C., Guclu, A., Aslami, H., Binnekade, J.M., deBoer, L., Boszhard, L., Paulus, F., Middelhoek, P. te Velde, A.A., Vandenbroucke-Grauls, C.M.J.E., Schultz, M.J. and Zaat, S.A.J. (2008). Medical-grade honey kills

antibiotic-resistant bacteria in vitro and eradicates skin colonization. Clin. Infect. Dis.,Vol. 46, pp1677-1682.

■ Kwakman, P.H.S., te Valde, A.A., de Boer, L., Speijer, D., Vandenbroucke-Grauls,C.M.J.E., and Zaat, S.A.J. (2010). How honey kills bacteria. FASEB J., Vol. 24,pp2576-2582.

■ Kwakman, P.H.S., te Valde, A.A., de Boer, L., Vandenbroucke-Grauls, C.M.J.E., andZaat, S.A.J. (2011) Two major medicinal honeys have different mechanisms ofbactericidal activity. PloS One, 6(3): e17709.

■ Lerrer, B., Zinger-Yosovich, K.D., Avrahami, B., and Gilboa-Garber, N. (2007).Honey and royal jelly, like human milk, abrogate lectin-dependent infection-preceding Pseudomonas aeruginosa adhesion. ISME J., Vol. 1, pp149-155.

■ Mavric, E., Wittmann, S., Barth, G., and Henle, T. (2008). Identification andquantification of methylglyoxal as the dominant antibacterial constituent of Manuka(Leptospermum scoparium) honeys from New Zealand. Mol. Nutr. Food Res., Vol. 52,pp483-489.

■ Merckoll, P., Jonassen, T.O., Vad, M.E., Jeansson, S.L., and Melby, K.K. (2009).Bacteria, biofilm and honey: A study of the effects of the honey on ‘planktonic’ andbiofilm-embedded wound bacteria. Scand. J. Infect. Dis. Vol. 41(5), pp341-347.

■ Okhiria, O.A., Henriques, A.F.M., Burton, N.F., Peters, A., and Cooper, R.A (2009).Hooney modulates biofilms of Pseudomonas aeruginosa in a time and dosedependent manner. J. ApiProd. ApiMed. Sci. Vol. 1(1), pp6-10.

■ Potera, C. (1999) Forging a link between biofilms and disease. Science, Vol. 283,pp1837-1839.

■ Rasmussen, T.B., Bjarnsholt, T., Skindersoe, M.E., Hentzer, M., Kristoffersen, P.,Kote, M., Nielsen, J., Eberl, L., and Givskov, M. (2005). Screening for quorum sensinginhibitors (QSI) by use of a novel genetic system, the QSI selector. J. Bact., Vol.187(5), pp1799-1814.

■ Singh, P.K., Parsek, M.R., Greenberg, E.P., and Welsh, M.J. (2002). A componentof innate immunity prevents bacterial biofilm development. Nature, Vol. 41(6888),pp552-555.

■ Stewart, P.S., and Costerton, J.W. (2001). Antibiotic resistance of bacteria inbiofilms. Lancet, Vol. 358, pp135-138.

■ Tapiainen, T., Sormunen, R., Kaijalainen, T., Kontiokari, T., Ikaheimo, I., and Uhari,M. (2004). Ultrastructure of Streptococcus pneumoniae after exposure to xylitol. J.Antimicrob. Chemother., Vol. 54, pp225-228.

■ Truchado, P., Lopez-Galvez, F., Gil, M.I., Tomas- Barberian, F.A., and Allende, A.(2009a). Quorum sensing inhibitory and antimicrobial activities of honeys and therelationship with individual phenols. Food Chemistry, Vol. 115, pp1337-1344.

■ Truchado, P., Gil-Izquierdo, A., Tomas-Barberan, F., and Allende, A. (2009b).Inhibition by chestnut honey of N-Acetyl-L-homoserine lactones and bioiflmformation in Erwinia carotovora, Yersinia entercolitica, and Aeromonas hydrophila. J.Agric. Food Chem., Vol. 57, pp11186-11193.

■ Wolcott, R.D., and Rhoads D.D. (2008). A study of biofilm-based management insubjects with critical limb ischaemia. J. Wound Care, Vol. 17(4), pp145-155.

references

One other interesting attribute of honey is its ability toinhibit quorum sensing. In a survey of 29 unifloral honeys apigmented reporter bacterium (Chromobacter violaceum)was employed to detect quorum sensing agonists. Chestnuthoney and linden honey showed the highest quorum sensinginhibition activity (Truchado et al., 2009a).

The capacity of chestnut honey and its aqueous andmethanolic extracts to inhibit quorum sensing controlledevents in one plant pathogen and two human pathogens wasfurther investigated by quantifying N-acetyl-L-homoserinelactones (AHL) produced in supernatants of test bacteriaexposed to honey. The aqueous extract of chestnut honey wasfound not only to inhibit AHL production in each of the threetest bacteria but to cause degradation of AHLs. The quorumsensing inhibitory activity was thought to be contained withinthe carbohydrate fraction (Truchado et al., 2009b). Thesefindings suggest that chestnut honey may be used to controlpathogens in plants, animals and humans by attenuatingvirulence and preventing biofilm formation and infection. Awound dressing containing chestnut honey is already availablein Slovenia, but whether it controls biofilms in wounds is notyet known.

ConclusionAt the present time the industrial and clinical implications

of biofilms in unwanted situations are far-reaching. Thereduced susceptibility of biofilms to antimicrobial agentsconfounds the use of conventional antibiotics and antisepticsand requires some novel interventions. The ability of honey tointerrupt quorum sensing provides a means to prevent anddisrupt biofilms in diseased plants and animals, and theeradication of antibiotic-resistant strains from wounds bytopical application of honey might also help to reduce crossinfection in healthcare establishments. There is a need tocharacterize the active component(s) in honey and todetermine how quorum sensing is inhibited. The developmentof a new class of antimicrobial agent might be possible. Honeyno longer seems to be just a quaint folk remedy for woundsthat has no place in modern clinical practice.

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Bacteriophages’function inwound healing

When we encountered the idea of usingbacteriophages to eradicate human infection, theepiphany was exciting and almost overwhelming.

Imagine, with antibiotics failing, an entity almost as old astime itself being harnessed by humans to eradicate infectiousdisease. Bacteriophages may be the most important “postantibiotic era” treating agent that medicine can utilize tointercede in this growing global crisis. This gives us hope; yetour hope is counterbalanced by the complexity of chronicinfections.

Bacteriophages possess incredible potential; however, wenow realize the nature of infectious diseases is much morecomplex; so a large amount of hard work remains to be doneto harness their potential.

Clinical Microbiology is dominated by KochClinical microbiology continues to be dominated by the

early work performed by Robert Koch in the late 1800s.

Koch’s famous postulates, especially the pillar of clinicalmicrobiology that “one organism produces one infection”,have led to the clinical bias that only “a single organism”that is causing the infection must be targeted, and all othermicroorganisms are just contaminants. When Koch firststarted his quest for pure culture (predicated on the idea ofone organism causing one disease), he looked for culturemedia which would select for the one organism. Otherscientists of his day found, “No matter how ingenious themachinery, how careful the researchers, they kept endingup with beakers of mixed bacteria. The inability to getanything but mixed cultures led many scientists to believethat bacteria had to be in mixed groups in order to thrive”(Hager, 2006). Now we can say with almost certainty that thisis a foreshadowing of the biofilm infections we understandtoday.

The genius of Robert Koch was to “make sense out of thechaos” (Nobel Foundation, 2007). For episodic planktonic

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diseases, Koch was mostly right because planktonic bacteriacompete and tend to be monoclonal infections. However,chronic infections tend to have a complex, polymicrobialdiversity, something which is inherent in biofilms. It is thiscomplexity which challenges bacteriophage therapy. But theuse of bacteriophages can still play an important role inmanaging human infection.

Bacteriophages and biofilmsBacteria developed the ability to organize into biofilms 3.25

billion years ago. A well-vetted and widely accepted theory isthat bacteria selected for biofilm phenotype mode of growthin the environment mainly to protect themselves from UV lightand bacteriophages (Stoodley et al., 2002). The ancient dancebetween the predator bacteriophages and their bacterial preyis billions of years old with the bacteria mutating (droppingout epitopes, developing restriction enzymes, etc.) and thebacteriophages exerting countermeasures (depolymerase,adhesin mutations, etc.). The colony defences of biofilms limitthe predatory effectiveness of bacteriophages. So the narrowhost spectrum of the bacteriophage in the context of thepolymicrobial diversity of biofilms makes it impossible for asingle bacteriophage to eradicate the biofilm.

Bacteria pursue two different strategies forinfection

Costerton et al., (1999) first suggested that bacteria caninfect human tissue through a biofilm strategy. The molecularunderpinnings of this theory have been elucidated,culminating in the observation by Kim et al., (2010) thatbacteria infect host tissue in two very different ways. Bacteriacan choose to infect in a planktonic mode of growth, in whichthe bacteria will upregulate virulence factors killing the hosttissue. The bacteria then digest the dead tissue to obtainnutrition and propagate in the host environment. This mostclosely correlates with acute infection. The second method ofinfection described by Kim et al., (2010) is for bacteria toorganize in a biofilm phenotype mode of growth,downregulating virulence factors and causing host cellsenescence to produce a sustainable host niche. These biofilminfections then obtain nourishment through hostinflammation, yielding plasma which percolates through thebiofilm community. In Kim’s words, “Bacterial pathogens usea variety of mechanisms that balance breaching theepithelial barrier with maintaining the epithelium inorder to promote bacterial colonization. These complexstrategies represent a new paradigm of bacterialpathogenesis.”

Wound bioburdenIn collaboration with the Center for Biofilm Engineering in

2008, we were able to establish that biofilms are present inchronic wounds and not in acute wounds. Once we understoodthat biofilm-forming bacteria on the surface of the wound wasthe sine qua non of chronic wounds, it became important forus to understand the importance of the wound bioburden innon-healing wounds. In other words, is the wound biofilm aprimary or secondary factor in the recalcitrance of chronicwounds to healing?

One of the first and most exciting findings in the evaluationof wound biofilms, using molecular methods, was theincredible microbial diversity in chronic wounds. The diversity

added a robustness to the wound bioburden which madehealing these wounds much more challenging. But it alsobecame clear that it wasn’t just the species present, but howthese species coordinate with each other to act upon the hostto maintain their niche (Wolcott et al., 2008).

Our group has produced large cohort studies (level 2evidence) which demonstrate that when wound biofilm issuppressed, chronic wounds improve their wound healingtrajectory: 48% of wounds heal in six months withouttargeting biofilms, whereas 90% heal when a biofilm isspecifically targeted (Dowd et al., 2011). This is sufficientevidence to suggest that the biofilm is a primary barrier towound healing.

Bacteriophages and/or their components have long beenknown to demonstrate a power against many of the individualbacteria which constitute a wound biofilm. Now it is up toscientists and clinicians to try to exploit this power to suppresswound biofilm formation to a level which allows host healing.

BacteriophagesBacteriophages are ubiquitous and more abundant than any

other nucleic acid-based entities. They display incrediblevariations in how they adhere to their target host cell, breachthe outer defences, replicate, and eventually lyse their host toinfect new cells. Added to this complexity are vastly differentstrategies for phage infections possessed by lytic versustemperate bacteriophages. However, through persistence thetherapeutic use of bacteriophages is emerging.

The therapeutic use of bacteriophages was popularized byFelix d’Herelle in the early 1900s. Soon after the discovery ofthe bacteriophage, d’Herelle, Frederick Twort, George Eliavaand others utilized bacteriophage’s predatory propensitiestowards a variety of human pathogens. This was met with onlylimited success and, with the advent of antibiotics,bacteriophage therapy decreased in popularity across thedeveloped world, except for in Eastern European countries.Bacteriophages produced at the Eliava Institute in Tbilisi,Georgia, have been used to treat tens of millions of patientswith conditions as diverse as wound infections, prostatitis,trauma, osteomyelitis, pharyngitis, dysentery, and many otherinfectious diseases. Bacteriophages seem to have had somesuccess, yet it is only in recent years that this efficacy hasbeen scientifically documented.

As with any new agent, there have been concerns withinthe US FDA as to the safety of bacteriophages. Our grouppursued a Phase I randomized control trial to establish thesafety of bacteriophages in an acceptable randomized controltrial format (Rhoads et al., 2009). After a significant amountof negotiation with the FDA, we were permitted to pursue astudy protocol which utilized a cocktail of lytic bacteriophagestargeting Pseudomonas aeruginosa and Staphylococcusaureus. The protocol limited the number of study patients tojust 40 (20 in the treatment group) with careful observationsat each visit to rule out any adverse events. In the 20 patientsthat were treated with bacteriophages, there were no adverseevents. Since only a small number of patients could beincluded, no information about the efficacy of phage therapycould be obtained. However, utilizing the currently acceptedstudy method of a randomized control trial, our study diddemonstrate conclusively to the FDA that lytic phage cocktailsare indeed safe in treating human patients.

The FDA has imposed two significant requirements for the

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use of bacteriophages in human treatments. First, only lyticphages can be used as they have a very low incidence oftransferring potentially dangerous genetic material to thetargeted host bacterium. This transferred genetic materialcould result in increased pathogenicity. It is our opinion thatthe risk of using lysongenous phages is minimal and couldactually offer some advantages over lytic phages.

The second requirement of the FDA is that allbacteriophages be sequenced and must demonstrate safetyand efficacy through all three phases of clinical trials. Thisadds significant cost to the use of bacteriophages. This isprobably the most significant regulatory hurdle that must beovercome for the general use of bacteriophages as humantherapeutic agents. It is our view that it would be moreappropriate to treat bacteriophages like vaccines: that is, oncea bacteriophage is proven safe and effective, any newtreatment for phage should be approved by an administrativeprocess rather than clinical trials.

Phage therapyWe began utilizing phage therapy in 2005 after a visit to

Tbilisi, Georgia. Our colleagues in Tbilisi verified the safety oflytic bacteriophages along with case-after-case demonstratingthe therapeutic efficacy of phages. We first started treatingchronic wound patients with a commercially availablepreparation from the Eliava Institute. Piobiophage is a phagecocktail that is poorly defined but is mainly comprised ofbacteriophages isolated from chronic wounds. The cliniciansin Tbilisi, Georgia, have the advantage of being close to a verycheap source of large volumes of this cocktail, and as a result,they have achieved outstanding clinical results.

Despite the limited supply of this cocktail, we have seensome clinical response. Several patients showed rapid andcomplete clearing of the slough of their wounds within 24 to48 hours, followed by rapid progression of their wounds tocomplete healing. A second group of patients showed a veryrapid but incomplete response to the treatment. By the end oftwo weeks the wound healing had stalled and a thick, waxytype of slough had developed on the wound. However, themajority of wounds treated with Piobiophage had no responseat all.

This seems puzzling in that we specifically chose woundsthat routine clinical culture information indicated bacterialspecies would be targeted by the cocktail. We had only beenworking with biofilm phenotype bacteria for about two yearsat this time, but made the partially correct assumption thatbiofilm colony defences must be playing some role in thisincomplete efficacy of phage therapy.

We started exploring bacteriophages relative to biofilmsand found that bacteriophages indeed struggle with infectingtheir host bacterium when it is encased by a biofilm matrix. Inthe laboratory, bacteriophages were more successful when thematrix of the biofilm was disrupted or any of the other colonydefences were degraded prior to application of thebacteriophage. Yet when we applied the bacteriophages tochronic wounds post-debridement, while concurrentlyspecifically targeting the biofilm defences, there was nosignificant improvement in clinical outcome.

It was during this time that we developed moleculartechniques for identifying and quantifying the bacteria presentin wound biofilms. We found that wound biofilms containhighly diverse bacterial species, often more than 40 species in

a single wound, and this was the reason for the lack ofefficacy of our bacteriophage therapy. We now know that themajor constituents of the wound bioburden seem to varybetween different geographic locations. The bacteriophagecocktail from Tiblisi is tailored towards their most commonorganisms, and we were probably trying to treat differentorganisms. But not all wound biofilms are highly diverse.There are a small percentage of wounds which are primarily asingle species such as Ps. aeruginosa, Staph. aureus,Serratia marcescens. Those occasional wounds that showeddramatic improvements in wound healing, associated with ouruse of bacteriophage therapy, were comprised of a singlesusceptible bacterial species. But bacteriophage treatment canbe successful against some biofilms, so we must assume thatbacteriophages possess effective strategies for infectingbiofilm bacteria.

After our sputtering start into the clinical use ofbacteriophages, we realized that bacteriophage therapy willneed to become much more sophisticated to be useful ineveryday clinical wound care practice. Becausebacteriophages have such a narrow host range, it is necessaryfor wounds to be fully diagnosed not only in terms of thebacterial species present, but also how much of each speciesis present.

We currently use a combination of PCR and sequencingtechnologies to fully diagnose wound bioburden. Whenwounds are identified with a preponderance of Ps.aeruginosa or Staph. aureus we include bacteriophagetherapy with other simultaneous treatments. We found that byfrequently debriding the wound this not only degrades theprotective biofilm matrix (EPS) but it also forces the biofilmto reconstitute itself, becoming more metabolically active andtherefore more sensitive to treating agents (Wolcott et al.,2010). With frequent debridement, we include targeting thewound biofilm with anti-biofilm agents (quorum sensinginhibitors, etc.) and other agents such as antibiotics and/orbiocides. Theoretically, there is some question regarding theuse of antibiotics alongside bacteriophages, but in thelaboratory as well as clinic use there seems to be a synergy

Figure 1. Phage epitheleal island

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■ Costerton, J.W., Stewart, P.S., and Greenberg, E.P. (1999). Bacterial biofilms:A common cause of persistent infections. Science, Vol. 21; 284(5418),pp1318-1322.

■ Dowd, S.E., Wolcott, R.D., Kennedy, J., Jones, C., and Cox, S.B. (2011).Molecular diagnostics and personalised medicine in wound care: assessmentof outcomes. J. Wound Care, Vol. 20(5), pp232-241.

■ Hager, T. The Demon Under the Microscope. First ed. New York, NY:Harmony Books; 2006.

■ Kim, M., Ashida, H., Ogawa, M., Yoshikawa, Y., Mimuro, H., andSasakawa, C. (2010). Bacterial interactions with the host epithelium. Cell Host Microbe, Vol. 22;8(1), pp20-35.

■ Nobel Foundation: Robert Koch The Nobel Prize in Physiology or Medicine1905. The Official Web Site of the Nobel Foundation. http://nobelprizeorg/nobel_prizes/medicine/laureates/1905/koch-lecture html 2007 [cited 2007Aug 8].

■ Rhoads, D.D., Wolcott, R.D., Kuskowski, M.A., Wolcott, B.M., Ward, L.S.,and Sulakvelidze A. (2009). Bacteriophage therapy of venous leg ulcers inhumans: results of a phase I safety trial. J. Wound Care,Vol. 18(6), pp237-243.

■ Stoodley, P., Sauer, K., Davies, D.G., and Costerton, J.W. (2002). Biofilms ascomplex differentiated communities. Annu. Rev. Microbiol., Vol. 56, pp187-209.

■ Wolcott, R.D., Rhoads, D.D., and Dowd, S.E. (2008). Biofilms and chronicwound inflammation. J. Wound Care, Vol. 17(8), pp333-341.

■ Wolcott, R.D., Rumbaugh, K.P., James, G., Schultz, G., Phillips, P., and YangQ. (2010). Biofilm maturity studies indicate sharp debridement opens a time-dependent therapeutic window. J. Wound Care, Vol. 19(8), pp320-328.

references

case history

Randy D WolcottUniversity of Oklahoma, USA

between the concurrent uses of these multiple strategies.Occasionally, the bacteriophages are extremely successful,

rapidly collapsing the predominant Ps. aeruginosa or Staph.aureus populations. This allows the host to regain itsadvantage. Once the wound bed has been freed of the dead,devitalized, and senescent tissue, new blood vessel formationand wound contracture rapidly occurs with the wound healingmore like an acute wound than a chronic wound and in somecases; the healing can be quite dramatic (Figure 1).

The future of bacteriophage therapyFor phage therapy to become clinically useful in the

management of chronic wounds, a large library ofbacteriophages must be developed that address the mostprevalent bacteria identified in the wound biofilm. Forexample, anaerobes are a major component of wound biofilm,and it will be a difficult task to develop bacteriophages forthese microorganisms. Also, phage components have thepotential to be developed into adjunctive therapies whichdegrade biofilm defences. For example, phage enzymes, whichbreak down specific bonds of the complex sugars whichmakeup the EPS of a biofilm, could cause disruption ofchronic infections not accessible to debridement. Anotherstrategy is to utilize the adhesins that target different specificbacteria as a drug delivery system. Several researchers arealready producing large quantities of phage lysozymes (lysins)against various bacterial species. Lysins work on either side ofthe cell membrane but they do still maintain their very narrowhost range.

There is a lot of work left to be done, but with our currenttools and a clear vision of what can and needs to beaccomplished, bacteriophage therapy could soon be a veryimportant part of managing chronic wounds and indeed allchronic infections caused by biofilms.

The patient is a very pleasant 60-year-old white male withlymphedema in the bilateral lower extremities, uncontrollednon-insulin dependent diabetes mellitus and non-healingwounds on his lower extremities for many years. The patient’sfirst visit to our clinic was 26 June 2002 with a very painfulhighly exudative wound. He failed to respond tocomprehensive wound management. The patient wasreadmitted in May of 2004 with a dramatic increase in pain.The wounds were much deeper and more exudative. Thepatient had multidrug resistant Ps. aeruginosa bacteriaintermediately sensitive to Amikacin and resistant to all otherantibiotics. He was depressed, and ended up losing his job.The patient was started on biofilm based wound managementalong with bacteriophage therapy with specific phages againstPs. aeruginosa. The patient showed dramatic improvement inhis wounds over the course of four weeks.

Teaching Point: The patient has multidrug resistant Ps.aeruginosa and only responded to phage therapy. Of interest,his wound developed epithelial islands throughout the centreof the wound, which coalesced and filled the wound in, whichis a different healing pattern than normally seen.

37

In the 27th of a series of articles about statistics for biologists, Anthony Hilton & Richard Armstrong discuss:

StatNote 27Investigation of bacterial strains based on DNA profiles:principal components analysis (PCA)

IntroductionIn StatNotes 24 (Hilton & Armstrong, 2011a) and 25

(Hilton & Armstrong, 2011b), multiple linear regression, astatistical method that examines the relationship between asingle dependent variable (Y) and two or more independentvariables (X), was described. The principle objective of suchan analysis was to determine which of the X variables had asignificant influence on Y and to construct an equation thatpredicts Y from the X variables. ‘Principal componentsanalysis’ (PCA) and ‘factor analysis’ (FA) are also methods ofexamining the relationships between different variables butthey differ from multiple regression in that no distinction ismade between the dependent and independent variables, allvariables being essentially treated the same. Originally, PCAand FA were regarded as distinct methods but in recent timesthey have been combined into a single analysis, PCA oftenbeing the first stage of a FA (Norman & Streiner, 1994). Thebasic objective of a PCA/FA is to examine the relationshipsbetween the variables or the ‘structure’ of the variables and todetermine whether these relationships can be explained by asmaller number of ‘factors’.

In StatNote 26, (Hilton & Armstrong, 2011c) theapplication of classificatory methods (cluster analysis) to theanalysis of the DNA profiles from a sample of eight unknownisolates of MRSA was described. The most commonlyemployed hierarchical clustering method is the un-weightedpair group method using arithmetic averages (UPGMA)(Clifford & Sokal, 1975). The result of the analysis is adendrogram (tree diagram) which classifies the bacterialstrains into clusters. A major problem with this approach,however, is whether or not classification or clustering of thedata is appropriate. The strains may consist of a number ofdifferent distinct groups or species or they may mergeimperceptibly into one another because their DNA profilesactually vary continuously. An alternative to cluster analysis isto examine the spatial relationships between the strains usinga ‘non-hierarchical’ method of analysis. This StatNotedescribes the use of PCA/FA in the analysis of the differencesbetween the DNA profiles of different MRSA strainsintroduced in StatNote 26.

ScenarioWe return to the scenario described in StatNote 26 in

which the genetic relationship between eight unknown isolatesof MRSA was studied. Bacterial strains were incubated for 18to 24 hours at 37°C in Brain-Heart Infusion (BHI) broth.

Following incubation, the bacterial cells were harvested and20 milligrams (wet weight) of cells were re-suspended in 1mlNET-100 (0.1 M Na2EDTA (pH 8.0), 0.1M NaCl, 0.01M Tris-HCl (pH 8.0)) and mixed with an equal volume of molten lowmelting point chromosomal grade agarose (0.9% (w/v) in NET-100; BioRad,UK). The prepared blocks were incubated for24h at 37°C in 3ml lysis solution (6mM Tris pH 7.6, 100mMEDTA pH 8, 100mM NaCl, 0.5% lauroyl sarcosine and 1mg/mllysozyme) with 20 units of lysostaphin (Sigma, UK). The initiallysis solution was removed and the blocks were incubated for48h at 50°C in 3ml ESP (0.5M EDTA pH 9, 1.5 mg/mlproteinase K (Sigma, UK) and 1% lauroyl sarcosine). Theblocks were washed at room temperature twice for 2hfollowed by two 1h washes using TE buffer (10mM Tris and1mM EDTA, pH 8). A portion of each agarose block (1×1×9mm) was digested with 20 units of SmaI (Roche, UK) in0.1ml buffer for 16h at 25°C. The digested DNA samples weresubjected to PFGE (CHEF Mapper system, BioRad, UK) underthe conditions outlined by Bannerman et al., 1995. Gels werestained with 1µg/ml of ethidium bromide for 45min anddestained for 45min in distilled water. Gels were visualizedunder UV illumination and photographed using theGeneGenius Bio Imaging System (Syngene, UK).

DataThe data comprise the band distances migrated in

millimeters from the origin of the gel for each of the DNApreparations and are presented in Table 1 of StatNote 26.Hence the data comprise a series of X variables (bacterialstrains) and there is no dependent Y variable.

How is the analysis carried out?Theory

The ‘variables’ in a PCA/FA are defined by a number of‘criteria’. If the variables are bacterial strains, called a ‘Q-typeanalysis’ (Pielou, 1969), the criteria could be the distancetravelled by the various DNA fragments on the gel. Hence, ifthere are ‘s’ criteria, each strain can be represented by a pointplotted in a Euclidean space defined by ‘s’ dimensions, i.e.,each criterion can be considered as an axis orthogonal to allthe other criteria and each strain as a point in this s-dimensional coordinate frame. Conceptually, in a PCA/FA, theoriginal ‘s’ dimensional geometric frame is reduced to two(2D) or three dimensions (3D) such that the original spatialrelationships between the strains are preserved as far aspossible. Strictly PCA is just one method, albeit the most

38

features

common, for selecting these fewer dimensions.A simple geometric model of the procedure is shown in

Figure 1. Imagine that each of 21 variables were defined byonly two criteria and the data plotted as a 2D scatter plot.Now suppose that we wish to simplify this 2D display byprojecting the points on to a single line. In carrying out thisprocedure, there is an inevitable loss of information regardingthe spatial relationships between the variables. The loss ofspatial information would be minimized if the line wasorientated through the cluster of points to preserve as muchof the original spatial variability as possible. In PCA/FA, if thenumber of original axes is ‘s’, a smaller number of axes or‘factors’ are extracted which account for significantproportions of the original variance in the data. If theextraction procedure is PCA, then the extracted factors arecalled principal components (PC). The first principalcomponent (PC1) accounts for the maximum variance whilethe remaining PCs account for decreasing proportions of theremaining variance. The PC is analogous to the single linedrawn through the points in Figure 1. Individual variablessuch as strains could then be plotted in relation to theseextracted PCs. Such plots summarize the similarities anddifferences that exist between the variables; variables closetogether in the plot being more similar in the measuredcriteria than those furthest apart. Such an analysis canindicate whether variation between bacterial strains wascontinuously distributed or clustered into subgroups. In otheruses of PCA/FA, the variables may not be bacterial strains butthe criteria used to define them (sometimes called a R-typeanalysis) and the intention may be to determine whether thevariables (distances of the DNA bands) can be grouped into asmaller number of underlying ‘factors’ which can best explainthe patterns of variation among the strains.

Most of the major statistical packages such as SPSS andSTATISTICA will offer FA and PCA often as part of a

‘multivariate analysis’ option. We will illustrate the analysis ofour data using STATISTICA software.

AnalysisThe original ‘raw’ data set comprises the variables (the

columns or strains) defined by a number of criteria (the rowsor band distances). The first stage in a PCA/FA is thecalculation of the correlation of each variable with all of theother variables. If the measured variables are clearly related toa smaller number of underlying ‘factors’, then this may beapparent by inspection of the correlation coefficient matrix.This is because all the variables that measure one factor wouldcorrelate strongly with each other and not with the variablesassociated with another factor. In practice, however, it is verydifficult to extract the actual factors by visual examination ofa correlation matrix since all variables are likely to show somedegree of correlation with other variables.

The next stage of the analysis involves checking whether itis worth carrying out a PCA/FA with the existing data, i.e., arethere sufficiently strong correlations between the variables toanalyse them factorally? The simplest of these tests involvesexamination of the ‘strength’ of the correlations within thematrix. If there are few correlations greater than about r =0.30, then it is probably not worth proceeding any further(Tabachnick & Fidell, 1989), i.e., the correlations betweenvariables are too weak for them to be combined into fewerfactors. A second test involves examination of the ‘partialcorrelations’ between the variables. If there are only threevariables in the study (X1, X2, X3), the partial correlationcoefficient between any two of them, say X1 and X2, is thecorrelation between them in a cross-section of individuals allhaving the same value of X3. In other words, the effect of X3 isremoved from a test of the correlation between X1 and X2. Inthe case of a PCA/FA, if the variables correlate with each otherbecause they are related to a smaller number of underlyingfactors, the partial correlations should be small (Guttman,1954). Another statistical test employed is ‘Bartlett’s Test ofSphericity’ which uses χ2 as a test statistic. If the value of χ2 isnot significant (P > 0.05), then no correlations are presentand the analysis should not proceed. A final test is that of‘sampling adequacy’ and is a measure of the degree ofcorrelation within the data set as a whole and of the individualvariables. If the sampling adequacy for the whole data set is<0.50, it is better not to proceed with the analysis. Similarly,if the sampling adequacy for an individual variable is <0.50,that variable is unlikely to show much correlation with theother variables in the study and it may be better to proceedwithout it.

The PCs are extracted so that first, they are uncorrelatedwith each other, and second, that each successive PC accountsfor a decreasing proportion of the remaining variance. Hence,PCA/FA tries to ‘explain’ the variance of a group of variablesin terms of a smaller number of uncorrelated PCs.

The analysis will extract as many PCs as there are variablesincluded in the analysis. An important question is how manyPCs should actually be extracted from the data and retained forexamination? There is no objective statistical method availablewhich can determine the number of PCs. Instead, moststatistical programs employ one or more rules, termed‘stopping rules’, to determine how many PCs should beretained. In a PCA/FA, each PC is associated with an‘eigenvalue’ which is the per cent of the total variance in the

Figure 1. A hypothetical example in which variables aredefined by only two criteria (X,Y), resulting in a two-dimensional (2D) scatter plot. In theory we could reduce the2D display of the patients to 1D by projecting the points sothat they lie on a single line (PC)

39

Bacterial strains

A

B

C

D

E

F

G

H

I

J

A

1

1

0.98

1

1

0.68

0.48

0.98

0.70

0.70

B

1

0.98

1

1

0.68

0.48

0.98

0.70

0.70

C

1

0.98

0.98

0.62

0.52

1

0.63

0.63

D

1

1

0.68

0.49

0.98

0.70

0.70

E

1

0.68

0.48

0.98

0.70

0.70

F

1

0.20

0.62

0.99

0.99

G

1

0.52

0.24

0.24

H

1

0.63

0.63

I

1

1

J

1

Table 1. Simple correlation matrix (Pearson’s correlation coefficient ‘r’) between the bacterial strains. There are eight SmaI genomic digests ofMeticillin-resistant Staphylococcus aureus (MRSA) while wells C and H carry a SmaI chromosomal digest from Staph. aureus strain NCTC 8325 asa control and molecular weight marker. The majority of the correlations exceed 0.30 suggesting that the data are suitable for PCA/FA

data explained by that PC. First, the ‘Kaiser’ criterion selectsall eigenvalues greater than 1. This criterion has twodisadvantages: (1) it is essentially arbitrary and (2) it tends toselect either too many or too few PCs depending on thenumber of variables in the study. Second, in ‘Cattell’s ScreeTest’, the eigenvalues for each factor are plotted in descendingorder. The change in the eigenvalue is rapid at first and thenlevels off and the last factor is chosen before the flat portion ofthe curve. Third, the 75% stopping rule selects all PCs whosevariance when added up sums to 75% and is also likely toextract many PCs. Whichever method is used, it is commonlyobserved in a PCA/FA that the first PC will contain adisproportionate amount of the variance, and the othersrelatively small amounts. Hence, in many studies, it is oftennot worthwhile to examine more than the first three PCs.

The next stage involves determining which of the measuredvariables are associated with each PC and this is strictly wherethe factor analysis part of the method begins. PCA/FAcalculates the correlations between each variable and theextracted PC and these are known as the ‘factor loadings’, ahigh value indicates a strong relationship between the variableand the PC. Ideally, each of the variables should load ontoonly one PC, i.e., the loadings should be close to unity orzero. In practice, variables are often complex and load ontomore than one PC and this can make their interpretation moredifficult. Another problem is that some variables may havepositive loadings and others negative loadings on a PC. Insome types of PCA/FA, rotation of the PCs is often advocatedto produce a solution that is easier to interpret. Rotation canbe carried out in such a way that the PCs remain uncorrelated(an ‘orthogonal’ solution) or this condition can be relaxed andsome degree of correlation between the PC can be accepted(an ‘oblique’ solution). Rotation of the PCs will often producea more equitable distribution of the variance between theextracted PCs and also result in individual loadings that arecloser to unity or zero.

InterpretationAn important step in a PCA/FA is to attempt to interpret

what the extracted PCs actually mean with reference to theproblem or hypothesis posed. The first stage of this analysisinvolves determining which variables load significantly ontoeach PC. A simple procedure would be to accept as significant

Extracted PCs

Strain

A

B

C

D

E

F

G

H

I

J

% Variance

PC1

-0.977435

-0.977435

-0.952375

-0.977486

-0.977435

-0.804656

-0.498039

-0.952375

-0.823068

-0.823068

78.861

PC2

-0.161167

-0.161167

-0.255144

-0.166691

-0.161167

0.572954

-0.489264

-0.255144

0.549259

0.549259

14.069

Table 2. Unrotated factor loading matrix: the correlation betweeneach of the bacterial strains and the extracted principal components(PC) and the percentage of the total variance explained by each PC

Extracted PCs

Band

1

2

3

4

5

6

7

8

9

10

11

12

13

14

PC1

0.26

0.51

0.39

0.66**

-0.13

-0.04

-0.16

-0.21

-0.42

-0.57

-0.69**

-0.80**

-0.31

0.88***

PC2

0.54

-0.08

-0.18

0.40

0.32

0.13

0.28

0.38

0.14

0.22

0.26

0.55

-0.96***

-0.44

Table 3. Correlations between the band distances and the factorloadings of the bacterial strains on PC1 and PC2 (** P < 0.01, ***P < 0.001)

40

Dr Anthony Hilton1 and Dr RichardArmstrong2

1Biology & Biomedical Sciences and 2VisionSciences, Aston University, Birmingham, UK

Ant

hony

Hilt

onRi

char

d A

rmst

rong

■ Bannerman, T.L., Hancock, G.A., Tenover, F.C. and Miller, J.M. (1995).Pulsed-field gel electrophoresis as a replacement for bacteriophage typing ofStaphylococcus aureus. J. Clin. Microbiol., Vol. 33, pp551–555.

■ Clifford, H.T. and Sokal, R.R. (1975). An Introduction to NumericalClassification, Academic Press, New York, NY.

■ Hilton, A. and Armstrong, R.A. (2011a). StatNote 24: Multiple linearregression. Microbiologist March, Vol. 12, No. 1, pp40-43.

■ Hilton, A. and Armstrong, R.A. (2011b). StatNote 25: Stepwise multipleregression. Microbiologist June, Vol. 12, No. 2, pp38-39.

■ Hilton, A. and Armstrong, R.A. (2011c). StatNote 26: Classification ofbacterial strains based on DNA profiles. Microbiologist September, Vol. 12,No.3, pp37-39

■ Guttman, L. (1954). Some necessary conditions for factor analysis.Psychometrica, Vol. 19, p149.

■ Norman, G.R. and Streiner, D.L. (1994). Biostatistics: The bare essentials.Mosby, St Louis.

■ Pielou, E.C. (1969). An Introduction to Mathematical Ecology. John Wiley &Sons.

■ Tabachnick, B.G. and Fidell, L.S. (1989). Using multivariate statistics (2nd Ed),New York, Harper and Row.

references

any variable whose loading was larger than a certain value,e.g., >0.30 or >0.50 but this is an arbitrary procedure anddoes not take into account sample size. A more rigorousmethod is to test the loadings statistically using ‘Stevensmethod’ (Norman & Streiner, 1994) and is given for a samplesize of ‘N’ by the equation:

Critical value = 5.152/√(N-2) (1)Hence, any variable whose factor loading exceeds this

critical value may be regarded as being significantly correlatedwith a PC.

In the present application of PCA/FA, bacterial strains arethe variables (Q-type analysis) and the result is a scatter plotof the strains in relation to the extracted PC. The objective istwofold: (1) to describe the pattern of variation betweenbacterial strains and (2) to identify those features of the DNAprofiles that best correlate with the distribution of the strains.

The matrix of correlations between the MRSA strains isshown in Table 1. The majority of the correlations exceed 0.30suggesting that the data are suitable for PCA/FA. Two PCswere extracted from the data, using Cattell’s Scree Test,accounting for approximately 93% of the total variance in thedata (Table 2). Hence, reducing the original 10D frame to 2Dhas resulted in the loss of approximately 7% of the originalspatial information. A plot of the bacterial strains in relationto PC1 and PC2 is shown in Figure 2. The data suggest thatthe data from wells I/J, C/H, and A/B/E immediately formthree groups which are each identical according to banddistances. Furthermore, D is the strain most closely related toE/B/A and F is most closely related to J/I. Strain G appears tobe the most unrelated to the others. In addition, thecorrelations between the band distances for each strain andthe factor loadings of the strains on PC1 and PC2 are shownin Table 3. Bands 4 and 14 were positively correlated withPC1 and bands 11 and 12 negatively correlated with PC1 andhence, these are the DNA band distances which are the mostimportant in determining the distribution of the strains.Moreover, band 13 was negatively correlated with PC2.

The interpretation of the PCA/FA data in this example is inclose agreement with that of the dendrogram analysisdescribed in StatNote 26. However, the PCA has a number ofadvantages over that of classification: (1) no assumptions aremade that the data are actually classifiable, (2) therelationship between strains and clusters of strains is spatiallydisplayed which facilitates discussion of the implications ofthe analysis, and (3) the analysis identifies the criteria, in thiscase the band distances, which best differentiate between thestrains.

ConclusionPCA/FA are methods of analysing complex data sets in

which there are no clearly defined X or Y variables. They havemultiple uses including the study of the pattern of variationbetween individual entities such as bacterial strains and thedetailed study of descriptive variables. In most applications,variables are related to a smaller number of factors or PCsthat account for the maximum variance in the data and hence,may explain important trends among the variables. Noassumptions are made before the analysis that the variablescan actually be classified and this may be a considerableadvantage in the analysis of more complex data sets such asDNA band data of bacterial strains which may be morecontinuously distributed.

Figure 2. The resulting pulsed-field gel electrophoresis (PFGE)patterns of the eight SmaI genomic digests of Meticillin-resistant Staphylococcus aureus (MRSA); wells C and H carry aSmaI chromosomal digest from Staph. aureus strain NCTC 8325as a control and molecular weight marker plotted in relationto PC1 and PC2

features

41

members

News from the SfAM Postgraduate andEarly Career Scientist Committee

PECS NEWSWe would like to announce thatSimon Gould has stepped down asChairman of the PECS Committee andthank him for all his work.Congratulations to Emmanuel Adukwuon his new role as Chairman. Wewould also like to welcome two newmembers of the PECS Team, DannySewell and Jenni Drever-Heaps. If youwould like to let us know about afellow student or early career scientistwho has recently been awarded aPhD/prize/award please emailpecs@sfam.org.uk.

How did you prepare before startingto write up?

One of the best things my supervisordid for me was to insist I write a

transfer report about halfway into myPhD even though at the time it wasn’t arequirement. This allowed me toexperience ‘writing up’ before the main‘writing up’. It also provided a very robust‘skeleton’ on which I could build thethesis on. With hindsight, I wouldencourage PhD students to always bewriting something. Sometimes whenreading a completed thesis andcomparing the clarity and structure toyour writing we forget that it took a lotof drafts to get there. It doesn’t have tobe perfect, just written. Leaving anywriting for the final year is never a goodidea in my opinion. Many institutionsnow insist on progressing studentssubmitting lengthy pieces of work at leastonce a year to ensure that they arealways writing.

When I write, I tend to start with whatI consider the ‘easier’ parts of the thesis.So for each chapter, I would put in themethods section, followed by the results.The sense of accomplishment from seeingthe chapter develop encouraged me towrite more. The writing process can bedifficult, it calls for organizational skills.You must plan your work and set goalsand targets for yourself. Discussing thesetargets with your supervisors gives youthat extra kick to stick to your plan. Thethesis itself is a big project but it can bemanaged if it’s broken into smallerprojects and you can celebrate when youpass each milestone. I found taking shortbreaks from writing helped. Reading asection after a few days (well hourssometimes) gave me some distance thatenabled me to spot any errors, ask myselfquestions and rewrite with more clarity.

I learnt that the writing process usuallytakes longer than you estimate in yourhead. You have to give time to send yourdrafts to your supervisory team, receivetheir comments and make thosecorrections. Remember that you are nottheir only priority. These were things Ididn’t really consider and contributed tothe writing taking longer than I actuallythought it would.

What is your advice regardingmanaging your references?

I’ll say do what works best for you.Most students I know use

referencing systems like EndNote. I’m not‘techy’ so I managed mine manually. Idon’t think this is the best way, itcertainly isn’t the easiest for most peoplebut it worked very well for me. Eachchapter had its own folder with thepapers I was using and I constantly editedthe reference list at specific writing timesto ensure that all my cited referencesended up on the reference list. So far, Ihaven’t noticed any mistakes!

What kind of preparation are youdoing for the viva?

I discussed with students who hadrecently completed their PhDs and

survived the viva. I’ve been told to makesure I really understand what I’ve writtenin my introduction section! I’ve beenreading through the thesis and trying toanticipate any questions I may be asked.Also trying to answer the question, ‘Whatis the contribution this body of work hasmade in your field?’ Making notes onconcepts/mechanisms I have brieflydiscussed but may require furtherexplanation. Creating a list of anymistakes e.g. spelling mistakes, figureannotations that I observe as I amreading the thesis and submitting this tothe examiner during the viva.

Any other advice?

In addition to the questions you’veasked, I would say that students

should be aware of the guidelines of theirinstitution regarding submission and theoral examination. What forms need to befilled prior to submission/to arrange yourviva? How many copies of your thesisneed to be submitted? These are littleadministrative things but can lead tounnecessary delays if not dealt with.

Lastly, I would say all the best. It is adaunting but very achievable task. Thework has been done already, but youneed to write it up in a way that givesyou and the work credit.

Amara AnyoguPECS Secretary

For the latest PECS pages we wantedan insight into writing up so PECS

Communications Officer, Irene Freire-Martin posed some questions to

PECS Secretary Amara Anyoguabout her experiences

Irene Freire-MartinPECS CommunicationsOfficer

writing up

42

members

The Germ Wars initiative began in2010 with a small pilot projectwith three Dundee schools,

engaging 80 Primary 6 (age 10 to 11)pupils with microbiology. This projectwas very successful with pupilsbecoming fascinated by the subject, andteachers building up their confidence toteach microbiology in the future. It wasclear that this project was a success, andthanks to the kind support of SfAM itwas rolled out to 10 Dundee City

Public Engagement Grant: Germ Wars

Council schools this year, with 250pupils taking part!

‘Germ Wars’ is a highly engagingbook in which the author, GillArbuthnott, uses fun illustrations and asound scientific knowledge to introducechildren to the sometimes mysteriousworld of microbiology! The book hasfour fabulous sections: What areGerms?, Diseases you never want tomeet!, Fighting back! and The future…what happens next? While this book

mainly deals with the horrors ofmicrobiology, including Black Death,small pox, and the terror of biologicalweapons such as Anthrax, it also tellsthe story of Edward Jenner, which is aproven favourite with the pupils! Thebook not only tells the story of smallpox eradication, but also gives thescience behind vaccination, givingchildren a basic understanding of ourimmune system. The book then goes onto tell the story of Alexander Fleming,

43

“I just wanted to say thankyou for another terrific setof sessions in Dundee. It’sobvious that the pupilshave found the work youhave done with them really

inspiring, and I’m honoured to be partof such a great team! I’m alreadylooking forward to next year...”

Gill Arbuthnott, author of ‘Germ Wars’

“…after the outreach visit from DSC Ithought one of my pupils had beencaptured and replaced by a new boy!He was completely engaged and hadenjoyed the session so much it hadpositively impacted on his behaviourwhich was normally not that great.”

“Children learned how easily germscan spread, that there are good andbad bacteria, and science is fun!”

“I asked the children to vote on theirfavourite topic of the whole year, andthe resounding favourite was GermWars!”

“I learned MILLIONS that my brain isactually tired, it’s that full!”

“This was a great project, I learned allabout small pox, bacteria and wallabymilk! I would definitely do it again!”

“I learned that there are thousands ofbugs in your gut!”

“I learned about bacteria Inever even knew about!”

“I learned that you do getgood germs!”

Howard Florey and Ernst Chain and therace to make and mass producepenicillin during wartime. The bookends with a section on the future ofantimicrobial medicines, introducing thehot topic of antibiotic resistance, andfuture possibilities such as phagemedicine and wallaby milk. Gill is afantastic writer, and has a knack ofknowing exactly what the children willengage with, always managing to tell agreat story while including soundscience knowledge.

The project ran under the sameformat as last year’s, starting with aCPD evening for teachers before Easter.The session introduced them to theproject, allowing familiarization with ourmicrobiology workshop, which wouldlater be delivered to their class, and alsoprovided them with an activity pack fullof active learning ideas and a list ofresources to support them further.Teachers were given a copy of the‘Germ Wars’ book, under which theproject is based, to allow them time toexplore the book and plan out theirlessons.

The kind funding from the Societyallowed all 250 children to be giventheir own copy of the book to keep as amemento for life, and these were handedout after the Easter holidays. Theclasses were able to either dedicate theirterm to microbiology as a main topic orhave it as an extra, and feedback thisyear shows that three of the classesdevoted their term to the project!

In June, Dundee Science Centre staffvisited each class and delivered our‘Befriending Bacteria’ workshop whichaimed to show children the good side ofmicrobes, their importance for oureveryday health and well-being and howwe can look after our good bacteriathrough our diet, helping to build ourimmune system and stay healthy!Additionally, we used art to help childrenunderstand the differences in shapesand sizes of different microbes, and letthem design their own to conclude thesession.

Finally, classes visited DundeeScience Centre to meet Gill Arbuthnott.In this session she talked to the childrenabout the book and told some more funscience stories before signing theirbooks.

Full evaluative techniques, includingLikert scales were used to evaluate thisproject. At the start of the CPD, teacherstold us that they did not feel confident

about teaching microbiology: whenasked to rate their confidence on aLikert scale (in which 1 is lowest and 8is highest), an average of 5.6 was cited,however, by the end of the project,average teacher confidence hadincreased to 7.3. This is fantastic, withcomments that they would definitelyapproach microbiology in future andhad learned lots of new knowledge andskills through the project.

At the end of the project pupils filledout a short feedback form, whichprovided a fantastic insight into thechildren’s feelings about, not only theproject, but microbiology as a whole.Pupils were asked to score the overallenjoyment of the project, with a finalaverage score of 7.3 out of 8! A smallnumber of pupils did not enjoy it, withjust 2% of pupils scoring below 5. Whenasked if they would like to learn moreabout microbiology, 22% scored lowerthan 5, with an average score of 6 out of8. These results highlight how wellchildren can engage with this subjectand that with increased training forteachers this subject can be taught veryeffectively in the primary classroom.

Pupils were also asked a range ofquestions such as favourite and worstparts of the project. 54% highlightedtheir visit to meet Gill as a favouritepart, while 23% commented that theyloved using the UV light box to learnmore about epidemiology and how far asneeze can travel. 60% of pupils saidthey enjoyed all of it and did not have aworst part, while the remainder eitherdidn’t like all the gruesome bits, or saidthe book was too short.

The project also had some majorimpacts and extensions that wereunexpected, including a group of girlsfrom one school taking Germ Wars totheir Brownie pack. When DundeeScience Centre staff went out to theschool they presented a big poster madeby the pack about MillenniumDevelopment Goal 6, detailing all theinformation they’d found out aboutmalaria. Some children had worked athome with their parents to research anarea they were interested in, taking thelearning outside the classroom anddemonstrating the far reaching impactsthis three-month immersive project canhave.

Dundee Science Centre hasimmensely enjoyed the opportunity toengage 250 children with microbiology,and see the long-term impacts this

Katie BlackettDundee Science Centre

project has on not only pupilsunderstanding, but teacher’s knowledge,skills and confidence to teach thissubject throughout future years!

We hope you enjoyed the read, andwill leave you with some amazingcomments from both contributors andlearners.

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members

Sponsored Lecture GrantUCC Hosts Young Microbiologist Mini-Symposium: Microbe Signalling,Organization and Pathogenesis – 2009

which are often integrated with eachother and within global regulatorynetworks and subject to the prevailingenvironmental conditions as well as thepresence and activities of otherorganisms. QS signal molecules,although largely considered as effectorsof QS-dependent gene expression, arealso emerging as multifunctionalmolecules that influence life,development and death in single andmixed microbial populations and impactsignificantly the outcome of host-pathogen interactions.

Professor Williams went on tomention that in pathogenic bacteria, QSrepresents an exciting target for novelantibacterials: the inhibition of QS inbacteria such as Ps. aeruginosaattenuates virulence and rendersbiofilms more susceptible toantimicrobial agents and host defences.The development of potent, safe QSinhibitors offers considerable scope inthe battle against multi-antibiotic-resistant bacteria and chronic biofilm-centred infections.

The conference also had two otherkeynote lectures given by eminentscientists during the meeting. TheEMBO lecture given by Professor JeffErrington FRS (Newcastle University)was entitled “Life without a wall or adivision machine in B. subtilis:

implications for the origins of life” andthe ASM Lecture given by ProfessorTony Romeo (University of Florida) wasentitled “The Csr system: A globalregulatory circuit that governs bacterialbiofilm development”.

During the conference, seniordelegates adjudicated on the shortstudent talks and posters. Prizes forthese were sponsored by the Journals -Biochemical Journal, MolecularMicrobiology and Nature ReviewsMicrobiology. Javier López Garrido(University of Seville) a PhD studentfrom Professor Josep Casadesús’laboratory was awarded the 1st PosterPrize: “Regulation of Salmonellapathogenicity island 1 by DNA adeninemethylation: Evidence forposttranscriptional control by theRcsBCD signalling system”. NataliaTschowri (Freie Universität Berlin) aPhD student from Professor RegineHengge’s laboratory was awarded 1stPrize Young Scientist Award for her talkentitled: “The BLUF-EAL protein YcgFacts as a direct anti-repressor in a bluelight response of Escherichia coli.”

The University College Cork hostedthe first Young Microbiologist Mini-Symposium on Microbe Signalling,

Organization and Pathogenesis (21 to 22April 2009). The Conference took placein Brookfield Health Sciences ComplexUCC. This two-day internationalconference was intended to bringtogether graduate students, postdoctoraland young principal investigatorresearchers from leading laboratoriesaround the world to discuss theircurrent research. Over 110 scientistsgathered for the conference whereyoung microbiologists shared theirresearch findings. This event wasorganized by Dr Robert Ryan (UniversityCollege Cork) and Dr Sarah Coulthurst(University Of Dundee). The symposiumwas funded under the British Councilsprogramme for International Networkingfor Young Scientists.

There were four seminar sessions,covering the following highly topicalareas of prokaryotic molecularmicrobiology:

1) Gene regulation and intracellular signalling.

2) Structure, biogenesis and transport across membranes.

3) Microbe-microbe interactions.4) Host-microbe interactions —

pathogenesis and commensalism.Each of these sessions was chaired

by distinguished academics from aroundEurope but the main core of the talkswere given by young researchers forleading laboratories around the world.

As part of this conference the Societyfor Applied Microbiology awardedfunding to Professor Paul Williams fromthe University of Nottingham to speakon “Quorum sensing and Pseudomonasaeruginosa: a tale of regulatorynetworks and multifunctional signalmolecules”.

During his lecture Professor Williamsdescribed how bacteria employsophisticated cell-to-cell communicationor ‘quorum sensing’ (QS) systems forpromoting collective behaviours thatdepend on the actions of one or morechemically distinct diffusible signalmolecules. He went on to describe thatmany bacteria have multiple QS systems

Robert RyanUniversity College Cork

Professor Jeff Errington and a delegate

Picture by kind courtesy of Ms Phrueksa Lawongsa, BIOMERIT Research Centre, University College Cork

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am I eligible — can I apply?Yes — if you are FULL Member who can offer an undergraduatemicrobiology student the chance to obtain work experience. If youwould like to read about the experiences of students who havebenefited from this grant, you can do so below.

For further information visit: http://www.sfam.org.uk/en/grants--awards/students-into-work-grant.cfm

Students into Work Grant reports

Identification of Bacillus spp. causing rope formation in wheaten breadPrior to entering my final year of

study for a BSc degree in Food Quality,Safety and Nutrition at Queen’sUniversity Belfast, I was given theopportunity to work in a ContainmentLevel 2 Pathogen Laboratory within theInstitute of Agri-Food and Land Use. Myinterest in food microbiology came aboutafter completing a module in this areaduring my degree course, and as theopportunity arose to undertake a 10week summer placement in foodmicrobiology research I was very keen toget started.

I had just completed a year-longindustrial placement in a local bakerycompany and during my time thereproblems were occurring concerningrope in wheaten bread. Rope is acommon quality issue in bread productsand has been known about for manyyears. Rope is visible as clear, thin,thread-like strands and is caused bygrowth of Bacillus spp. It isaccompanied by unpleasant odours,followed by enzymatic degradation of thecrumb causing it to become soft andsticky due to the production ofextracellular polysaccharides. Threedifferent shaped wheaten loaves weretested to see if there was a differencebetween: the bacterial species present ineach loaf, pH changes during shelf lifeand if their shape had an impact on thebake core temperature of the loaves, andhence on Bacillus spp. remaining viablein the wheaten bread. My main objectiveduring the summer placement was todetermine the species of Bacilluscausing rope in the three different typesof wheaten bread. The wheaten breadsamples were tested on the day ofproduction and then four days later tocompare which Bacillus spp. werepresent immediately after baking and

which dominated at the end of theproducts shelf life. Data generatedduring the summer placement projectwould complete my final year honoursproject when I started back at university.

A series of experiments werecompleted which gave me the experienceof working with several new methodsand technologies. Around 120 bacterialisolates were obtained from the wheatenbreads sampled. These were initiallygrouped according to: colony size andappearance, Gram reaction, presence ofspores, and oxidase, catalase andamylase test results. This resulted in 38different bacterial ‘types’ and DNA wasextracted from a representative of eachof these types using a freeze-boil methodand DNA concentration was measuredusing a biophotometer. A presumptiveidentification of Bacillus spp. was thenconfirmed using Bacillus-specificprimers and PCR. The API® 50 CHsystem was used to identify some of theisolates on the basis of results of 50biochemical tests studying carbohydratemetabolism.

Speciation of the Bacillus isolateswas then attempted by RAPD, which is

another type of PCR technique thatamplifies random DNA sequences whichresult in a banded profile for eachisolate. The isolates were also subjectedto 16S PCR and denaturing gradient gelelectrophoresis (16S PCR-DGGE) usingprimers EC1055+GC clamp andEC1392. The 16S PCR-DGGE methodallowed the separation of PCR productson a 35 to 70% urea gradient gel whichgenerally yielded a single band perisolate. UVP gel analysis software wasused to help group the wheaten breadisolates with similar 16S PCR-DGGE orRAPD profiles, in comparison to profilesof reference Bacillus strains.Unfortunately, RAPD-PCR using primerOPA3 was of little help in identifying themajority of isolates. 16S PCR-DGGEresulted in fewer different profiles andgreater correlation with referenceBacillus profiles. Instead of there beinga dominant Bacillus spp. present whenrope occurred in wheaten bread,between four and six different Bacillusspp. were present, including B.licheniformis, B. pumilus and B.subtilis.

Aside from working on the Bacillus

Figure 1. RAPD-PCR profiles of Bacillus isolates obtained at the end of product shelflife from different shaped wheaten breads: lanes 1-7 rectangular, lanes 8-16 squareand lanes 12-21 oval

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Elaine EmoQueen’s University Belfast

project, I was also given the chance toparticipate in other experiments carriedout in the lab. This included workingwith MAP (Mycobacterium aviumsubsp. paratuberculosis) cultures. Igained an insight into methods such asphage amplification assay, phage titre,immunomagnetic separation (manualand the automated Bead Retrievermethod), and the ELISA technique. Ienjoyed learning about these methods asI had never heard of them before. It wasdifficult at the start getting to grips withthese procedures but with practice Ihave mastered these new skills.

This experience in a foodmicrobiology research laboratory hasgiven me the opportunity to work closelywith PCR and other techniques that Iwould not have gained experience ofduring my degree course due to theircomplexity. This insight into foodmicrobiology research has made medetermined to pursue a career in thisarea and I hope to undertake a PhD aftergraduation next year. I am very gratefulfor having been given this opportunity towork in this area of research and wouldlike thank SfAM for a Students into WorkGrant. I would also like to express myappreciation to my supervisor, Dr IreneGrant, for her time in teaching me newskills and for all her help and supportthroughout.

Surveillance of humanviruses by wastewatersampling

Following my honours yearstudying Infectious Diseases at TheUniversity of Edinburgh, I was providedwith the opportunity to spend thesummer researching the incidence ofviruses in wastewater and I wasdelighted to have a SfAM Students intoWork Grant to make this possible.

There is a long history of wastewaterbeing used to screen for humanpathogenic viruses, most especially forthe monitoring of poliovirus. My projectwas designed to develop a methodologysuitable for screening wastewater whichwould enable the identification andgenetic characterization of a range ofknown and recently discovered entericviruses that may be circulating withinthe community.

Four positive sense RNA viruses werechosen for screening, all of which aremembers of one of the major families ofenteric viruses, the Picornaviridae.Human enteroviruses (HEVs) andhuman parechoviruses (HPeVs) arefrequently associated with asymptomaticinfection but can cause seriousconditions such as aseptic meningitis,respiratory and enteric disease and, informer times before universal poliovirusimmunization, poliomyelitis. We alsoscreened for human cardiovirus (HuCV)and human cosavirus (HCoSV), bothrecently discovered viruses with largelyunknown epidemiologies and infectionfrequencies in the UK or elsewhere.

The Enterovirus genus is comprisedof four different human species (A-D)and has recently been expanded toinclude the rhinoviruses (A-C). Manywell-known and well characterizedviruses belong to this genus, forexample polioviruses, coxsackievirusesand echoviruses.

Parechoviruses were originallyclassified as enteroviruses, but were re-classified after sequence analysisshowed them to represent a distinctgenus within the picornavirus family.There are currently 14 genotypesrecognized through the sequencedivergence they show in the capsidregion of their genome. Type 1 wasoriginally recognized through anenterovirus screening programme in the1960s and has been found throughoutthe world. Other genotypes that arefrequently detected in Western countriesare types 3 and 6, the former associatedwith neonatal sepsis and severe CNSdisease; nothing is known about thecirculation and clinical impact of type 6infections.

Identification of serotypes of HEVand HPeV is regularly undertaken inhospitals and permits epidemiologicalsurveillance, monitoring of theemergence of more pathogenic variantsand investigating infection sources.However, it is not known whether or notthe genetic variants infecting individualsasymptomatically in the communitycorrespond with those found indiagnostic samples from symptomaticpatients.

The Cardiovirus genus consists oftwo species, encephalomyocarditis virus(EMCV) and theiloviruses. EMCVconsists of a single serotype whereasthere are four theiloviruses, includingthe recently discovered HuCV. The even

more recently discovered cosavirusesare a highly heterogeneous group ofviruses classified into a new genuswithin the picornaviruses, currentlyhaving five highly divergent species (A-E) recognized. It was originallyidentified in 2008 from the stools ofPakistani children with acute flaccidparalysis, discovered through apoliovirus screening programme,although its possible link with severeneurological or other disease has yet tobe clearly established.

Samples were collected from theVeolia Wastewater Treatment works justoutside Edinburgh city centre, whichtreats sewage from the Midlothian areaalong with run-off from roads. Samplingof solid waste was performed weekly for10 consecutive weeks. In the lab,phosphate buffered saline (PBS) wasadded to the samples and vortex mixed.The liquid portion was filtered threetimes through progressively smallerpores to remove large particles andconcentrate any viruses present. RNAwas extracted from the filtrate, the RNAwas reverse transcribed and the cDNAscreened for the four viruses of interestusing a sensitive nested amplificationprocedure with specific primers. Positivesamples were sequenced, aligned withrelevant sequences from Genbank usingthe Simmonics computer programmeand phylogenetic trees wereconstructed. Twelve separate reversetranscriptions and PCRs were performedon each RNA sample. Not all of thePCRs were positive and analysis of thesequences indicated that a singlesequence was present in each case, thuscircumventing the need for cloning.

This methodology proved quitesuccessful in detecting the viruses ofinterest with positive samples beingfound every week, most of which weresufficiently divergent from each other toindicate that they were from differentinfected individuals in the community.

HEVs were screened using primerswhich amplify the VP4 genome region ofthe viral capsid. This region can be usedto assign isolates into different speciesand serotypes. Over the course of thestudy, 39 different positive samples werefound and phylogenetic analysis showedthey were comprised of species B and Centeroviruses. Amplicons werepredominantly identified as species C(35/39) and clustered most closely withcoxsackievirus A1 (26/35), A19 (6/35)and A22 (3/35). The three species B

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Joseph CalvertViral Evolution Group, Centre of InfectiousDiseases, The University of Edinburgh

that they represent new genotypes ofSAFV. In addition to HuCV, we identifiedthree sequences closely related toanother species of Cardiovirus, Theravirus, which has, to date, only beenfound in rats and mice. This highlights apotential drawback of this type ofcommunity surveillance in that virusesshed by rodents and other animals mayalso be detected in the tested samples.

A region of the 5’ untranslated regionwas amplified for HCoSV and 36sequences were found. The cosavirussequences from the current studyallowed the variants to be identified asspecies A (20/36) and D (16/36).However this genome region can only beused for species identification, notserotyping which requires sequencesfrom the VP1 region to be amplified.

This summer project has enabled thedevelopment of a methodology forcollecting wastewater and determiningthe types of picornaviruses within it, aswell as increasing the number ofsequences of each virus type. The high

isolates were most closely related toechoviruses 6 or 11. The VP4 primersalso amplify rhinoviruses and a singlerhinovirus species C sequence wasidentified.

The presence of HPeV wasdetermined using primers which amplifythe VP3/1 region, enabling genotypeidentification. In total, 35 HPeVsequences were found, all of which werefrom genotypes 1 or 6.

For HuCV a region of the conservedpolymerase was amplified. This cannotbe used to assign isolates into differentgenotypes as picornaviruses frequentlyundergo recombination in this genomeregion. However, analysis stronglysuggested the sequences belongedpredominantly to Saffold Virus (SAFV)type 1. In addition, two distinct clusterswere identified, one of which is 5%divergent from SAFV-1 (5/25) and theother of which is 8% divergent fromSAFV-2 (8/25). This level of divergenceindicates that these sequences belong toa new polymerase clade and it is likely

Archaea represent one of thethree primary domains of life. Althoughmany archaeal species resemble bacteriawhen viewed using a microscope, theyare evolutionarily distinct and areactually more closely related toeukaryotes. Archaea were considered tobe exclusively ‘extremophiles’, restrictedto environments extreme in temperature,salinity or anoxia. However, since theearly 1990s, molecular methodologieshave revolutionized our understanding ofthese organisms and they are nowrecognized as a significant proportion ofmicrobes in most, if not all, ‘non-extreme’ environments.

The archaeal domain has beentraditionally divided into two kingdoms:the Euryarchaeota and the

Crenarchaeota (although otherkingdoms have been proposed such asthe thermophilic Korarchaeota).Although once considered to beexclusively sulfur-dependent (hyper)thermophiles, a distinct but relatedlineage (‘Group 1’ Crenarchaea ) is nowrecognized as one of the most abundant(if not the most abundant) kingdom ofprokaryotes in the biosphere, where theymay constitute around 6% of allmicrobial biomass in aquatic andterrestrial environments. Despite theirubiquity however, the ecological functionof these organisms remained elusiveuntil very recently.

A number of ground-breaking studiesindicated that these organisms have thepotential ability to oxidize ammonia (the

detection frequency of bothcardioviruses and cosaviruses wasunexpected and the first evidence forthe extensive circulation of these newlydiscovered viruses in the UK. Finally, thesurveillance data will be of considerablelong-term value in providing theopportunity to compare and contrastvariants causing disease with those thatare present in the community. From apersonal perspective, I have gainedvaluable experience within the lab whichI will be able to draw upon as I begin aresearch Masters in MedicalMicrobiology studying broader aspectsof public health in the same department.I wish to thank Dr Carol McWilliamLeitch for the opportunity to studywastewater, Professor Simmonds for theopportunity to work in the ViralEvolution Group and SfAM for a grantenabling me to have this opportunity.

am I eligible — can I apply?It is not only our Student Members who require our help. Seniormicrobiologists often find difficulty in funding attendance atmeetings. If you are in this position you are eligible for this fund.

For further information visit: http://www.sfam.org.uk/en/grants--awards/presidents-fund.cfm

President’s Fund reports

The effect of soil pH on crenarchaeal communitiesfirst step in nitrification) and thereforemay have a major role in the globalnitrogen cycle. Metagenomic analyses insoil and the oceans found genesencoding putative ammoniamonooxygenase subunit homologueswithin crenarchaeal genome fragments.Archaeal ammonia oxidation activity wassubsequently confirmed by thecharacterization of an ammonia oxidizingcrenarchaeon, Nitrosopumilusmaritimus, isolated from a marineaquarium. This organism growschemolithoautotrophically, usingammonia and inorganic carbon as thesole energy and carbon source,respectively. These characteristicsindicate that these organisms are similarto ammonia-oxidizing bacteria such as

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Nitrosomonas europaea. Intriguinglyhowever, quantitative studies have shownthat these ‘ammonia-oxidizing archaea’are numerically dominant over theirbacterial counterparts in mostenvironments studied to date. It istherefore possible that archaea, and notbacteria as previously assumed, are thecentral players in ammonia oxidation inthe environment.

In most soil systems, crenarchaealcommunities are dominated by onespecific evolutionary lineage, theubiquitous Group 1.1b lineage. This isone of the evolutionary lineages that hasbeen implicated as capable of ammoniaoxidation. A number of molecular studieshave indicated that this ubiquitous groupis conspicuously absent from, or presentin relatively low numbers, in some acidicsoil environments, including coniferousforest soils. In these habitats,crenarchaeal communities can bedominated by organisms with 16S rRNAgene sequences placed within the relatedbut distinct Group 1.1c. Unlike the Group1.1b, the ecological role of this group isunknown with no evidence to suggest thatit is involved in nitrogen cycling.

Previous studies have indicated thatsoil pH may be a major factor indetermining microbial communitycomposition and experiments have beenperformed to investigate whether theabundance and diversity of lineage 1.1ccrenarchaea are also pH-dependent. Todetermine the impact of soil pH, I havebeen examining crenarchaealcommunities across an establishedgradient ranging from pH 4.5 to 7.5. Theabundance of the different crenarchaealpopulations was assessed by quantitativePCR (qPCR) targeting the 16S rRNAgene, revealing that soil pH has no effecton the overall numbers of crenarchaeaor bacteria. In contrast, the 16S rRNAgene abundance of crenarchaeal 1.1corganisms declined with increasing pH.These observations strongly support thehypothesis that pH is a major driver of1.1c crenarchaeal abundance. Theresults are intriguing and raise a numberof questions regarding the physiologyand ecological function of theseorganisms. For example, the presence ofthe ammonia monooxygenase genes hasnot been verified in Group 1.1ccrenarchaea. Also, as nitrification istypically restricted at low pH due to lowavailability of ammonia, it is possiblethat these organisms do not oxidizeammonia to generate energy, as this

Laura Lehtovirta University of Aberdeen, UK

Illustrating theimportance of goodhygiene practice

A report from the CDC indicatesthat little progress has been made incontrolling foodborne illness in the USAsince 2004 and that enhanced measuresare required to educate consumers aboutinfection risks and prevention measures.In an effort to better understandconsumer kitchen sanitation and foodhygiene practices, we comparedconsumer questionnaire responses withbehaviours observed on video, in adescriptive study of 30 households. Theresults highlighted the differencesbetween individuals’ reported beliefs andactual practice. For example, whilst100% of the participants reported thathandwashing after handling ground beefis an important practice, 30% of themwere not compliant in practice. Of thosewho did wash their hands, 40% washedfor 10 seconds or less, although 70% ofall participants reported 20 seconds aseffective. When asked, 67% ofparticipants thought it was important touse a meat thermometer but only oneparticipant actually used a thermometerwhen cooking a hamburger. While 90%of participants were aware of the linkbetween E. coli and ground beef, 70%had never heard of Campylobacter.When asked about risky behaviours, 90%attached little or no risk to preparingfood at home, while at the same time30% indicated that thawing frozen beef atroom temperature for 12 hours was onlymoderately risky. We believe that

information from this and other similarstudies can inform consumer food safetyeducation.

Another topic of great current concernis the emergence of MRSA. In a bacterialstudy of 32 hand and food contactsurfaces in 35 healthy homes, we isolatedMRSA from a variety of surfaces in 9 ofthese homes. We analysed for a numberof factors that might serve as predictorsof MRSA and a positive correlation wasfound between the isolation of MRSAfrom surfaces and the presence of cats inthe home. This provides further evidencefor the potential for infectiontransmission through inanimate surfacesin our homes.

In 2007, two of our students werefortunate enough to benefit fromPresident’s Fund student travel awards,allowing them to attend the annual SfAMconference in Cardiff and present theirresearch posters on the bacterial load inpre-packaged, ready-to-eat spinach(Asjeric & Scott, 2007; Bruen & Scott,2007). The following year, a new projectwe worked on was an assessment ofdifferent patterns of consumer use andthe impact of the effectiveness of the lowtechnology, slow-sand water filters thatSimmons students are building andinstalling in a rural community inNicaragua. To date (2008) we haveconstructed a slow-sand filter in ourlaboratory and we are in the early stagesof priming it with local river water toencourage the formation of a biofilmlayer of Schmutzedecke, which providesa biological treatment layer, supported byunderlayers of sand and gravel. Wellmanaged slow-sand filter systems arecapable of reducing the bacterialcontamination in water by 99%. We willbe testing for faecal coliforms in the riverwater, the filtered river water and thefiltered river water after storage underdifferent patterns of usage. For example,we will compare the water quality whenthe filter is used daily as opposed tointermittent use. In addition, we arepreparing a questionnaire survey tool toestablish patterns of use of these filtersystems in the community in Nicaragua.For this reason, I was very grateful to theSfAM President’s Fund for giving me theopportunity to attend the 2008 annualconference in Belfast, on the topic of‘Water in Work, Rest and Play’.

Elizabeth Scott Simmons College Center for Hygiene andHealth in Home and Community

would require adaptation by lineage 1.1cto very low concentrations of ammonia.My current research is attempting tounderstand the ecological function ofGroup 1.1c crenarchaea, and whetherthey are involved in nitrification or otherimportant ecological functions.

I am grateful to SfAM for awardingme this grant from the President’s Fund,which allowed me to attend the 12thInternational Symposium of MicrobialEcology in Cairns, Australia in August2008. This has enabled me tocommunicate my research findings tothe science community as well as learnfrom other leading research groups inmy field.

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Soil autotrophic ammonia oxidizingbacteria (AOB) are a monophyletic groupwithin the β-subgroup of the divisionProteobacteria whose main ecologicalfunction is the oxidation of ammonia tonitrite: an important, rate-limiting stagein the nitrogen cycle. Nitrogen isfundamental to all life on earth as amajor constituent of protein and nucleicacid and is assimilated and excreted indifferent forms by different organisms.Successful crop production is dependenton the presence of sufficient bioavailablenitrogen in soil and the activities of soilmicroorganisms largely dictate this.Ammonia oxidizing crenarchaea alsocontribute to the oxidation of ammoniato nitrite, which is further oxidized bynitrite oxidizers to nitrate, a combinedprocess referred to as nitrification.Excessive rates of nitrification to themore soluble nitrate form lead toleaching of nitrogen from soils and adecrease in agricultural productivity. Agood understanding of the rate-limitingammonia oxidation step is therefore animportant requirement for agriculture,particularly in terms of nitrogenfertilizers addition and conversion.

During the last few decades advancesin molecular technology and developmentof new techniques have revolutionizedmany aspects of the life sciences.Application of community profiling toolsin microbial ecology has revealed hugediversities of previously unseenorganisms responsible for importantprocesses including nitrogen cycling(Morgan, 1991). Techniques include PCRamplification of community DNAbiomarkers with subsequent resolution byDGGE or terminal restriction fragmentlength polymorphism analysis (T-RFLP).Studies utilizing such techniques haveshown evidence of diverse AOBcommunities in soils exhibiting changesin structure in response to pH and otherconditions (Stephen et al., 1998). Therecent emphasis in microbial ecology hasbeen on molecular characterization andobservation of natural communities but,despite the knowledge of high moleculardiversities, it is difficult to resolve thephysiological and functional diversitywithin a community. Without thisknowledge it is difficult to predict how aparticular community will respond tochanges caused by land managementregimes. AOB strains isolated in pureculture provide an opportunity to

characterize this physiological diversitytowards a better understanding of theecological dynamics in soil communitiesand the significance of their diversity.

The majority of reports in theliterature have characterizedphysiological aspects of particularstrains. The aim of my work is toquantitatively characterize variousecologically relevant physiological traitsof six soil AOB, two Nitrosomonas andfour Nitrosospira strains, previouslyisolated to pure culture, and to assesstheir diversity. Physiological responsesrelevant to soil ecology include: ammoniaoxidation rate at different ammoniaconcentrations, tolerance of high levelsof ammonia and the ability to regainammonia oxidation activity quickly afterperiods of starvation. Many enzyme-mediated processes follow the same typeof saturation kinetics whereby theincrease in reaction rate responds less asthe substrate concentration increasesuntil the rate approaches a theoreticalmaximum, at which point saturation hasbeen reached. This has been modelled byMichaelis and Menten (1913) amongstothers and can be applied to specificenzymes in whole cells whose substratesare acquired externally providing twouseful parameters with ecologicalimplications. The first is the Michaelisconstant (KM) corresponding to thesubstrate affinity of an enzyme orpopulation and the second is themaximum reaction velocity (VMAX) perenzyme or unit biomass. Under mostnitrogen based fertilizer regimes thedominant members of the communitywould be expected to have a high VMAX

whilst fallow or pristine land may bedominated by organisms with low KM, anadaptation to low ammoniaconcentrations.

An organism may gain certainadaptations in evolving to fill anecological niche. However, in becomingwell suited to one niche it may becomeless suited to others, because of physicalor chemical constraints (Bohannan et al.,2002). This concept could help explainhow members of diverse communities,such as AOB communities, can co-exist.With respect to ammonia concentration,a hypothetical trade-off could beadaptation to either high or low ammoniaconcentrations because of physiologicalconstraints. Two ecological strategieswould result: low KM and VMAX equivalent

David Williams University of Aberdeen, UK

to an ‘autochthonous’ lifestyle or a highKM and VMAX equivalent to a‘zymogenous’ lifestyle. If certain groups,identified by molecular techniques to bepresent within a soil community, areassociated with particular physiologicaltraits, predictions about the overallcommunity function and potentialresponse to land use could be made.Such predictions could help adaptation ofland use strategies for mitigation of soilnitrogen loss through nitrification. In asoil dominated by AOB with a high VMAX

and rapid recovery following starvation, afertilizer regime in which large amountsof ammonia quickly enter the soil may beliable to loss by leaching through rapidnitrification. A slow nitrogen-releasefertilizer may therefore be preferable.

In this study some significantdifferences were found between ammoniaoxidation recovery potential followingstarvation and KM and VMAX values of thesix AOB strains. Molecular communityanalyses have shown close relatives ofthese strains to be dominant underdifferent conditions (Webster et al.,2005). These observations suggest thephysiological traits characterized haveecological relevance.

I would like to thank the SfAM forproviding the President’s Fund Grantwhich contributed towards mypresentation of this work at theInternational Symposium for MicrobialEcology, 2008.

References■ Bohannan, B.J.M., Kerr, B., Jessup, C.M.,Hughes, J.B., and Sandvik, G. (2002). Trade-offsand coexistence in microbial microcosms.Antonie van Leeuwenhoek, Vol. 81, pp107-115.

■ Michaelis, L., and Menten, M. (1913). Diekinetik der invertinwirkung. BiochemischeZeitschrift, Vol. 49, pp333-369.

■ Morgan, J.A. (1991). Molecular biology: newtools for studying microbial ecology. Sci. Prog.,Vol .75, pp265-277.

■ Stephen, J.R., Kowalchuk, G.A., Bruns, M.-A.V., McCaig, A.E., Phillips, C.J., Embley, T.M.,and Prosser, J.I. (1998). Analysis of β-subgroupproteobacterial ammonia oxidizer populations insoil by denaturing gradient gel electrophoresisanalysis and hierarchical phylogenetic probing.Appl. Environ. Microbiol., Vol. 64, pp2958-2965.

■ Webster, G., Embley, T.M., Freitag, T.E., Smith,Z., and Prosser, J.I. (2005). Links betweenammonia oxidizer species composition, functionaldiversity and nitrification kinetics in grasslandsoils. Environ. Microbiol., Vol. 7, pp676-684.

Physiological and functional diversity among ammonia oxidizing bacteria

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Prepared Media products

SAS portable and installed remote controlled microbial air samplers DataTrace wireless, intrinsically safe miniature data loggersRaven Biological Indicators

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For more information about advertising your productsor services in Microbiologist please contact theSociety Office on 01234 326846 or visit the websiteat www.sfam.org.uk

51

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Serious Technologyf o r S e r i o u s S c i e n c e .

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New medium for fasterscreening of CRE, includingNDM-1

Oxoid Brilliance™ CRE Agar, is a new mediumfor faster and easier detection of carbapenem-resistant Enterobacteriaceae (CRE). It is recognizedthat the only way to stop the spread of infectionsby resistant bacteria, such as CRE, is to rapidlyidentify and isolate any hospital patients who areinfected with the resistant strain. Laboratories canuse Brilliance CRE Agar to screen patient samplesfor CRE and provide clinicians with results in just18-24 hours. This faster time to result acceleratesthe start of infection control measures, includingaccurate antibiotic prescription, for better patientoutcomes.

Brilliance CRE Agar is easier to use and readthan the Hodge test traditionally used to detectresistance to carbapenems. Brilliance CRE Agarcontains a modified carbapenem, at levelsrecommended by both the CLSI and EUCAST,ensuring reliable detection of numerous CREstrains, including those with the NDM-1mechanism of resistance. Chromogeniccompounds in the medium differentiate betweenresistant E. coli (pink colonies) and Klebsiella,Enterobacter, Serratia or Citrobacter (KESC)organisms (blue colonies). The colonies of otherresistant organisms appear white and are easy tospot against the novel pigmented agar.

further information

Visit: www.oxoid.comTel: +44 (0) 1256 841144Email: val.stroud@thermofisher.com

corporatenewsThe latest news, views and microbiologicaldevelopments from our Corporate Members

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Tel: +44 (0) 1224 711100Fax: +44 (0) 1224 711299

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New Autoclave Indicator Bags

For added assurance Sterilin now offer a high temperature bag with an autoclave indicator.

• The word ‘AUTOCLAVED’ appears above ‘Hazardous Waste’ following a routine autoclave cycle

• Strong disposal bag with blue biohazard printing - ideal for use in all laboratories for hazardous waste disposal

• For use at high temperatures up to 135ºC - for the inactivation of particularly resistant waste

• Convenient ‘tissue-box’ cartons - for easy bag dispense

• No need to affix autoclave tape - no training required for new starters in the laboratory

Tel: +44 (0) 844 844 3737 Fax: +44 (0) 1495 242 242e-mail: info@sterilin.co.uk www.sterilin.co.uk

Sterilin is part of

Thermo Fisher Scientific

55

From nuts and spice to foodon ice!

Lab M’s fungal media range suits wide varietyof foodstuffs. Lab M’s specialist range of culturemedia for the isolation of yeasts and moulds istailored to ensuring the recovery of theseorganisms from a broad range of foodstuffs,providing the convenience of purchasing from asingle supplier.

The most recent inclusions in Lab M’s range ofmore than 20 yeast and mould products areDichloran Rose Bengal Chloramphenicol Agar (ISO)and Dichloran 18% Glycerol Agar (ISO). Bothcomply with the requirements of the relevant ISOstandards and both products are supplieddehydrated as complete formulations. These areready to prepare without the need for anyadditional antibiotic supplementation.

DRBC Agar(ISO) is used to enumerate viableyeasts and moulds in foodstuffs whose wateractivity is greater than 0.95, while DG18 Agar(ISO) is intended for food or animal feed productsthat have a water activity of 0.95 or less. Eachmedium has been carefully designed to balancethe need to encourage growth of the selectedorganisms with the requirement to control growthenough to allow efficient colony counting. Fordetails of Lab M’s complete range of yeast andmould media please contact us.

further information

Visit: www.labm.comTel: +44 (0)161 797 5729Email: info@labm.com

Comprehensive veterinaryvaccines productdevelopment service

VLA Scientific offers a range of services forlivestock pharmaceutical and vaccine research anddevelopment. Our services extend from datageneration during initial research through to batchtesting on finished products.

We have a wealth of experience of in vitro andin vivo techniques with expertise in infectiousdisease modelling, efficacy and safety testing.Benefits include access to comprehensive librariesof bacterial, viral and parasitic organisms and ourextensive facilities enable us to work withorganisms requiring containment ACDP level 3and SAPO category 4.

In addition to these services, our clients haveaccess to world leading livestock disease experts inpathology, epidemiology, microbiology,parasitology and virology who can advise onrecent research findings as well as modeldevelopment and study design.

Creating anaerobicconditions...quicklyAND cheaply

The Whitley Jar Gassing Systemcreates perfect conditions for growinganaerobes in just two minutes for lessthan 18 pence and microaerophiles in15 seconds for less than three pence(compared with about £1.60 for everygas generating kit used).

This latest iteration of the WhitleyJar Gassing Systems enables you to:

■ reduce the cost of creating microaerobic conditions by 98% and anaerobic conditions by 89%;

■ use the colour, touch screen control panel to monitor conditions in real time;

■ obtain graphic evidence that your required conditions have been met;

■ print a hard copy audit trail for accreditation purposes;

■ train operators quickly and easily — it is very simple to use;

■ incorporate PIN code protected user access levels for additional security;

■ use your existing anaerobic jars by ordering the adaptor kit;

■ take out a fully comprehensive maintenance and breakdown cover to prolong the life of your investment.

If your laboratory cannot justify the purchase ofan anaerobic workstation then consider theWhitley Jar Gassing System.

further information

Visit: www.dwscientific.co.ukTel: + 44 (0)1274 595728Email: sales@dwscientific.co.uk

Our vaccine development services include: ■ Primary research■ Antigen development■ Antibody and antigen detection ■ Testing for attenuation■ Extraneous agents testing■ Efficacy, safety and bioequivalence testingWhere necessary, our services are performed in

accordance with Good Laboratory Practice, GoodManufacturing Practice and Good Clinical Practice(veterinary) with a minimum site standard for allactivities of ISO 9001 and ISO17025 for most ofthe testing work.

further information

Visit: www. vlascientific.comTel: +44 (0)1932 357641Email: salesdesk@ahvla.gsi.gov.uk

56

commercial

Cherwell Laboratoriescelebrates 40 years inbusiness

Cherwell Laboratories was founded in 1971 byLawrence Whittard B.V.Sc. as a veterinarydiagnostic laboratory, and over 40 years hasevolved into a manufacturing and distributioncompany offering a fully integrated range ofproducts for environmental monitoring and thevalidation of sterilisation processes.

At the heart of the business is a modern cleanroom manufacturing suite with fully automatedplate filling for Redipor prepared media, most ofwhich is available gamma irradiated. CherwellLaboratories also offers the SAS range ofmicrobiological air samplers and the DataTracerange of miniature, wireless data loggers.

Cherwell Laboratories has recently taken overthe UK distributorship for the Raven Laboratoriesand Apex range of biological indicators, chemicalintegrators and process indicators for industrialand healthcare applications. The comprehensiverange is ideal for developing, monitoring andvalidating a range of sterilisation processes andincludes spore strips and suspensions for use withconventional culture media, self-contained andindustrial use biological indicators.

After 40 years of growth and adaptation weare proud to remain family owned with loyalemployees dedicated to supplying high qualityproducts with excellent customer service.

further information

Visit: www.cherwell-labs.co.ukTel: +44 (0)1869 355500Email: sales@cherwell-labs.co.uk

TCS Biosciences

Here at TCS Biosciences Ltd, we have over 40years experience in supplying the needs ofmicrobiologists worldwide. As Europe’s leadingsupplier of donor animal blood and sera forinclusion in plated media, we have built areputation for quality, versatility and outstandingcustomer service.

Selectrol® discs are first generation micro-organisms that are manufactured under licencefrom the Health Protection Agency CultureCollections (HPACC). Selectrol® strains are fullytraceable and guaranteed to be first generationderivatives of the original NCTC or NCPF strain.Presented as a water soluble freeze-dried disc,Selectrol® is versatile in its application for use witheither plated or liquid media.

Our in-house Selectrol® quality control testinglaboratory is UKAS accredited and our growingrange encompasses nearly 70 strains, many of

which have been added as a direct result ofcustomer requests.

As Selectrol® organisms are guaranteed to befirst generation microorganisms, they are ideal foruse in accredited laboratories. Selectrol® batchesare tested for a range of identification andcharacterization attributes and certificates ofanalysis for each batch can be accessed via ourwebsite: www.tcsbiosciences.co.uk.

further information

Visit: www.tcsbiosciences.co.ukTel: +44 (0)1296 714222Email: sales@tcsgroup.co.uk

Microbiologics QCmicroorganism products

Microbiologics offers the most diversified line ofcost-effective, reliable, and convenient QCmicroorganism products in the market! All of ourQC microorganism strains are traceable to theoriginal culture collection because we are alicensed manufacturer. We offer a variety ofqualitative and quantitative microorganismpreparations designed specifically for thepharmaceutical, cosmetic, food and environmentalindustries.

Products such as EZ-Accu Shot™ and EZ-AccuShot™ Select for Growth Promotion Testing andEZ-PEC™ for Preservative Efficacy andAntimicrobial Effectiveness Testing are designed forpharmaceutical testing laboratories. Food testinglaboratories use EZ-FPC™, EZ-SPORE™ andEpower™ microorganism preparations for dailyprocess controls, method validation andverification, and proficiency testing. QualitativeKWIK-STIK™ and LYFO DISK®, and quantitativeEpower™ microorganisms are commonly used forQC testing throughout the water andenvironmental industry.

Microbiologics is FDA registered and conformsto CE Mark regulations. We have also received ISO9001 certification, as well as ISO 17025 and ISOGuide 34 accreditations. As a ISO Guide 34accredited manufacturer, Microbiologics providesCertified Reference Material microorganism strainsto meet ISO 17025 requirements. ChooseMicrobiologics for superior quality, traceability andunsurpassed Customer Service for less!

further information

Visit: www.microbiologics.comTel: 00+1 320 229 7083Email: info@microbiologics.com

57

New swab for faecalspecimens

Medical Wire’s new FecalTranswab® with liquidCary Blair medium has attracted considerableinterest from clinical microbiologists. Althoughdeveloped for use with automated systems, it is

UK based Neogen Europe Ltdoffer world class food safetytesting

Neogen Europe Ltd is dedicated to thedevelopment and marketing of novel diagnostictest kits that address topical concerns about thequality and safety of agricultural and foodproducts.

We offer quality test kits and reagents to detectpathogens, micro organisms, toxins, allergens,pesticide residues, contaminants and GMOs.Neogen also offers a range of sanitation productsto detect the amount of food residue and otherorganic matter, such as bacteria, yeast andmoulds, that remain after cleaning. Innovativeproducts allow rapid, on-site analysis providingreal-time data at low unit cost.

As a subsidiary of Neogen Corporation, weoffer customers worldwide even greater choiceand innovation to meet the growing challenges ofproviding food products that are both safe andnutritious.

further information

Visit: www.neogeneurope.comTel: +44 (0) 1292 525610Email: info_uk@neogeneurope.com

recognised that it could also have usefulapplications using conventional techniques.Validation studies have shown that is suitable for awide range of fecal pathogens, includingSalmonella, Shigella, Clostridium difficile, E. coliO157, Campylobacter, and also viruses such asRotavirus and Norovirus. Further identificationusing toxigenic and PCR techniques can beperformed readily and without interference.

The swab comes with a screw cap vial withliquid Cary Blair medium. The swab itself is amoulded stick with breakpoint and soft foam tip.It can be used to take the specimen directly fromthe rectal canal, or dipped in the patient’s stoolspecimen. The swab is then snapped into the vial,and when closed the swab is “captured” by thecap, and will be removed with the cap whenopened manually or mechanically. Cary Blairmedium was specifically designed for the recoveryof enteric bacteria, and has an alkaline pH toinhibit overgrowth. The liquid format allows easyand direct aliquotting onto plates or broths forfurther processing.

further information

Visit: www.mwe.co.ukTel: +44 (0) 1225 810361Email: sales@mwe.co.uk

Leatherhead Food Research

Food microbiology at Leatherhead provides abroad range of microbiological food analysescovering food spoilage and pathogenic micro-organisms for our customers.

We are able to determine the levels of micro-organisms in foods, predict their ability togrow/survive in your products and verify theirbehaviour through real-time analysis. Leatherheadcan evaluate and confirm the shelf life of yourproducts, and advise you on the necessary recipealterations, packaging and process conditions thatwill help you achieve a safe shelf life or even allowyou to extend your product’s shelf life.

Using our expertise in microbial recovery fromcomplex matrices, we are able to help you validateyour heat process and provide you with data onmicrobial susceptibility to the application of thatheat.

If you are looking to reduce the level of heatapplied to your products, make energy savingsand/or enhance the sensory characteristics of yourproducts, we are able to advise you on the use ofalternative preservation technologies that willensure the safety of your products.

Leatherhead’s Food Safety services aresupported by expertise in Food Innovation, Sensoryand Consumer Science, Nutrition, GlobalRegulatory Services and Market Intelligence.

further information

Visit: www.leatherheadfood.comTel: +44 (0)1372 822298Email: help@leatherheadfood.com

58

Are you a CorporateMember of the Society?If so, this section ofMicrobiologist is foryou. Here you canpublish short pressreleases, acquisitionnotices, news of newstaff appointments,technical developmentsand much more.

Each CorporateMember of the societymay publish up to 200words on a topicrelated to their field ofactivity in each issue ofMicrobiologist. Forfurther informationplease contact LucyHarper by email at:lucy@sfam.org.uk

Both CorporateMembers and OrdinaryMembers of the Societywill find a wealth ofuseful information andresources in thissection.

information

commercial

New primary collectiontubes for automatedurinalysis

Thermo Fisher Scientific has introduced new arange of Sterilin Primary Urine Tubes.

Automation has dramatically increased theaccuracy and throughput of urine analysis inlaboratories but standard urine collection vesselsare too large to fit into the racking of currenturinalysis systems, however, meaning samplesmust be decanted into suitable tubes in thelaboratory. This is not only time consuming, but italso presents opportunities for spillages and errors.

Sterilin Primary Urine Collection Tubes fitperfectly into the racking allowing faster, simplerand more efficient processing of samples. Theclear plastic tubes have a rounded base, which isessential for efficient mixing of samples in theanalyser, and are supplied with or without boricacid preservative, depending on the needs ofindividual laboratories.

Sterilin Primary Urine Tubes are easy to use, areavailable with a urine collection cup for easiersample collection and are 95kPa-compliant withleak-free screw caps for safe and securetransportation of samples. In addition, each tube isCE marked and labeled for accurate recording ofpatient details.

further information

Visit: www.sterilin.co.ukTel: +44 (0) 844 844 3737Email: info@sterilin.co.uk

Keep track of winter bugswith a cost effective andreliable surface samplingsponge kit

As winter approaches there’s always an increasein cold and flu outbreaks and it’s prudent tomonitor hygiene levels on food contact,environmental, pharmaceutical and clinicalsurfaces. Technical Service Consultants ready touse Sterile Sponge Sampling Kits are a simple,safe, cost-effective method for quantifying themicrobial load.

Many surfaces contain traces of disinfectantsand/or sanitisers, but it is also possible that theywill contain a small number of bacteria too. Leftalone, these cleaning fluid traces will not kill themicroorganisms, but will prevent any growth (thebacteriostatic effect). Detection therefore requiresa neutralising solution to be effective.

TSC’s Sponge Sampling Kits include neutralisingbuffer, providing optimal microorganism recoveryfor sampling and significant compliance

advantages over ‘do it yourself’ kits. Mostcommon disinfectants including peroxides,phenols, quaternary ammonium compounds(QAC’s), amphoterics, chlorines and associatedhalogens are neutralised, providing the end userwith total testing assurance.

The kits are available in a blue sterile screw capcontainer with glove for easy sampling or sealedwithin a medical grade bag with a sterile minigripbag in which to place the sample sponge.

further information

Visit: www.tscswabs.co.uk/Tel: +44 (0)1706 620600Email: sales@tscswabs.co.uk

Polyphasic identificationservices

The NCIMB microbial identification serviceprovides fast, confidential and accurateidentification of bacteria and certain commonfungi to Good Manufacturing Practice standards.

We isolate and identify bacteria from diversesources, having particular expertise in theenvironmental and pharmaceutical sectors. Recentexamples of material we have examined include:

Industrial and pharmaceutical contaminantsincluding factory or plant slimes, injection, processand cooling waters, clean room contaminants andisolates from personnel monitoring

Process contaminants from produce such assoup, butter, soft drinks and horticultural products.

Client specific investigations or projects arisingfrom diverse activities such as skin flora analysis,identification of fish pathogenic organisms inaquaria and confirmation of presence or absenceof key strains in live products such as yoghurts orfermented foods.

We service a wide range of blue chipcompanies both from the UK and overseas whoregularly inspect and audit our facility. Clients canhave a wide range of analytical needs.

NCIMB, as custodians of the Major UKrepository for environmental bacteria is ideallyplaced to provide an Identification Service withimmediate access to a unique source of authenticreference material for comparative purposes.

We apply three core techniques in ourPolyphasic approach to bacterial identifications:Genotypic identification, Phenotypiccharacterisation, and Strain to strain comparisonand differentiation.

further Information

Visit: www.ncimb.comTel: +44 (0)1224 711100Email: enquiries@ncimb.com

Accurately ID microorganisms in just 2 – 4 hours

RapID™ Manual Identification Systems

Save budget: Rapid results allow rapid decisions

Save time: One-step inoculation, aerobic incubation

Save resources: Web-based ERIC application for results interpretation

www.oxoid.comTel: 01256 841144 Fax: 01256 329728Email: oxoid.info@thermofisher.comPart of Thermo Fisher Scientific