Novel Strategies for the Delivery of Antimicrobials into Bacterial Cells

A Review

Alva Jay Smith B1009075 Supervisor: Jem Stach Word Count: 7520

110090753

1

Abstract

The rise of antibiotic resistance has prompted a tremendous amount of research into the identification

and description of antimicrobial compounds. Through in depth study of the literature describing these

breakthroughs, I have identified that whilst many new, important antimicrobials (and the

development of techniques to discover them) have been outlined, these and indeed existing

antimicrobials with proven methods of inhibition lack entry into the bacterial cell cytoplasm. The

cytoplasmic targets of a plethora of antimicrobials are well known but the bacterial cell wall of Gram-

negative bacteria is proving problematic to overcome for a variety of reasons. Delivery of

antimicrobials is the next step in tackling dangerous pathogens but finding efficient ‘vehicles’ to aid in

the transport of antimicrobials across the bacterial cell membrane is disproportionate to the number

of active compounds discovered thus far which cannot access their targets in the cytoplasm. This

review hopes to explain why antibiotic drugs are difficult to deliver to the resistant Gram-negative

bacterial cytoplasm and describe major advances in the delivery of antimicrobials such as membrane

active peptides, hybrid compounds and phage and pyocin therapy, all in great detail.

Introduction

Gram-negative bacteria are the primary cause of most deadly nosocomial and (ever increasing)

community acquired (CA) bacterial infections. Media attention focused on methicillin resistant

Staphylococcus aureus in recent years has brought resistant bacteria to the forefront of public

awareness but has not yet identified the most resistant and dangerous pathogens. Gram-negative

bacteria especially those of the Enterobacteriaceae family cause urinary tract infections (UTIs), blood

poisoning, healthcare associated pneumonias and various incurable intra-abdominal infections

originating from the gut. Gram-negative pathogens which are becoming increasingly resistant to

modern antibiotics and are clinically important, include Escherichia coli, Clostridium spp, Pseudomonas

spp. and Acinetobacter spp.

Many antibiotics and antibacterial compounds have potent intercellular action; they act by inhibiting

key processes: peptidoglycan synthesis, DNA replication, cell division and protein synthesis

(translation) (Barna and Williams 1984; Chopra and Roberts 2001; Waksman and Lechevalier 1962).

However, most of these compounds have difficulty crossing the bacterial cell wall, particularly in

Gram-negative bacteria. Therefore, in this review I will explore novel methods and strategies for the

delivery of effective antimicrobial compounds into the most resistant of bacteria, outlining current

strategies and suggesting antimicrobial therapies based on emerging technologies from recent

literature published in scientific journals and older studies which I believe to be vital in discovering

110090753

2

novel strategies for the delivery of antimicrobials into bacterial cells. Many of these techniques have

huge scope to be used in combination with active antimicrobials which lack entry to the bacterial

cytoplasm and should be taken into consideration when designing new anti-infective therapies.

Gram-negative bacteria have a plethora of antibiotic resistance mechanisms

In the wake of prolonged overuse of antimicrobials we are now becoming increasingly exposed to

environments which harbour lethal multi-drug resistant bacteria. In his review Resistance in Gram-

Negative Bacteria: Enterobacteriaceae (2006), Paterson describes antibiotic inhibition with emphasis

on chromosomally and plasmid mediated extended spectrum beta-lactamases (ESBLs) which inhibit

B-lactam antibiotics through hydrolysis – although beta-lactamase inhibitors can be used in

combination therapies with antibiotics, resistant varieties over produce beta-lactamases and may or

may not have mutated less porins for transport of antibiotics. AmpC beta-lactamases

(cephalosporinases) provide another important enzymatic defence mechanism (Jacoby 2009) and

coupled with organisms that reduce influx or enhance efflux (Webber and Piddock 2003), these

mechanisms present antibiotics with an armoury of defences. The top eight death causing

microorganisms, the antibiotics/antifungals which combat them and their various methods of

resistance can be found illustrated in figure 1.

The biggest obstacle facing the antimicrobial resistance research community is the outer membrane

(OM) of Gram-negative bacteria. This selective membrane utilises numerous beta-barrel structure

motif proteins and general diffusion porins which contain an inwardly folded extracellular loop, which

together with the opposite barrel wall, form the so called eyelet or constriction zone, determining the

size exclusion limit and other permeation properties of the barrel (Delcour 2009). These mechanisms

encompass a narrow size exclusion limit making it difficult for large peptide antimicrobials to

overcome the Gram-negative bacterial OM. It can be assumed that it is the OM rather than the

cytoplasmic membrane in Gram-negative bacteria that provides antibiotic exclusion as Lazzaroni and

Portalier’s study (1981) into E.coli cells with a leaky periplasmic (OM) membrane discovered that E.

coli mutants which leaked a large amount of periplasmic enzymes were also particularly sensitive to

peptidoglycan synthesis inhibiting antibiotics such as carbapenems and ampicillin usually reserved for

Gram-positive bacteria. Other antimicrobials like mitomycin C (DNA cross-linker), rifamycin (RNA

synthesis inhibitor) and chloramphenicol (protein synthesis inhibitor) also demonstrated action

against leaky periplasmic E. coli. This evidence provides researchers with an empyrean objective: how

to overcome the OM and access Gram-negative cell cytoplasm to enable active compounds to inhibit

cellular processes?

110090753

3

Figure 1. Diagrammatic representation of the top eight death causing micro-organisms in the USA

(2012). Numbers to the left of the false colour electron microscopy (FCEM) images represent the

number of deaths caused in the year 2012. Species names can be seen above the images in either

purple (Gram-positive micro-organisms) or red (Gram-negative micro-organisms). Text to the right of

images lists antibiotics, past and present, known to combat infections caused by the related

bacterium/fungi - (*) indicates emerging or established resistance to that particular antibiotic. Text

below the illustrations describes known resistance mechanisms.

110090753

4

Antimicrobial drug discovery

Modern research has given birth to efficient screening techniques, allowing large pharmaceutical

establishments and small research teams to search for new antimicrobial compounds in a streamlined

fashion. Although resistance to antibacterial compounds is becoming more common, it is believed

that most antimicrobial compounds are yet to be discovered. The potential antimicrobial compounds

derived from bacteriophage genomics alone amount to an almost incomprehensible number - if every

species of bacteria on the planet hosts one or more bacteriophage pathogens, it can be assumed that

novel antibacterial compounds lie undiscovered within the genomes of such pathogens, owing to their

mechanism of bacterial killing. In their renowned article published in Nature Biotechnology, Lui et al.

(2004) describe applying the concept of phage-mediated bacterial growth inhibition to antibiotic

discovery. The team identified 31 novel polypeptide families that inhibit bacterial growth when

expressed in S. aureus. One peptide in particular (77ORF104), of which the cellular target is DnaI (an

essential protein). Apart from utilising phage genomes to determine antibiotic compounds, one can

reduce the amount of target protein in a bacterium via RNAi (antisense RNA inhibition) making it more

sensitive to inhibition, thus aiding in the discovery of low abundance compounds from natural product

libraries. There is a large pool of literature surrounding this phenomenon including the Forsyth et al.

study (2002) and Goh et al (2009).

Natural products are an important weapon in the fight against bacteria, as they tend to have

polypharmological activities in addition to being ‘tailor made’ by evolution to overcome resistance. In

contrast, compounds that are made by design (semi-synthetic) give rise to resistant strains of bacteria

owing to the fact that they regularly only inhibit one target – this means that a point mutation in the

target protein is commonly all that is needed to render the compound obsolete. In their science article,

Haydon et al. (2008) describe an FtsZ (bacterial cell division protein) inhibitor with potent action

against S. aureus. However, due to the high occurrence of resistance, this compound must be

administered in combination therapies to avoid whole population resistance.

There are many bacterial targets available for inhibition by antimicrobial compounds which can now

be more easily discovered owing to RNAi and other antibiotic discovery techniques. However, Gram-

negative pathogens still present many conserved resistance mechanisms to antimicrobial compounds

which inhibit novel targets. For example, platensimycin – a FabF inhibitor, which shows potent, broad

spectrum Gram-positive activity in vitro and exhibits no cross-resistance to other key antibiotic

resistant bacteria including MRSA and vancomycin-intermediate Staphylococcus aureus, shows no

antibacterial activity against wild type Escherichia coli. Interestingly, platensimycin does exhibit

antibacterial activity against efflux-negative (tolC) E. coli indicating that efflux mechanisms and not

110090753

5

compound specificity limit the effectiveness of platensimycin in E. coli and possibly other Gram-

negative bacteria. Other antibiotic discovery methods include genome mining (Lautru et al. 2005),

transcriptional profiling of conditional mutants (Freiberg et al. 2005) and fusion/hybrid compounds

which will be described in more detail, later in this review.

Membrane active peptides

As previously mentioned, the outer membrane of Gram-negative bacteria provides an effective,

inherent mechanism for the exclusion of large antimicrobial peptides and other antibacterial agents.

Overcoming this barrier is essential for any compound to provide antimicrobial action and has been

the target of a plethora of recent research (Savage 2001; Schwechheimer et al 2013). Derived from

the peptides of multicellular organisms, membrane active peptides show the most promise in

overcoming the Gram-negative bacterial cell wall and beg the question, could we design anti-infective

drugs based on their properties?

The mechanism of the killing action of membrane active peptides is not understood in great detail but

three models for their activity have been proposed. (1) The Barrel-Stave model (Oren and Shai 1999)

where peptides attach to the membrane via electrostatic interaction then adopt an α-helical

conformation and self-assemble into bundles. The bundles then insert into the membrane and form

pores by placing their hydrophobic part in contact with the hydrophobic portion of the bacterial

membrane. (2) The Toroidal-Pore model which is similar to the barrel-stave model except α-helical

peptides keep their hydrophilic part in contact with the hydrophilic head groups of the lipid membrane

and bend the membrane to form pores. (3) The Carpet model (Brogden 2005). At first, the membrane

has a curvature of zero and the peptides bind preferentially to the lipid head groups. The peptides

then reorientate/realign to let their hydrophilic surface face head groups. Once a critical local

concentration is reached, transient holes are formed in the membrane and a positive curvature is

generated.

In light of these three pools of thought regarding the mechanism of membrane active peptides, all are

in agreement: these compounds act by the formation of ion channel pores that span membranes

without requiring a specific target receptor. Whilst the exact killing action exhibited on bacteria is

unknown, it is thought to be a direct result of one of two processes: damage to the bacterial cell

membrane results in the collapse of transmembrane electrochemical gradients and upon this collapse,

microorganisms lose their source of energy allowing increased water and ion flow across the

membrane resulting in cell swelling and lysis. Or, peptides act via a multi-hit mechanism that involves

more than one target (Shai 2002). Many reports including Wade et al. (1990) show that the site for

110090753

6

the antibacterial action of these compounds is the cytoplasmic membrane as the bactericidal action

exhibited on these microorganisms indicates they must be initially able to cross or disintegrate the

membranes of Gram-negative bacteria.

In addition to this, membrane active peptides are safe to use in a clinical setting, bacterial membranes

are organised in such a way that the outermost bilayer is heavily populated with lipids and as a result

present negatively charged phospholipid head groups on their surface. In contrast, animal membranes

are composed mostly of lipids with no net charge. The presence of cholesterol also reduces the activity

of membrane active peptides on a cell (Zasloff 2002). So, these cationic membrane active peptides

will only exhibit action on bacterial cells as opposed to animal and plant cells – showing great potential

for use in a clinical setting.

Membrane active peptides encompass a large family of molecules with proven bactericidal action

against Gram-positive and some Gram-negative species. In particularly, cecropin P1 has been found

to exhibit antimicrobial action against all members of the Enterobacteriaceae family except the

Pseudomonas genus (Giacometti et al. 1998).

Unfortunately, as with all antimicrobial compounds, resistant bacteria can arise, however, owing to

their mode of action, only a handful of resistance mechanisms have been outlined for membrane

active peptides. One identified mechanism is most prevalent in species belonging to the Psuedomonas

genus - the production of alginate acts as an auxiliary bacterial membrane, a diffusion barrier to

cationic antibacterial agents (Chan et al. 2005). However due to their potent antimicrobial activity and

ability to disrupt the cell membrane of Gram-negative bacteria, I believe these compounds show great

promise for the delivery of antimicrobial compounds and have the potential to be used as adjuvants.

In addition to this, the very nature of these compounds means that bacteria cannot easily acquire

resistance without changing the structure of the outer membrane. The membrane active peptides

represent a conserved theme in host antimicrobial defences throughout nature and have the potential

to be used in conjunction with other compounds as fusion/hybrid antibiotics thus allowing intra

cellular access to Gram-negative bacteria.

From critical analysis of the literature surrounding membrane active peptides I have discovered

extraordinary examples of α-helical cationic antibacterial membrane active peptides which are

unaffected by common methods of bacterial resistance. A case in point is the novel membrane-active

antimicrobial peptide polybia-CP - designed, produced and discussed by Wang et al. (2012). Polybia-

CP is a compound derived from the venom of the social wasp Polybia paulista and shows antibacterial

action against both Gram-positive and Gram-negative bacteria, permeabilizing the bacterial cell

110090753

7

membrane. The ability of polybia-CP to permeabilize multiple drug resistant enterohemorrhagic E. coli

(EHEC) was determined by NPN, a hydrophobic fluorescent probe that fluoresces weakly in an aqueous

environment and strongly when it enters a hydrophobic environment such as the interior of a

membrane (Loh et al. 1984). NPN is normally excluded from Gram-negative bacterial cells but upon

treatment with polybia-CP the compound shows strong fluorescence. In addition, after treatment with

polybia-CP E. coli cells demonstrated visual morphological changes and peptide induced dye leakage.

Solely administered, polybia-CP demonstrates an impressive minimum inhibitory concentration (MIC)

to a range of Gram-negative and Gram-positive bacteria. However, minimum bactericidal

concentration (MBC) requires 4-8 times increase of the MIC, making it difficult to attain feasible

treatment in a clinical setting following infection by resilient bacteria namely P. aeruginosa. In light of

this information polybia-CP provides an excellent demonstration of why membrane active

antimicrobial peptides should be used as adjuvants in antimicrobial chemotherapy for the entry of

antibacterial compounds in bacterial cells. Wang et al. (2012) proved this using DNA intercalating dye

P1, which does not bind to DNA in untreated Gram-negative bacterial cells. After treatment with

polybia-CP, P1 was visualised bound to DNA through confocal laser-scanning microscopy. In theory,

quinolones - which target DNA replication and repair by binding DNA gyrase complexed with DNA,

driving double strand DNA break formation and cell death (Kohanski et al. 2007) - could be used in

conjunction with polybia-CP to access DNA in Gram-negative bacterial cells because of increased

target availability due to bacterial cell wall permeabilization by polybia-CP.

Most membrane active peptides share similar morphology. Amphiphilic secondary structures in which

cationic amino acid side chains (i.e., arginine, lysine and histidine) are orientated on one face of the

molecules while hydrophilic side chains are on the opposing face. This morphology has been dubbed

“facially amphiphilic” (Epand et al. 2010) and is an important blueprint which should always be

considered when designing Gram-negative active compounds. Ceragenins, which share facially

amphiphilic morphology (owing to their bile acid scaffolding) and exhibit antibacterial action on Gram-

negative bacteria give strong evidence for the use of this structure. The most potent ceragenin – CSA-

13 even exhibits antibacterial action on the notoriously problematic, multi-drug resistant Gram-

negative pathogen Pseudomonas aeruginosa. Figure 2 shows a diagrammatic representation of a

membrane active peptide with important morphological features highlighted – CSA-13 mimics this

morphology, providing membrane activity (Chin et al. 2007).

110090753

8

Ceragenins have many advantages over membrane active peptides including their resistance to

proteolysis and amenability to large scale synthesis. Furthermore, they selectively associate with

bacterial cell membranes and disintegrate the antibiotic excluding OM of Gram-negative bacteria. This

permeabilization extenuates the activity of other antibiotics, making these compounds good

candidates for combination therapies.

Hybrid Compounds

For years, the capability of hybrid compounds to overcome and reduce the incidence of resistant

bacteria has been noted. Compounds which inhibit more than one target allow for a decline in the

number of spontaneous resistance mutants by taking the product of the amount of resistant

spontaneous mutants from both compounds so resistance rarely arises. However, I propose that

instead of joining two known antibiotics, as outlined in the expert review by Pokrovskaya and Baasov

(2010), we should investigate antibiotic compounds whose killing action can be combined with cell

wall permeabilizing compounds to overcome antibiotic resistance and gain entry to the Gram-negative

cell cytoplasm; especially that provided by compounds conferring a facially amphipathic morphology.

In theory, compounds which cause OM disruption grant at least some entry into the Gram-negative

bacterial cell cytoplasm, allowing other antibiotics access to their targets and conferring bactericidal

Figure 2. Chemical structure of the tryptophan-rich membrane-active antimicrobial peptide gp41w-

FKA. Important morphological structures are labelled: top – cationic amino acid side chain, bottom –

hydrophobic residues, and centre – helical conformation of the central backbone. Adapted from

Haney et al. (2012).

110090753

9

activity. The best antimicrobials to be used as candidates for fusion with OM disrupting compounds

would be those with proven polypharmological capabilities, namely natural compounds produced by

bacteria. Although ceragenins provide potent Gram-negative OM disruption and bactericidal action

in addition to other benefits, they are synthetically produced and as a result cannot feasibly be

produced as a hybrid compound with antimicrobials (Isogai et al. 2009). Instead, I would recommend

these compounds be used only in combination therapy with other antibiotics to aid in the disruption

of the bacterial OM so providing better access to drug targets in bacteria. Further research would

require screening the efficacy of known antibiotics which lack entry into the Gram-negative cell

cytoplasm in conjunction with ceragenin therapy.

Production of hybrid peptide antibiotics is widely documented and represents and a more realistic

goal for future research. This would involve combining membrane active peptides with peptides that

have proven antibiotic activity but lack efficient transport into the Gram-negative cell cytoplasm – all

critical processes could be inhibited, with many essential proteins available for targets (highlighted in

figure 3, 1a-1d). Antibiotics which cannot overcome the OM of some resistant Gram-negative bacteria

include aminoglycosides, penicillins, cephalosporins, fatty acid synthase inhibitors like triclosan and

cerulenin, and quinolones (Mingeot-Leclercq et al. 1999; Ruiz 2003; Schweizer 2001). Creation of

hybrid antimicrobials requires the careful identification of gene clusters which code for a desired end

product and usually take the form of a polyketide synthase. After this, a synthetic gene cluster is made

and inserted into an appropriate bacterium for the in vivo synthesis and extraction of the hybrid

antibiotic. The process is complicated but end results are achievable (Ichinose et al. 2003). Other

methods are available for the creation of hybrid peptide antibiotics too. Chemical synthesis, as

demonstrated by Zhang et al. (1998) who created functionally active cell-permeable peptides via

single step ligation of two peptide molecules. Another peptide ligation technique - solid-phase peptide

synthesis is well documented (Merrifield 1969). These methods infer that non-invasive cellular import

of synthetic compounds can be achieved by incorporating a membrane permeable, and therefore

presumably a membrane disrupting, sequence. Membrane active hyrbid compounds can also be

created in vitro - Mehravar et al. (2011) isolated membrane-active metabolites produced by soil

actinomycetes.

In my opinion, future research should concentrate on the identification of membrane active

compounds which are or can be expressed in a bacterial vector. The next step is to create synthetic

gene clusters which include peptide antibiotics that cannot cross the Gram-negative OM and can also

be expressed in a bacterial vector. Cross referencing of these three variables should reveal a hybrid

compound that is able to overcome the resistant Gram-negative OM, exhibit antibiotic action and be

110090753

10

produced on a large scale via heterologous expression in recombinant bacteria. I suggest that one of

the three membrane active metabolites identified by Mehravar et al. (2011) produced in 3

Streptomyces strains be combined with an innate Streptomyces peptide antibiotic such as one of the

coronamycins (Ezra et al. 2004) by one of the previously outlined techniques. This should increase the

chance of a successful hybrid peptide ligation and effective antibiotic production. If such a compound

were produced resistance mechanisms which usually inhibit either of the two components individually,

such as degradative enzymes, would be rendered ineffective due to a probable change in

conformation of the active site as demonstrated in figure 3 (4). This has huge implications for ESBL

producing resistant Gram-negative bacteria whose list of available resistance mechanisms is

substantially shortened with this theoretical hybrid cell wall permeabilizing antibiotic.

Figure 3. Diagrammatic representation of (1-5): (1) Known targets available to antibiotics; (1a) fatty

acids indicative of FAS inhibitors; (1b) DNA indicative of DNA damaging antibiotics e.g. quinolones;

(1c) ribosome indicative of protein synthesis inhibiting antibiotics; (1d) peptidoglycan structure

indicating target for cell wall synthesis inhibitors; (2) Most antibiotics cannot cross the OM of Gram-

negative bacteria; (3) Degradative enzymes (green) to certain antibiotics are produced by bacteria –

the antibiotic is complementary to the enzyme and fits into its active site; (4) Slight conformation

changes to the antibiotic when hybridized to a cell wall permeabilizing molecule (yellow) result in

lack of complementarity between it and the degradative enzyme – also able to disrupt outer

membrane of Gram-negative cell wall; (5) Disrupted cell wall allows for entry to the cytoplasm for

hybrid compound.

Gram negative bacterium cell cytoplasm

110090753

11

Membrane active compounds have huge potential for use as fusion/hybrid antibiotics, but apart from

my theoretical suggestion, hybridising compounds for increased antibacterial action has been the

focus of research dating back to 1998. I believe hybrid molecules as a strategy for the delivery of

antimicrobial compounds into bacterial cells will quickly be exhausted unless biochemical research

reveals a plethora of synthetically viable compounds which can be hybridized to overcome the OM of

Gram-negative bacteria whilst exhibiting antibacterial action. Thus we must look for other strategies

for the delivery of these compounds - starting with manipulation of the bacterial pathogen

bacteriophage.

The history of phage therapy

Phage have been recognised as antimicrobial agents since early in the 20th century. But since the dawn

of penicillin, have been brushed aside and are rarely used in western medicine today. However, the

rise of antimicrobial resistance among bacteria has once again caused the world’s antimicrobial

resistance research community to study these natural predators of bacteria.

The first acknowledgement of phage was in 1897 when British chemist E.H. Hankin reported that

water straight from the sewage ridden Ganges could kill the cholera pathogen. It was not until twenty

years later the cause of the bactericidal activity was suggested when British bacteriologist Frederick

W. Twort described an “ultramicroscopic virus” that by some means killed bacteria. French scientist

d’Herre gave the bacteria killing virus fame after he and his wife isolated “anti-shiga” microbes from

the faeces of patients with dysentery and grew them using bacterial hosts. D’Herre dubbed these

microbes ‘bacteriophage’ and was also first to appreciate their potential in antimicrobial therapy.

After characterization and improved bacteriophage culture methods it was possible to use

bacteriophage as an antimicrobial therapy, dubbed ‘phage therapy’. Since one bacteriophage will only

target one type of bacterium, therapies can be tailored once an infective bacterium is identified.

During World War Two and the ensuing Stalin era of the Soviet Union, this technique was employed

to cure patients of various antimicrobial resistant bacteria with great success (Stone 2002).

Following the early success of bacteriophage, which can be grown easily and in great numbers, a huge

decline in clinical use by the western world has occurred – small molecule antibiotics became cheaper

and easier to manufacture in large homogenous quantities. In my opinion, the profit driven business

of pharmaceutical corporations (particularly in the USA) has encouraged the use of antimicrobial drugs,

which are made in large quantities following massive investment. As a result there has been a lack of

research and production of cheaper effective therapies such as bacteriophage. Another complication

that should be considered whilst assessing the administration of bacteriophage to patients are

110090753

12

society’s potential ethical issues with the technology. Lack of understanding could lead to fear of the

virus particles and therefore proper education should be given to the public about this potentially

lifesaving therapy.

Whilst a lack of proper understanding of phage therapy continues in the western world, research

conducted in studies from the former Soviet Union is abundant, harbouring excellent success rates in

the treatment of resistant bacteria residing in mainly external infections. Phage study successes

include: Markoishvilli et al. (2002) reporting a 70% success rate in curing ulcers and wounds infected

with medically relevant resistant bacteria including the Gram-negative organisms E. coli and

Pseudomonas spp, Lazareva et al. (2001) reporting reduced septic complications, better temperature

normalization and two fold reduction in bacteria when treating resistant infection with phage in tablet

form, Perepanova et al. (1995) curing 92% of patients who had acute and chronic urogenital

inflammation caused by resistant bacteria and Miliutina and Vorotyntseva (1993) who studied the

efficacy of phage in combination with antibiotic therapy, discovering that combination therapy was

only effective against resistant infections vs. antibiotics alone. The success of phage therapy in the

former Soviet Union can be additionally determined by noting the production of phage related medical

products including PhageBioDerm, a novel wound-healing preparation consisting of a biodegradable

polymer impregnated with and antibiotic and lytic bacteriophages, recently licenced in the Republic

of Georgia (former soviet union) (Markoishvili et al. 2002).

Exploiting phage

110090753

13

Lytic phage kill bacteria during their normal life cycle and as a result require no modification to be

used as an antibacterial therapy - the mechanism is well understood. After location of their specific

bacteria, phage inject pathogenic DNA to shut down bacterial processes and commence with the

replication of themselves simultaneously. Phage proteins, known as lysins, initiate bacteriolysis during

which a number of clones of the original bacteriophage are released into the environment. See figure

4 for a detailed representation of the T4 phage life cycle - how tailed-phage infect, replicate and

escape infected cells upon bacteriolysis.

Firstly phage need to locate and identify their specific targets, this is known as adsorption. As

previously mentioned, one type of phage can only usually infect one species of bacteria. Specificity

allows phage to be used on the body without fear of affecting an individual’s natural flora. Adsorption

usually occurs between a receptor protein on the bacteriophage tail fibre and a particular recognisable

target on the bacterial surface, usually a protein or lipopolysaccharide. These are encoded by a largely

Figure 4. Life cycle of T4 lytic phage as described in Josslin (1970). (1) Attachment and injection of

DNA; (2) transcription of early genes; (3) replication and the beginning of capsid head formation; (4)

transcription of late genes; (5) head assembly; (6) tail assembly; (7) attachment of the head to tail;

(8) attachment of tail fibres – complete phage; (9) cell lysis and release of the mature phage.

4

110090753

14

conserved section of the genome within species and genera (Mahony et al. 2013). Until recently, these

tail fibres were little understood. The Bartual et al. study (2010) gave insight into the structure of tail

fibres when they determined the blueprint of the bacteriophage T4 long tail fibre receptor-binding tip.

It is described as “an unusual elongated six-stranded antiparallel beta strand needle domain

containing seven iron ions coordinated by histidine residues arranged collinearly along the core of the

biological unit. At the end of the tip, the three chains intertwine forming a broader head domain,

which contains the putative receptor interaction site.” This structure suggests a framework for

mutations to expand or modulate receptor binding-activity. If targets change on the bacterial outer

membrane then phage may be able to ‘adjust’ it’s recognition site through the process of natural

selection.

Interestingly, experiments involving hybrid phage have revealed the receptor-binding region of phage

tail fibres to be around 89 amino acids long (Montag et al. 1990). Future research should be aimed at

the identification of particular targets on the bacterial cell surface and genomic isolation of these 89

amino acids and their place in the phage genome so that they can be replicated by a genetically

modified phage. Outer membrane proteins/antigenic proteins which are recognised by bacteriophage

could be prepared from cell envelope suspension by differential Sarkosyl solubility. Outer membrane

proteins can then be identified from spots resolved by two-dimensional electrophoresis and LC-

MS/MS (Dumetz et al. 2008). This technology has recently been employed for developing vaccines

against flavobacteriosis infection in aquaculture and if achieved for bacteriophage targets, we could

tailor lytic phage therapy to target any bacterial species, including multi-drug resistant bacteria.

Identification of the part of the phage genome which codes for the 89 amino acids would be the result

of determining the amino acid sequence and cross referencing the thousands of combinations of

nucleic acid order with the bacteriophage genome.

Entry into the Gram-negative cell cytoplasm is inferred by bacteriophage who have evolved a ‘hole

punch’ mechanism for the transport of their own genome into bacteria. After receptor binding, a

recognition signal is sent to the baseplate causing the short tail fibres to extend and bind irreversibly

to the outer core region of the lipopolysaccharides. This is then followed by contraction of the outer

tail sheath, penetration of the bacterial membrane by the hollow inner tail tube and injection of the

viral DNA from the bacteriophage. See figure 5, adapted from Bartual et al. (2010) for a diagrammatic

demonstration of this mechanism. Viral DNA either inserts itself into the genome – known as the

lysogenic cycle, or, if conditions permit, viral DNA will commence the lytic cycle creating many virus

particles to be released into the environment upon bacteriolysis.

110090753

15

Figure 5. Bacteriophage T4 and its long tail fibres, annotations of the capsid head and protein tail are

provided - adapted from Bartual et al. (2010). (A) Bacteriophage before initiation of the ‘hole punching’

mechanism and delivery of viral DNA; (B) after this event has taken place. Receptor region of long tail

fibre is highlighted by grey box. This mechanism could provide an efficient vehicle for the delivery of

compounds other than DNA in the future.

In addition to the ‘hole punching’ mechanism and phage ability to adhere to specific bacteria phage

genomes can be exploited to aid in antibiotic discovery. In a previously mentioned example published

in Nature Biotechnology (2004), Lui et al. demonstrated identification of 31 novel peptide families

from the Stapholococcus spp. phage. This technique could be applied to genomes of phage which

infect Gram-negative bacteria, thus identifying Gram-negative active compounds. However, these

compounds would probably lack entry to the Gram-negative cell cytoplasm without phage, their

normal transport being from the phage body. In addition, a plethora of research has been conducted

concerning the bacteriophage lysins. These are phage-encoded peptidoglycan cell wall hydrolases that

accumulate in the bacterial cytoplasm during a lytic infection cycle. Compounds have been developed

using phage lysins to clear in vivo populations of Gram-positive bacteria. Nonetheless, a major

limitation of this approach is that lysins cannot normally pass the OM of Gram-negative bacteria

without the aid of accessory proteins or cell wall disrupting agents. The OM shields periplasmic

peptidoglycan so that lysins cannot exhibit their killing action. Genetic screens are available to search

for these compounds in the phage genome (Schuch et al. 2008) and can be applied to any phage.

More recently, research has turned towards the synthetic structural engineering of phage lysins that

can target Gram-negative pathogens and overcome the OM. Pesticin, a type B bacteriocin which

requires the gene products of tonB, exbB, exbD and fyuA is produced in times of stress by Yersinia

pesticis. It is harboured on a 10kb plasmid called pPCPI (Schuenemann et al. 2011) with the pesticin

immunity protein and plasminogenic activator. Interaction between FyuA (which is an outer

membrane, TonB-dependant iron transporter protein (Noinaj et al. 2010) expressed on some

110090753

16

pathogenic Gram-negative bacteria) and pesticin, is required for entry of the compound into Gram-

negative cell periplasm. Pesticin is a soluble protein with two domains, one that binds to FyuA and the

other which shares homology with the T4 phage lysozyme. Upon determining the crystal structure of

pesticin, Lukacik et al. (2012) went on to design a hybrid compound which shared the FyuA domain of

pesticin fused with the N-terminus of the T4 lysozyme. This hybrid phage lysin showed bactericidal

activity against pathogenic E. coli and was not inhibited by the pesticin immunity protein due to a

change in conformation. Figure 6 shows a cartoon representation of the mode of action of pesticin

versus this hybrid compound.

Figure 6. Mode of action of pesticin and the hybrid phage lysin as described by Lukacik et al. (2012).

(1) Pesticin – a 2 component molecule consisting of a FyuA binding domain and a lysozyme like domain)

enters the periplasm through binding to FyuA protein in Gram-negative outer membrane (OM). After

traversing the outer membrane, pesticin degrades the bacterial peptidoglycan layer to exhibit cell

death. This traversing depends on the presence of an outer membrane virulence protein FyuA. Pesticin

can be inhibited by bacteria expressing the pesticin resistance protein in the periplasm. (2) The

lysozyme like domain was substituted for a similar T4 lysozyme during creation of the hybrid

compound and can traverse the Gram-negative bacterial OM in the presence of FyuA. Translocation

of this hybrid compound also results in the killing of cells via peptidoglycan degradation, activity is not

affected by pesticin resistance protein. (3) Without additional means of disrupting the OM, externally

added T4 lysozyme cannot effect killing.

110090753

17

In addition, FyuA outer membrane protein is only expressed when pathogenicity occurs and therefore

this hybrid compound would not exhibit killing action on the normal gut flora, for example, due to a

lack of transport into the bacterial cell cytoplasm. Some modern antibiotics currently offer an ‘all or

nothing’ approach to eliminating infection, during which healthy bacteria can also be compromised

leading to increased production of resistant populations. With the FyuA targeting system only

pathogenic bacteria would be killed, allowing normal function of the body’s healthy microbial

populations. Figure 7, taken from Lukacik et al. (2012) demonstrates this hybrid phage lysins

bacteriolytic action against E.coli cells.

Figure 7. Cyro-electron micrograph taken directly from Lukacik et al. (2012). Cells were exposed to (A)

no treatment; (B and C) treatment with hybrid T4 lysozyme/FyuA binding compound. Extensive

membrane breakdown of the treated samples is apparent. As a result the cell is weakened and,

consequently spreads and flattens on the thin film of buffer while largely preserving its 3D surface

area so it projects a larger 2D surface area. As the cell is much thinner, membrane vesiculation occurs

all over the cell, not just at the edges. Inset (B and C), this vesiculation is shown with increased

magnification. Inset (A) – increased magnification highlights the outer membrane (OM), periplasm (PG)

and inner membrane (IM).

Phage lysins seem to yield considerable potential, yet they will always need hybridization with

accessory compounds to aid with transport to the Gram-negative periplasm. The above example is

impressive but identification of specific binding/transport proteins is difficult – they are usually part

of antibiotic compounds produced by bacteria. In contrast, targeting FyuA for antimicrobial therapy

offers new hope. Recent findings indicate that fyuA appears to be associated with the persistence of

urinary tract infections and multi-drug resistance in animal and human infections (Platell et al. 2012).

110090753

18

Since bacteria expressing FyuA are pathogenic and not a part of the normal microbial flora, we can, in

theory, selectively kill the detrimental bacteria and leave the rest unharmed.

The future of phage therapy

Phage can be exploited in a variety of ways, with huge scope for overcoming antibiotic resistance and

the Gram-negative OM. Future therapies must base their technology on recent innovations in phage

therapy. The rise of the genomic era has given us the ability to genetically engineer phage and holds

much promise for the future. An excellent demonstration of phage potential is the Westwater et al.

(2003) study where the team used genetically modified (GM) phage to deliver lethal DNA encoding

bactericidal proteins. These proteins were ‘addiction molecules’ consisting of two components (one

with lethal action, the other usually transcribed as an antidote) which mediate programmed cell death

under particular conditions. A non-lytic phage delivery system (phage M13) transformed with helper

phage R408 (to introduce phagemids into the delivery system) transferred the lethal genes into the

bacterial cell cytoplasm. A high copy number plasmid containing the lethal genes was constructed and

placed under the control of a LacI/IPTG regulated promoter for screening purposes. When induced,

viable counts of bacterial populations dropped dramatically and did not recover. One drawback to this

research is that E. coli was used because of the wealth of molecular tools available. To utilise this

technology for other, more problematic Gram-negative bacteria, specific phage delivery systems must

be created to target conserved proteins in pathogenic bacteria, FyuA for example.

Phage can only deliver DNA sequences into bacteria. Anything the phage needs is then created utilizing

bacterial ‘machinery’. A distinct lack of ability to deliver antibiotics to resistant bacterial cells is ever

looming: why not reverse modern antibiotic resistance in bacteria by inserting sensitive genes? The

antibiotic streptomycin binds to a highly conserved region in 30S small ribosomal subunit in bacteria,

this inhibits translation of mRNA into proteins. Point mutations in resistant bacteria mean that

streptomycin can no longer bind. The 30S small ribosomal subunit is encoded by rspL gene, the most

common mutations in the gene are K88R and R68S but rpsL carries dominant sensitivity over its

homolog; mutation results in resistance but also generates a recessive gene. This knowledge can be

used to reinstate streptomycin sensitivity via phage insertion of a dominant gene (Edgar et al. 2012).

In this instance, phage λGT11 was used as a delivery vector. This phage can be directed to specific cell

types under particular conditions and enters the lysogenic (as opposed to lytic) cycle at 32°C – phage

genes form into circular plasmids and can be transferred through bacterial populations by conjugation.

Phage mediated insertion of the original, dominant gene infers sensitivity of resistant bacteria to

streptomycin. Bacteriophage which can be used as adjuvants for antibiotic therapy, like the above

example, provide great potential as a strategy for the delivery of antimicrobials to bacterial cells.

110090753

19

Reversing resistance relies on a previous understanding of antibiotic targets. Bactericidal antibiotics

such as quinolones induce hydroxyl radical formation that leads to DNA damage, which induces the

SOS response in bacteria for DNA repair. Increased resistance to these compounds arises from

evolution of a more efficient DNA repair response and it has been shown that bacterial killing from

quinolones can be enhanced by knocking out recA – a critical gene in the SOS response pathway

(Kohanski et al. 2007). Lu and Collins (2009) used non-lytic filamentous phage M13 which can

accommodate DNA insertions into the genome to deliver the lexA3 gene which disrupted the SOS

response and in turn made even resistant bacteria sensitive to bactericidal antibiotics. Furthermore,

the team show that engineering phage to target other gene networks and overexpress multiple factors

can also produce effective antibiotic adjuvants. The work as a whole establishes a synthetic biology

method for the rapid production of modified phage which target specified gene networks and puts

forward a valid argument for the creation of a bacteriophage genomic library. Current prices for a

bacteriophage genome carrying multiple constructs to target different gene networks are decreasing

rapidly, making the idea more feasible as time passes and prices drop (Baker 2011).

With my final note on phage therapy, I believe the technology will compensate for unavoidable

complications of antimicrobial therapy with antibiotics such as the appearance of multi-drug

resistance or substituted microbism. Moreover, traditional antibiotic drugs typically do not take

advantage of targets that need to be upregulated to achieve antimicrobial activity. Phage therapy has

at least the potential to inhibit up regulated targets as demonstrated by Lu and Collins (2009). In

addition, phage therapy in the traditional sense (non-GM phage) has been improved vastly thanks to

the arrival of the genomic era. The two technologies should evolve in conjunction with one another

as phage studies have already provided us with invaluable insights into bacterial DNA/cell biology

processes (Birge 2000).

Phage may be difficult to administer for internal infections because of protease susceptibility but

provide a valid argument for them to be considered as disinfectants and for external application. I

suggest that genomic isolation and synthesis of the ‘hole punching’ and delivery mechanisms from

phage could yield an important tool in the delivery of antimicrobials to bacterial cells.

Pyocins

Sharing a similar morphology and delivery mechanism with phage, and greatly understood with the

aid of genome sequencing, pyocins represent another technology with potential to deliver

antimicrobial compounds into even Gram-negative bacteria. Pyocins are produced by Psuedomonas

aeruginosa, look much like a bacteriophage tail (particularly R-type pyocins) (Figure 5) and portray a

110090753

20

similar recognition mechanism. In many respects, R-type pyocins are viewed as defective prophages

that produce noninfective particles containing only the tail region without the capsid head or DNA,

which have been adapted by the host as defensive antibacterial agents. Neither the host nor its

offspring are affected by the pyocins, although one exception is noted (Goodwin et al. 1972). The

potential of pyocins as antimicrobial agents has only recently been exposed, because at the time of

discovery, antimicrobial resistance was not as prominent and so there was no need for new

antimicrobial therapies. However, in the face of rising antimicrobial resistance, particularly in Gram-

negative bacteria, this avenue must now be explored.

The lipopolysaccharide tail fibres of pyocins are able to recognise competing P. aeruginosa strains as

well as many other Gram-negative pathogens, indicating a conserved target. Once adsorption occurs

between pyocins and target, penetration of the core through the outer membrane, cell wall and

cytoplasmic membrane immediately ensues, this is a process critical to bactericidal activity. After

adsorption, pyocins halt critical bacterial processes and promote the release of intercellular material,

indicating cell wall permeabilization. Upon permeabilization of the bacterial cell wall, the targets of

many antimicrobials would become available making pyocins valid for use as adjuvants with antibiotics,

providing another strategy for the delivery of antimicrobials. Protein sequences for pyocins can be

deduced from the genome sequencing of P. aeruginosa and by comparing these sequences with the

structure of similar phage, it is possible to assign functions to ORF’s coding for these proteins (Michel-

Briand and Baysse 2002). In theory, the components of the ‘hole punching’ mechanism could be

produced under the control of a strong constitutive promoter. In addition, R type pyocins are protease

resistant providing an added benefit over phage therapy.

The limited bacterial spectra of natural R-type pyocins ultimately compromises their ability for clinical

use. However, replacing pyocins tail fibres with phage PS17 tail fibres changes the specificity of the

pyocins to target the P. aeruginosa strain that which PS17 predates. Natural and retargeted pyocins

exhibit very narrow bactericidal action and thus yield little in the way of collateral damage to healthy

microbiotae, blocking the rise of antibiotic resistance through horizontal gene transfer. This is

achieved by “swapping tail fibres or fusing the N-terminal portion of the tail fibres of bacteriophages

that infect hosts other than R2-sensitive P. aeruginosa.” (Williams et al. 2008). In their experiments,

Williams et al. (2008) showed that fusing of the C terminus of the P2 (phage 2) tail fibre to the R2

pyocin protein Prf15 (tail fibre protein), changed pyocins killing spectrum to target Escherichia coli

strain C and multiple uropathogenic E. coli strains as well as creating pyocins that kill Yersinia pestis.

More evidence that the plasticity observed among bacteriophage tail genes can, with modern

molecular techniques, be exploited to produce non-natural, targeted microbial agents is detailed in a

110090753

21

separate study (Scholl et al. 2009). This team emphasised pyocins potential for killing clinically relevant

Gram-negative bacteria by switching pyocins tails with tails from phage ΦV10, the natural virus

predator of food-borne pathogen E.coli 0157:H7. Pyocin production is initiated by the SOS response

in bacteria so treatment with UV light is needed, but after this, bacteria produce around 100-200

particles per cell; a viable option for large scale production. These examples outline pyocin potential,

highlighting especially the new found ability to target any bacterial strain, provided a relevant phage

has been identified. Through genomic identification of the pyocin mechanism - which is rightly likened

to that of the bacteriophage tail – in combination with a bacteriophage capsid head containing

antimicrobial compounds I believe an efficient vehicle for the delivery of antimicrobial compounds

can be achieved. In absence of this technology at present, pyocins can provide entry to antimicrobial

targets via permeabilization of the bacterial cell membrane or exhibit antibacterial action themselves.

Conclusion

Finally, resistant bacteria continue to pose a threat to human health after the relative early success of

antibiotics. It is in these difficult times, where antimicrobial compounds lack entry to bacterial cells,

when we must look to novel, innovative research for answers and properly educate those who will be

treated to overcome any reluctance about potentially lifesaving therapies such as phage. New

technology aiding in the delivery of antimicrobials to bacterial cells currently captures much interest

and funding in the struggle to halt the ongoing rise of antimicrobial resistance. The most promising

ideas stem from old knowledge thought to be exhausted. Phage technology is a case in point, allowing

researchers to deliver any small DNA fragments, helping to permeabilize the Gram-negative cell wall.

As bacteria evolve more efficient resistance mechanisms we must endure to explore novel strategies

for the delivery of antimicrobials into those bacteria which are causing most incurable and deadly

infections. By further investigating technologies like phage and pyocin therapy, researchers have

discovered specific and powerful weapons in the fight against the rise of resistant bacteria. These

include the natural ability of these molecules to attack specific bacterial targets of our choosing.

However, I believe that an important aspect of these technologies has been overlooked and that is

the ability to use these advances in combination therapies, providing entry to bacterial cells for

antimicrobials. Indeed, the antibacterial effectiveness of combination chemotherapies has already

been well documented; clavulanic acid, which inhibits β-lactamases is proven to increase the

antimicrobial effects of penicillin through β-lactamase inhibition (Heerema et al. 1979) and brand

name products adhering to this combination chemotherapy are prescribed all over the world.

Using cell wall permeabilizing compounds like phage, pyocins or membrane active peptides provides

effective, novel strategies for the delivery of antimicrobials into bacterial cells, mostly through

110090753

22

disintegration of the bacterial cell wall and I believe that in the future, designs based on this

technology will be used to deliver antimicrobial compounds directly into the cytoplasm of bacteria,

taking advantage of the phage/pyocin ‘hole punching’ mechanism. For example the T7 phage virus

has already been used to implement a biotemplating technique for the creation of magnetic

nanoparticles which imitate phage morphology and biochemical activity (Lui et al. 2006).

Nanotechnology, phage and pyocin technologies, membrane active peptides and hybrid molecules

which can overcome the bacterial OM represent major advances in strategies for the delivery of

antimicrobials into bacterial cells. Future research should focus on the identification and isolation of

compounds which bind to conserved targets on the pathogenic Gram-negative bacterial OM with

emphasis on pore targets to aid in the efficient transport of antimicrobial compounds to the Gram-

negative cytoplasm. In addition, an international bacteriophage genomic library should be constructed

so that bacteriophage legs specific to problematic bacteria can be integrated on the pyocin body.

Unlike the original studies and other reviews which outline and describe strategies for the delivery of

antimicrobials into resistance bacterial cells singularly, this work hopes to encompass all of the most

important work, helping the reader to understand new technologies. Whilst some other delivery

strategies are described in the literature, this work only focuses on the most promising strategies

published in respected journals and papers. This review also suggests ways in which future research

should be directed to complement existing strategies, like the identification of conserved targets on

pathogenic bacteria for use with phage/pyocin tail recognition domains. To conclude, I hope that this

work outlines the importance of discovering novel strategies for the delivery of antimicrobials and the

danger of drug resistant pathogenic bacteria and can go some way to encourage use of these therapies

in conjunction, now, to slow the occurrence of new resistant bacteria.

References

Barna JCJ, Williams DH (1984) The structure and mode of action of glycopeptide antibiotics of the

vancomycin group. Annual Reviews in Microbiology 38(1): 339-357.

Bartual SG. Otero JM, Carcia-Doval C, Llamas-Saiz AL, Kahn R, Fox GC, van Raaij MJ (2010) Structure of

the bacteriophage T4 long tail fibre receptor-binding tip. Proceedings of the National Academy of

Science of America DOI:10.1073/pnas.1011218107.

Birge EA (2000) Bacterial and Bacteriophage Genetics. 4th edition. Springer-Verlag, New-York.

110090753

23

Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature

Reveiws Microbiology 3: 238-250.

Carmeli Y, Troillet N, Eliopoulos GM, Samore MH (1999) Emergence of Antibiotic-Resistant

Pseudomonas aeruginosa: Comparison of Risks Associated with Different Antipseudomonal Agents.

Antimicrobial Agents and Chemotherapy. 43(6): 1379-1382.

Cetinkaya Y, Falk P, Mayhall CG (2000) Vancomycin-resistant Enterococci. Clinical Microbiology

Reviews 13(4): 686-707

Chan C, Burrows LL, Deber CM (2005) Alginate as an auxiliary bacterial membrane: binding of

membrane-active peptides by polysaccharides. Journal of peptide research 65(3): 343-351.

Chin JN, Rybak MJ, Cheung CM, Savage PB (2007) Antimicrobial Activities of Ceragenins against Clinical

Isolates of Resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 51(4): 1268-

1273

Chopra I, Roberts M (2001) Tetracycline antibiotics: mode of action, applications, molecular biology,

and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews 65(2): 232-260.

Delcour AH (2009) Outer Membrane Permeability and Antibiotic Resistance. Biochimica et Biophysica

Acta (BBA) 1794(5): 808-816.

Dumetz F, Duchaud E, Claverol S, Orieux N, Papillon S, Lapaillerie D, Le Henaff M (2008) Analysis of the

Flavobacterium psychrophilum outer-membrane subproteome and identification of new antigenic

targets for vaccine by immunomics. Microbiology 154(6): 1793-1801.

Edgar R, Friedman N, Molshanski-Mor S, Qimron U (2012) Reversing Bacterial Resistance to Antibiotics

by Phage-Mediated Delivery of Dominant Sensitive Genes. Applied and Environmental Mircobiology

78(3): 744.

Epand RF, Pollard JE, Wright JO, Savage PB, Epand RM (2010) Depolarization, Bacterial Membrane

Composition, and the Antimicrobial Action of Ceragenins. Antimicrobial Agents and Chemotherapy

54(9): 3708-3713.

110090753

24

Ezra D, Castillo UF, Strobel GA, Hess WM, Porter H, Jensen JB, Condron MA, Teplow DB, Sears J,

Maranta M, Hunter M, Weber B Yaver D (2004) Coronamycins, peptide antibiotics produced by a

verticillate Streptomyces sp. (MSU-2110) endophytic on Monstera sp. Microbiology 150(4): 785-793.

Forsyth RA, Haselbeck RJ, Ohlsem KL, Yamamoto RT, Xu H, Trawick JD, Wall D, Wang L, Brown-driver

V, Froelich JM, Kedar GC, King P, McCarthy M, Malone C, Mislner B, Robbins D, Tan Z, Zhu Z, Carr G,

Mosca DA, Zamudlo C, Foulkes JG, Zyskind J (2002) A genome-wide strategy for the identification of

essential genes in Staphylococcus aureus. Molecular Microbiology 43(6): 1387-1400.

Goodwin K, Levin RE, Doggett RG (1972) Autosensitivity of Pseudomonas aeruginosa to its own pyocins.

Infection and Immunity 6: 889–892.

Gupta N, Limbago BM, Patel JB, Kallen AJ (2011) Carbapenem-Resistant Enterobacteriaceae:

Epidemiology and Prevention. Clinical Infectious Diseases 53(1): 60-67

Freiberg C, Fischer HP, Brunner NA (2005) Discovering the Mechanisms of Action of Novel Antibacterial

Agents through Transcriptional Profiling of Conditional Mutants. Antimicrobial Agents and

Chemotherapy 49(2): 749-759.

Giacometti A, Cirioni O, Gregananti G, Quarta M, Scalise G (1998) In vitro Activities of Membrane-

Active Peptides against Gram-Positive and Gram-Negative Aerobic Bacteria. Antimicrobial Agents and

Chemotherapy 42(12) 3320-3324.

Goh S, Boberek JM, Nakashima N, Stach J, Good L (2009) Concurrent Growth Rate and Transcript

Analyses Reveal Essential Gene Stringency in Escherichia coli. PLoS ONE 4(6): e6061.

doi:10.1371/journal.pone.0006061

Haney EF, Nguyen LT, Schibli DJ, Vogel HJ (2012) Design of a novel tryptophan-rich membrane-active

antimicrobial peptide from the membrane proximal region of the HIV glycoprotein gp41. Beilstein

Journal of Organic Chemistry 8: 1172-1184.

Haydon DJ, Stokes NR, Ure R, Galbraith G, Bennett JM, Brown DR, Baker PJ, Barymin VV, Rice DW,

Sedelnikova SE, Heal JR, Sheridan JM, Aiwale ST, Chauhan PK, Srivastava A, Taneja A, Collins I, Errington

110090753

25

J, Czaplewski LG (2008) An Inhibitor of FtsZ with Potent and Selective Anti-Staphylococcal Activity.

Science 321:1673-1675.

Ichinose K, Ozawa M, Itou K, Kunieda K, Ebizuka Y (2003) Cloning, sequencing and heterologous

expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards

comparative analysis of the benzoisochromanequinone gene clusters. Microbiology 149(7): 1633-

1645

Jacoby GA (2009) AmpC β-Lactamases. Clinical Microbiology Reviews 22(1):161.

Josslin R (1970) The lysis mechanism of phage T4: Mutants affecting lysis. Virology 40(3): 719-726.

Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ (2007) A common mechanism of cellular

death induced by bactericidal antibiotics. Cell 130(5):797-810.

Lambert PA (2002) Cellular impermeability and uptake of biocides and antibiotics in Gram-positive

bacteria and mycobacteria. Journal of Applied Microbiology 92: 46S-54S.

Liu C, Chung S, Jin Q, Sutton A, Yan F, Hoffmann A, Kay BK, Bader SD, Makowski L, Chen L (2006)

Magnetic virus via nano-capsid templates. Journal of magnetism and magnetic materials 302: 47-51.

Liu J, Dehbi M, Moeck G, Arhin F, Bauda P, Bergeron D, Calejo M, Ferretti V, Ha N, Kwan T, McCarty J,

Srikumar R, Williams D, Wu JJ, Gros P, Pelletier J, DuBow M (2004) Antimicrobial drug discovery

through bacteriophage genomics. Nature Biotechnology 22(2): 185-191

Lautru S, Deeth RJ, Bailey LM, Challis GL (2005) Discovery of a new peptide natural product by

Streptomyces coelicolor genome mining. Nature chemical biology 1(5) 265-269.

Lazareva EB, Smirnov SV, Khvatov VB, Spiridonova TG, Bitkova EE, Darbeeva OS et al. (2001) Efficacy

of bacteriophage use in complex treatment of the patients with burn wounds. Antibiotic Khimioterapy

46: 10-14

Lazzaroni JC, Portalier RC (1981) Genetic and biochemical characterization of periplasmic-leaky

mutants of Escherichia coli K-12. Journal of bacteriology 145(3): 1351-1358.

110090753

26

Little ML, Qin X, Zerr DM, Weissman SJ (2012) Molecular diversity in mechanisms of carbapenem

resistance in paediatric Enterobacteriaceae. International Journal of Antimicrobial Agents 39(1): 52-

57.

Lukacik P, Barnard TJ, Keller PW, Chaturvedi KS, Seddiki N, Fairman JW, Noiaj N, Kirby TL, Henderson

JR, Steven AC, Hinnenbusch BJ, Buchanan SK (2012) Structural engineering of a phage lysin that targets

Gram-negative pathogens. Proceedings of the National Academy of Science USA 109(25): 9857-9862.

Lui J, Dehbi M, Moech G, Arhin F, Bauda P, Bergeron D, Callejo M, Ferretti V, Ha N, Kwan T, McCarty J,

Srikumar R, Williams D, Wu JJ, Gros P, Pelletier J, DuBow M (2004) Antimicrobial drug discovery

through bacteriophage genomics Nature Biotechnology. 22.2: 185-191.

Markoishvili K, Tsitlanadze G, Katsatava R, Morris JG, Sulakvelidze A (2002) A novel sustained-release

marix based on biodegradable poly(ester amide)s and impregnated with bacteriophages and an

antibiotic shows promise in management of infected venous stasis ulcers and other poorly healing

wounds. International Journal of Dermatology 41:453-458

Martin MV (1999) The use of fluconazole and itraconazole in the treatment of Candida albicans

infections: a review. Journal of Antimicrobial Chemotherapy 44: 429-437.

Mehravar M, Sardari S, Owlia P (2011) Membrane-active metabolites produced by soil actinomycetes

using chromatic phospholipid/polydiacetylene vesicles. Indian journal of experimental biology 49(2):

946-952

Merrifield (1969) SOLID-PHASE PEPTIDE SYNTHESIS. Advances in Enzymology and related Areas of

Molecular Biology 32: 221-296.

Michel-Briand Y, Baysse (2002) The pyocins of Pseudomonas aeruginosa. Biochimie 84: 499-510.

Miliutina LM, Vorotynseva NV (1993) Current strategy and tactics of etiotropic therapy of acute

intestinal infections in children. Antibiotic Khimioterapy 38: 46-53.

110090753

27

Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM (1999) Aminoglycosides: activity and resistance.

Antimicrobial Agents and Chemotherapy 43(4): 727-737.

Oren Z, Shai Y (1999) Mode of Action of Linear Amphipathic α-Helical Antimicrobial Peptides. Peptide

Science 47(6): 451-463.

Paterson DL (2006) Resistance in Gram-Negative Bacteria: Enterobacteriaceae. The American Journal

of Medicine 119.6: S20-S28.

Pelarez T, Cercenado E, Alcala L, Marin M, Martin-Lopez A, Martinez-Alarcon J, Catalan P, Sanchez-

Somolinos M, Bouza E (2008) Metronidazole Resistance in Clostridium difficile Is Heterogenous.

Journal of Clinical Microbiology 46(9): 3028-3032

Perepanova TS, Darbeeva OS, Kotliarova GA, Kondra’eva EM, Maiskaia LM, Malysheva VF et al. (1995)

The efficacy of bacteriophage preparations in treating inflammatory urologic diseases. Minerva

Urologica e Nefrologica 5: 14-17

Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA (2007) Global Challenge if Multidrug-

Resistant Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 51(10): 3471

Platell JL, Trott DJ, Johnson JR, Heisig P, Heisig H, Clabots CR, Johnston B Cobbold RN (2012)

Prominence of an 075 clonal group (clonal complex 14) among Non-ST131 Flouroquinolone-Resistant

Escherichia coli Causing Gastrointestinal Infections in Humans and Dogs in Australia. Antimicrobial

agents and chemotherapy 56(7): 3898-3904.

Ruiz J (2003) Mechanisms of resistance to quinolones: target alterations, decreased accumulation and

DNA gyrase protection. Journal of Antimicrobial Chemotherapy 51: 1109-1117.

Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Billie J (1995) Mechanisms of resistance to azole

antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug

transporters. Antimicrobial Agents and Chemotherapy 39(11): 2378

Savage PB (2001) Multidrug resistant bacteria: overcoming antibiotic permeability barriers of gram-

negative bacteria. Annals of Medicine 33(3): 167-171.

Scholl D, Cooley M, Williams SR, Gebhart D, Martin D, Bates A, Mandrell R (2009) An Engineered R-

type Pyocin Is a Highly Specific and Sensitive Bactericidal Agent for the Food-Borne Pathogen

Escherichia coli 0157:H7. Antimicrobial Agents and Chemotherapy 53(7): 3074.

110090753

28

Schuch R, Fischetti VA, Nelson DC (2009) A genetic screen to identify bacteriophage lysins. Methods

in Molecular Biology 502: 307-319.

Schuenemann VJ, et al. (2011) Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid

of Yersinia pestis from victims of the Black Death. Proceeding of the National Academy of Science USA

108:E746–E752

Schwechheimer C, Sullivan CJ, Kuehn MJ (2013) Envelope control of outer membrane vesicle

production in Gram-negative bacteria. Biochemistry 52(18): 3031-3040.

Schweizer HP (2001) Triclosan: a widely used biocide and its link to antibiotics. FEMS microbiology

letters 202(1): 1-7.

Shah D, Dang MD, Hasbun R, Koo HL, Jiang ZD, DuPont HL, Garey KW (2010) Clostridium difficile

infection: update on emerging antibiotic treatment options and antibiotic resistance. Expert review in

anti-infective therapy 8(5): 555-564

Shai Y (2002) Mode of Action of Membrane Active Antimicrobial Peptides. Peptide Science 66(4): 236-

248.

Shinomiya T, Osumi M, Kageyama M (1975) Defective pyocin particles produced by some mutant

strains of Pseudomonas aeruginosa. Journal of Bacteriology 124: 1508–1521

Stapleton PD, Taylor PW (2002) Methicillin resistance in Staphylococcus aureus. Science Progress 85(1)

57-72.

Stone (2002) Stalins Forgotten Cure. Science 298(5594): 728-731

Tam VH, Chang KT, Garey KW (2010) Prevalence, Resistance Mechanisms, and Susceptibility of

Multidrug-Resistant Bloodstream Isolates of Pseudomonas aeruginosa. Antimicrobial Agents and

Chemotherapy 54(3): 1160-1164.

Wade D, Bowman A, Wahlin B, Drain CM, Andreu D, Boman HG, Merrifeild RB (1990) All-D amino acid

containing channel-forming antibiotic peptides. Proceedings of the National Academy of Sciences of

the United States of America 87(12) 4761-4765.

Waksman SA, Lechevalier HA (1962) The Actinomycetes. Vol. III. Antibiotics of Actinomycetes.

Wang J, Soisson SM, Young K, Shoop w, Kodali S, Galgoci A, Painter R, Parthasarathy G, Tang YS,

Cummings R, Ha S, Dorso K, Motyl M, Jayasuriya H, Ondeyka J, Herath K, Zhang C, Hernandez L, Allocco

110090753

29

J, Basilio A, Tormo JR, Genilloud O, Vicente F, Peleaz F, Colwell L, Kee SH, Michael B, Felcetto T, Gill C,

Silver LL, Hermes JD, Bartizal K, Berrett J, Schmatz D, Becker JW, Cully D, Singh SB (2006) Platensimycin

is a selective FabF inhibitor with potent antibiotic properties. Nature 441: 358-361.

Webber MA, Piddock LJ (2009) The importance of efflux pumps in bacterial antibiotic resistance. The

Journal of Antimicrobial Chemotherapy 51(1): 9-11.

Westwater C, Kasmna LM, Schofield DA, Werner PA, Dolan JW, Schmidt MG, Norris JS (2003) Use of

Genetically Engineered Phage to Deliver Antimicrobial Agents to Bacteria: an Alternative Therapy for

Treatment of Bacterial Infections. Antimicrobial Agents and Chemotherapy 47(4): 1301

Williams SR, Gebhart D, Martin DW, Scholl D (2008) Retargeting R-Type Pyocins to Generate Novel

Bactericidal Protein Complexes. Applied and Environmental Microbiology 74(12): 3868

Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415: 389-395

Zhang L, Torgerson TR, Liu XY, Timmons S, Colosia AD, Hawiger J, Tam JP (1998) Preparation of

functionally active cell-permeable peptides by single-step ligation of two peptide molecules.

Proceedings of the Nation Academy of Science of USA. 95: 9184-9189.