bacteriophages and their endolysins for control of ... · bacteriophages and their endolysins for...

Bacteriophages and their endolysins for control of pathogenic bacteria

R. Keary1, O. McAuliffe2, R.P. Ross2, C. Hill3, J.O’Mahony1 and A. Coffey1 1Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland. 2Biotechnology Department, Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland. 3Alimentary Pharmacobiotic Centre, University College Cork, Cork, Ireland.

Bacteriophages (phages) are viruses that specifically infect bacterial cells. Phages were first discovered in the early 1900s and soon after, the concept of phage therapy was conceived. Phage therapy involves the application of bacterial viruses to eliminate pathogenic bacteria. The results of early human trials were hampered by limited knowledge of phage biology, poor experimental design and in some cases a lack of understanding of the causative nature of the illness being treated. As a result, antibiotics, which became widely available in the 1940s, replaced phage as antibacterials. However, with an increase in antibiotic resistance in pathogenic bacteria in recent decades, it is now widely recognised that alternative bactericidal agents for the prevention and treatment of bacterial infections are urgently required. With this in mind and with a better understanding of phage biology, the concept of phage therapy has been revisited and expanded upon over the past twenty years. This chapter presents an overview of phage-based approaches which have been employed for the elimination of pathogenic bacteria.

Keywords Bacteriophage therapy; antibiotics; resistance; endolysins; enzybiotics; Gram-positive pathogens; biofilms; chimeric lysins; synergy.

1. Introduction

1.1. General overview of bacteriophages

Phages are ubiquitous in nature and far outnumber their bacterial hosts. They have been reported to be present in soil and sediment at a titre of approximately 109 viral particles per gram and in aquatic systems at titres between 104 and 108

per ml [1]. Replication of these viral particles and release of the progeny generally leads to death of the host cell. Phages have been estimated to kill 20-40% of marine bacteria every day [2]. Hence, they are a major player in bacterial evolution and ecological systems, and have a considerable role in biogeochemical cycles (carbon, nitrogen & phosphorous cycles). Phages can be readily isolated from environmental samples such as soil, sewage and water and their prominence in the environment means that humans are constantly exposed to phages without adverse effect. This fact, along with their specific bactericidal potential, are two important factors which justify utilising phages and phage-based products for their therapeutic and prophylactic potential.

1.2. Phage replication cycle

The infection cycle utilised by a phage is an important consideration when choosing a phage for antibacterial application. Phages are obligate, intracellular, parasites of bacterial cells. Once they have infected and replicated inside the host cell they can be released by a number of mechanisms. The main steps of infection for tailed phages are outlined in Fig.1. Phages are commonly classified as either virulent or temperate based on their replication cycle. When a virulent phage has infected a host cell it immediately begins to exploit the metabolic machinery of the cell and direct it towards replication of new virion particles. Once the new phage DNA has been packaged and the protein capsid is fully formed phage-encoded proteins, called holins and endolysins work together to cause lysis of the cell and the progeny are released (lytic cycle). A temperate phage has the ability to enter a lysogenic cycle, in which the phage DNA is integrated into the host genome. The DNA is replicated along with the host genome. It is inherited by daughter cells during each division and is referred to as a prophage. The prophage remains dormant until the lytic cycle is induced by some environmental stress such as UV light or by a chemical agent such as mitomycin C. Variations on these cycles, which include chronic and pseudolysogenic infection have also been observed [1]. In recombinant DNA technology, temperate phages, such as lambda, are commonly utilised for cloning because of their potential for genetic recombination. It is this same characteristic which makes them unsuitable candidates for phage therapy because of the potential for horizontal transfer of genes encoding virulence factors and/or antibiotic resistance [3, 4]. In addition, unlike virulent phages they do not lyse every organism they infect. Therefore, they are less efficient bactericidal agents. Obligate lytic phages are the ideal candidate for phage therapy applications.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

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1.3. Brief history of phage biology

In 1896 Ernest Hankin reported the antiseptic character of water from the Ganges and the Juma rivers in India against a wide range of bacteria including Vibrio cholera [5]. He attributed this phenomenon to some volatile chemical substance. Several other scientists including Nikolay Gamaleya and Frederick Twort made similar observations on the autolysis of bacteria [6, 7]. It is thought by many that these observations may have been the bacteriocidal effect of phages. However, it wasn’t until 1917,that French Canadian scientist Felix D’Herelle characterised the nature of these viruses and named them ‘bacteriophages’ [8]. He has also been attributed with coining the word ‘plaque’ (to describe the zone of clearing caused by phages on a bacterial lawn) and developed protocols for plaque assays and burst size studies. D’Herelle who was working in the Institut Pasteur in Paris, immediately recognised the potential of using phages as an antibacterial therapy. During the 1920s and 1930s phage therapy saw modest success. However, the commercialisation of penicillin in the 1940s lead to the widespread use of antibiotics and phage therapy was largely abandoned in Western countries. Fundamental phage biology remained an area of interest and has been paramount to establishing modern molecular biology and microbial genetics [9]. Most notably, studies on the biology of E. coli phages, lambda and T4, played an important part in many fundamental discoveries, including gene expression and DNA replication [10, 11]. In recent years, an interest in phage therapy in Western countries has been rekindled. This renaissance is mainly due to the widespread emergence of antibiotic resistance in pathogenic bacteria [12, 13] and the fact that discovery or development of new antibiotics has significantly declined. There are currently many active phage researchers around the globe advocating for the approval of phage therapy in Western medicine [14]. The renewed interest in phage therapy has also lead to the creation of the field of ‘enzybiotics’. This word was coined to descirbe a treatment which uses purified phage-encoded enzymes as antibacterial agents [15]. This chapter discusses how phage therapy and phage-enzybiotics have been demonstrated as effective agents for elinimation of a broad range of infectious bacteria including antibiotic resistant strains.

Fig. 1 Main steps of lytic and lysogenic phage infection cycles in Caudoviridae (tailed phage); a.) Adsorption: Phage binds to specific receptor molecules on the host cell. b.) Injection: DNA passes through the phage tail into host cell. c.) Reprogramming of host metabolic processes for phage gene expression and Replication of phage DNA. d.) Assembly: Phage DNA is packaged into preassembled icosahedral protein procapsid. e.) Cell lysis: Phage-encoded holin and endolysin proteins reach critical levels. Holin forms a pore in the cell membrane. Endolysin exits through the pore and hydrolyses the cell wall peptidoglycan, resulting in lysis and release of phage particles. The steps outlined thus far are common to both lytic and temperate phage. Step f, g and h are only associated with temperate phage; f.) Lysogenisation: phage DNA integrates into the host genome and is now referred to as a prophage. g.) Transmission: phage DNA remains silent. It is replicated along with the hosts genome and is transmitted to daughter cells. h.) Induction: Phage DNA excises from the host genome and enters the lytic cycle.


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2. Phage therapy

As natural killers of bacteria, phages are obvious candidates for exploitation as antibacterial agents. Phages have many intrinsic characteristics which make them attractive candidates for such applications. They cannot replicate in eukaryotic cells or incorporate their DNA into the genome of such cells. They are highly specific in their bactericidal potential. They generally target a single bacterial species and some phages are even strain specific. This means that there is little or no effect on the natural microbiota of the patient/ animal. This is a strong advantage over the many broad range anti-microbials (including antibiotics) which are in common use today. In addition, there have been no reports of purified phages having harmful effects on eukaryotic cells. Humans are exposed to phages in the natural environment every day without adverse side effects, indicating their non toxic nature. Phages are considered to be self-dosing, as they will multiply in the presence of the target bacteria and cannot multiply in its absence and thus get eliminated from the human/animal. Phages also have the ability to lyse bacteria present in a biofilm, mucous membrane and supurrative wounds. These are bacterial niches, which are unsuitable for treatment with antibiotics [16-20]. In addition, a synergistic relationship has been demonstrated between the action of phages and antibiotics [21, 22]. Dual phage-antibiotic therapies could lead to a reduction inthe emergence of antibiotic resistant strains. In order for phage therapy to become widely accepted as an effective antibacterial strategy, it is essential to recognise its potential shortcomings and look at how these may be addressed. As with antibiotics, bacteria have been reported to develop resistance against phage infection. However, this can be dealt with in a number of ways. Because phages are ubiquitious, an alternative phage, which adsorbs to different receptor molecules on the bacteria, can readily be isolated from the environment using simple, low-cost techniques. A noteworthy point in relation to phage resistance is that it has been associated with reduced bacterial virulence [23, 24]. Often this is because the phage receptor also acts as a virulence factor. The fact that phages are so specific in their choice of host can also be disadvantageous because an individual strain of phage can not be used as a generic treatment against various infections. Therefore, one is required to isolate and characterise the infectious bacterium and matching it with an effective lytic phage, before proceeding with treatment. In order to circumvent the limitations of phage specificity, mixtures of phages targeting various host strains have been successfully employed [25]. To date, phages are generally used where antibiotics have failed. It is therefore worth noting that antibiotic resistant bacteria have a low level of genetic variability, thus reducing the problem of phage specificity in this respect [26].

2.1. Clinical phage therapy applications

The potential to utilise phages for clinical treatment of infectious disease was recognised at the time of their discovery and it became widely referred to as ‘phage therapy’. The first clinical trial in this area was carried out by d’Herelle in 1919 in the Hôpital des Enfants-Malades in Paris [9]. It involved the treatment of severe dysentry in a 12 yr old boy. A single treatment with phage preparation resulted in complete recovery of the boy within a few days. A delay in publishing the results of this trial meant that in 1921 Bruynoghe and Maisin were in fact the first to offically report on a successful phage therapy trial in humans [27]. Their work involved local injection of an anti-staphylococcal phage preparation to cutaneous boils which resulted in reduction of the infectious agents and of the associated symptoms within 24-48 hrs [28]. As a result of such trials, the concept of phage therapy was initially recieved with great enthusiasm and quickly gained widespread recognition internationally. In 1918, George Eliava the director of a newly establised Institute of Microbiology in Tbilisi, Georgia, travellved to Paris to gain up-to-date expertise and equipment for the new Institute. During this time he worked with d’Herelle who trained him in phage therapy and subsequently Eliava brought d’Herelle’s theories and methodologies back to Tbilisi. The Eliava Institute in Tbilisi remains a major centre of phage therapy up to the present day. The commercial prospects, of such a natural antibacterial treatment, was also quickly recognised in the West and by the 1930s companies such as Eli Lily (USA) and Robert and Carrière, a french company later to become L’Oréal had begun to market phage preparations as antibacterial therapeutics [28]. This period of enthusiasm was relatively short-lived in Western countries and the decline is generally attributed to the following factors: lack of understanding of phage biology and phage-host interactions, poorly designed experiments and the discovery and widespread marketing of antibiotics in the 1940s, which proved a strong competitor (as they generally had a very broad spectrum and did not require medical practitioners to have any microbiological expertise). World War II was also an influential factor because although the use of phage therapy remained popular in the Former Soviet Union, the majority of the data produced was not accessible to scientists in the West. A comprehensive review, written in English, by Sulakvelidze et al. presents an invaluable account of phage therapy experience in the Former Soviet Union [28]. However, the main focus of phage therapy trials in the former Soviet Union was military application. Therefore, a lot of the data is poorly documented in scientific journals and due to military secrecy has remained inaccessible. In addition to Tbilisi, the Hirszfeld Institute in Wroclaw, Poland also became and still is an important centre for phage therapy. The research and application of phage therapy in these Institutes has focused mainly on preventing and treating enteric diseases, such as dysentry, and surgical and wound infections [28]. It has been reported that during the 1980s the Eliava Institute employed 1,200 people and often produced several tons of phage preparations a day against a variety of pathogenic bacteria which were used throughout the Soviet Union [28]. One of the most note-worthy studies


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performed in Tbilisi involved a thorough evaluation of the prophylactic effect of a therapeutic cocktail of phages against bacterial dysentry [29]. This study was performed during 1963 and 1964, with a group of 30,769 children, under seven years of age. They were divided into two groups depending on the side of the street they lived on. One group (17,044 children) were given an oral administratin of Shigella phage once a week over 109 days and the other group (13,725) did not receive phages. Clinical diagnosis showed that the occurence of dysentry was 3.8 times lower in the phage-treated group. This is one of the very few cases of large scale, placebo-controlled, randomized clinical trial which has investigated the efficacy of phage therapy. Other clinical studies from the Fomer Soviet Union which demonstrated the efficacy of phage therapy have included treatment of; surgical wound infections [30], urological infections [31], neonatal sepsis [32], staphylococcal lung infections [33], P. aeruginosa infections in cystic fibrosis patients [34] and ocular infections [35]. In Poland a more directed approach to phage therapy has been used, in that the bacterial infectious agents were isolated from patients and screened against their extensive phage bank in order to chose the most suitable phage mixture. The efficacy of this approach was demonstrated in a study by Zhukov-Verezhnikov et al. in 1978 [36]. In contrast to Georgia, phage therapy in Poland is based on ‘compassionate use’ i.e. phage therapy is only used in cases where antibiotics have failed. Detailed reports have been compiled on the treatment of each patient with phage therapy in the Hirszfeld Institute. As in Georgia, the results have been strongly in favor of phage therapy. A report published in English by Slopek et al. in 1987, reviewed the treatment of 550 patients treated in different clinics and health centres in three different cities in Poland [16]. The report addressed infections associated with a wide range of pathogens including; Staphylococci, Pseudomonas, Escherichia, Klebsiella, and Salmonella. An overall cure rate of 92.4% was reported. An improvement in condition was demonstrated in 6.9% of patients and only 0.7% of patients were found not to respond to the phage treatment. Despite there being an extensive body of work reported from Georgia and Poland demonstrating the safety and efficacy of phage therapy in eliminating antibiotic resistant infections the vast majority of these do not meet the criteria set out by modern US and European regulatory authorities. In order to aquire approval by the American Food and Drug Administration (FDA) any phage used for therapeutic purposes must be well characterised, including genome annotation, which indicates that the phage is an obligate lytic phage and does not contain known virulence factors. It must not be able to avoid clearance from the body and must be sourced from the natural environment. Phages which use host virulence factors as receptors are preferred so that if phage resistance develops the virulence of the host will concurrently be decreased [37]. Well characterised growth media must be used and phage lysates must be purified to remove harmful substances such as endotoxins released by lysed host cells [38]. Sterility and stability tests are required, as are animal trials which demonstrate the safety of the therapeutic preparation [37]. An in-depth review on preparation of phages for phage therapy has been published by Gill and Hyman [39]. The pharmacokinetics and pharmacodynamics of phage therapy differ significantly from those of antibiotic treatments, not least due to the ability of phages to replicate at the site of infection. Thus, the following must be considered for the design of a reliable clinical trial: time of treatment, dosage relative to the location of the infection and the route of admnistration and if a single phage or a mixture is being used for treatment [40]. In 2005, the Hirszfeld Institute in Worclaw established an official Phage Therapy Clinic which facilitates human phage therapy clinical trials which comply with current EU regulations. Recently, a report was published which analysed the safety and efficacy of the treatment of 153 patients with phage therapy in this clinic, between January 2008 and December 2010 [41]. Complete pathogen elimination and recovery was reported for 18% of the patients and a positive clinical response was seen in 40%. Less than 4% did not complete the treatment due to adverse events that may or may not have been attributed to the phage preparation. The overall conclusion from the data analysis was that phage therapy can provide effective treatment for a range of bacterial infections, against which all other available therapies have failed. Although most reports on phage therapy over the past ninety years have come from Georgia and Poland, there has been a constant interest in phage therapy in the West which has recently gained momentum. The first commercially available phage cocktails, produced by d’Herelle’s lab in Paris, were still produced in France up until the late 1970s. Likewise, a staphylococcal phage lysate (initially called Lincoln Bacteriophage Lysate and later known as SPL) has been commercially available in USA since about the 1950s. In 1994 Delmont, the company producing this phage lysate, ceased production of SPL for human use, pending additional human efficacy trials. In a document submitted to the FDA in 2000 Delmont Laboratories Inc. stated that clinical effectiveness was difficult to evaluate because the diseases it was being used to treat were inherently variable, the availability of licensed SPL made enrolment for clinical trials difficult and the comparative rarity of certain staphylococcal diseases also added to the difficulty [42]. However, SPL is still available for treatment of staphylococcal infections in dogs [43]. Furthermore it is interesting to note that, occasionally a small number of physicians in the west, turn to phage therapy when all other available therapies fail. Often these phage preparations have been sourced from Eastern Europe or at times phages have been isolated from the environment by screening with the identified infectious bacterial strain. In order to be more accessible for use in a clinical setting phage therapy will need to complete large scale clinical trials which comply with US FDA or European EMA guidelines [44]. In this regard, regulatory hurdles are high and substantial monetary investment is necessary. Despite these challenges a number of clinical trials are underway in the West. In the UK, Biocontrol Ltd. carried out clinical trials which investigated antibiotic resistant P. aeruginosa


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infection in the ear which causes chronic otitis. The poor efficacy of antibiotics in treating this disease is believed to be due to the production of a biofilm by the infectious agent. The group first performed an in vivo study on dogs with pseudomonad ear infections and subsequently used the successful outcomes from this study to gain approval for phase I/II human clinical trials. This clinical investigation was a randomised, double-blind, placebo-controlled clinical trial which involved 24 patients with chronic otitis caused by antibiotic-resistant P. aeruginosa strains susceptible to one or more of the six phages present in the cocktail used for treatment. Approval for the trial was granted by the UK Medicines and Healthcare products Regulatory Agency (MHRA) and the Central Office for Research Ethics Committees (COREC) ethical review process. Overall, significant clinical improvement and reduction in P. aeruginosa colonisation was seen in the phage-treated group but not in the placebo group. The results showed that the average bacterial count had decreased by 80% in the phage-treated group and a slight increase was observed in the control group. In terms of safety no treatment-related adverse event was observed. It has been reported that this company are now in preparation for large scale phase III clinical trials for phage treatment of otitis infections and are also pursuing other avenues of treatment for their phage preparation, including treatment of burn wounds and lung infections in cystic fibrosis patients [43]. In 2011, Biocontrol Ltd. merged with Targeted Genetics Inc. to create the company now known as AmpliPhi BioSciences Corporation. They have been granted a patent in Australia (2010) and in the US (2013) for ‘bacteriophage-containing therapeutic agents’ for treatment of infections associated with P. aeruginosa biofilms. Two recent studies have reported the efficacy of phages in controlling acute infection of P. aeruginosa in a murine lung model [45, 46]. In addition, it has been reported that phages were capable of causing a 3-4 log reduction of P. aeruginosa in a biofilm formed on a cystic fibrosis bronchial epithelial CFBE41o- cell line [45]. Another area which has seen success in relation to phage therapy, is the treatment of suppurative wounds infections. A physician initiated phase I trial was performed in a wound care centre in Texas [47]. This trial was approved by the FDA and involved a prospective, randomised, double-blind study to investigate the safety of using an 8-phage cocktail, produced by the company Intralytix, on patients with venous leg ulcers. The phages included were lytic against P. aeruginosa, S. aureus and E. coli. The study involved 42 patients, 39 of whom completed the trial (21=contol group, 18 = treatment group). The 50ml of phage preparation or sterile saline was administered via an ultrasonic debridement device. No adverse events were associated with the treatment. Other studies have also shown the efficacy of phage therapy in treating infections associated with burn wounds [48-50]. Staff at the Burn Centre of the Queen Astrid Military Hospital in Brussels are currently carrying out controlled clinical trials in burns patients. This trial is a collaboration between the Belgian military and scientists from Universities in Leuven and Ghent. As of yet no efficacy or safety data has been reported in relation to this trial. Also of significance to the treatment of burns was the report by Soothill that phages could be used to prevent destruction of a skin graft cause by pseudomonad infection [51]. The Néstle Corporation have completed two separate phase I clinical safety trials, in Switzerland and Bangladesh, both involving administration of E.coli phage T4 to 15 healthy volunteers. All treatments were reported as being well tolerated. This organisation are currently involved in clinical trials in Bangladesh which are looking at the safety and efficacy of phage therapy against diarrhoea associated E.coli infection in children. They are hoping to enroll 450 children with diarrhoea due to ETEC (enterotoxigenic E. coli) and/or EPEC (enteropathogenic E.coli) infections and the trial is estimated to be completed by 2014 [52]. This trial represents a major milestone for the advancement of phage therapy as it is the first large scale clinical trial which adheres to current European and American guidelines.

2.2. Phage therapy in veterinary medicine & aquaculture

Aside from prophylaxis and treatment of human infections, phage therapy has also been successfully employed to address a wide range of infectious diseases in animals. The benefits of performing animal trials are two-fold; they are often required for supporting data when applying for human clinical trials and secondly, they may also be persued for direct application in veterinary medicine. D’Herelle’s first phage therapy trials involved treatment and prevention of typhoid in chickens [53]. In one such trial a hundred chickens were infected with Salmonella serovar Gallinarum. Twenty of these chickens were treated with S. Gallinarum-specific phages and all of these birds survived while 75% of untreated birds died [54]. The revival of phage therapy in the west in the 1980s is often linked to the studies by Smith and Huggins in the Institute for animal Disease research in Houghton, in the UK. They performed a series of animal trials which looked at neuromusclar and intracerebral administration of phages to treat mice with experimentally induced E. coli septicemia and also investigated the control of diarrhoea associated E. coli infection in calves, piglets and lambs [24, 55, 56]. In these animal trials the phages were found to protect the animal against E.coli infection when phages were administered soon after challenge. However, after the onset of symptoms associated with infection (~16 hr post challenge) the phages were not effective in preventing the disease but it did reduce severity of symptoms and the rate of mortality. Better understanding of this time-dependant efficacy will help in the design of more effective phage therapy strategies. As well as looking at the benefits of phage treatment, Smith and Huggins also investigated the factors influencing the survival and multiplication of phages in calves and in their environment [57]. Also in relation to the control of E. coli infection by phages, Huff et al. [58-61] investigated the potential of phage therapy in the treatment of colibacillosis respiratory infection in broiler chickens and looked at the efficacy of treatment using the following routes of phage administration: in drinking water [61], as an aerosol [60] and as an intramuscular


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injection [60]. The aerosol and intramuscular injection were both found to be effective routes of phage administration while the drinking water route was not. More recent work by Oliveria [62] on treatment of colibacillosis involved treatment of naturally and experimentally infected flocks. In this study two diferent titres of phage were employed, 107pfu/ml and 109pfu/ml. Interestingly it was found that the lower phage titre was ineffective in controlling experimentally induced infections but was sufficient to decrease mortality rates below 0.5% in naturally infected flocks. In addition, studies have also been performed which explored the development of antiphage antibodies in Salmonella infected chickens treated with phages via oral administration [63] and no phage-neutralising antibodies were detected. A recent review by Carvalho et al. discusses the use of phage therapy for controlling Salmonella and Campylobacter, two important zoonotic pathogens [64]. From the commercial perspective, American phage-based company Intralytix has developed and licensed phage-based products effective against Salmonella (PLSV-1™) and Clostridium perfringens (INT-401™) in poultry [65, 66]. The aquaculture industry is another area where there has been considerable interest in using phage therapy. Pathogenic bacteria which have been targeted in this context include Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas plecoglossicida, Edwardsiella tarda, Aeromonas hydrophila, Vibrio harveyi, Aeromonas salmonicida, Streptococcus inia, Edwardsiella ictaluri and Flavobacterium columnare [67]. Antibiotics have traditionally been used to eliminate these pathogens but with the growing concern over the use of antibiotics in husbandry, phages are a worth while alternative. It is important to mention that animal phage therapy trials do not have the same extent of regulatory hurdles as human trials and are likely to set an important precedent for phage therapy in a clinical setting.

2.3. Phage biocontrol of phytopathogens

Phage treatment of crop-associated infections was first proposed in 1926 [68]. Since then many studies investigating phage therapy as a method of plant pathogen control have been carried out. A review of the early literature on phage therapy for treatment of phytopathogens was published by Okabe and Goto [69]. More recent reviews discussing this topic and its applicability in modern agricultural have also been published [70, 71]. Research in this area has included but is not limited to studies involving the following; crown gall, bacterial wilt and and bacterial spot in tomatoes, soft rot and potato scab in potatoes, fire blight in pome fruits, bacterial soft rot in Calla lily, bacterial wilt in tobacco plants, bacterial spot on stone fruits, leaf blight in onions, bacterial leaft spot in mungbeans, blight in walnuts, leaf and fruit spot in peaches, black sopt in peppers and cranker and black spot in citrus fruits [71]. One problem associated with using phage therapy in conrol of phytopathogens is the rapid destruction of phage particles by UV radiation [72]. Therefore, the timing of application is important when designing an effective field trial, with application in the evening being more effective. Other factors to be considered include: where the bacteria are located and if they are accessible to phages, the moisture levels in the environment and on the leaf (phages work better when there is some moisture through which it can diffuse) and the presence of other chemicals which may compromise the viabiliy of the phage population [73]. Agriphage produced by American company Omnilytics is a prime example of a phage product commercially available for the treatment of phytopathogens. Another application of phages in relation to horticulture is controlling the level of competitors of beneficial bacteria such as nitrogen fixing strains [74].

2.4. Phage biocontrol in the food industry

As well as being used to decontaminate animals and plants that will enter the food chain, phages can be applied directly to food for control of food borne pathogens. A major step forward, in phage-related biocontrol, occured in 2006 when the FDA approved, under generally recognised as safe (GRAS) criteria, two phage cocktails, namely ListShield (produced by Intralytix) and Listex (produced by MICREOS formerly EBI) for the prevention of Listeria contamination on ready-to-eat foods. In 2009 the European Food and Safety Authority (EFSA) approved phage-related preparations as additives to organic foods and in August 2012 the regulatory body Food Standards Australia and New Zealand (FSANZ) approved that phages could be used as a food processing aid. Applications of phage biocontrol in the food sector have been discussed in several comprehensive reviews [75-79].

3. Enzybiotics

Enzybiotics refers to enzymes which can be utilised as bacteriocidal agents. When the word was first coined it was in reference to phage-encoded bacteriolytic enzymes and thus it is sometimes used to specifically refer to such enzymes. However, now the word has a broader meaning encompassing all enzymes which are capable of killing bacteria. Apart from phage encoded enzybiotics, these include enzymes such as autolysins and lysozymes. The increasing interest in this field of research is demonstrated by the recent creation of databases such as phiBiotics [80] and EnzyBase [81] which provide data on the growing number of characterised enzybiotics. Phage encoded enzybiotics include two classes of peptidoglycan hydrolase enzymes namely endolysins and virion associated hydrolase. Endolysins are the better characterised of the two and thus are the focus of the following section. As mentioned previously, endolysins are phage-encoded proteins involved in the lytic cycle of phage replication (See section 1.2). They work in conjunction


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with a holin protein. The holin forms a pore in the bacterial cell membrane through which the endolysin exits in order to gain access to the peptidoglycan layer. Subsequently the endolysin cleaves bonds in the peptidoglycan which leads to lysis of the host cell. When endolysins are applied exogenously to specific Gram-positive pathogens they have direct access to the peptidoglycan in the bacterial cell wall and can cause rapid lysis, without the assistance of a holin. This characteristic lead to the exploration of phage lysins as novel antibacterials with potential applications in healthcare, veterinary, agriculture, food and biotechnology sectors. In order to take advantage of their bactericidal potential, phage-encoded endolysins are now being cloned and overexpressed in well-defined expression systems and subsequently purified to investigate their antibacterial activity. Phage endolysins demonstrate certain advantages over whole phage and antibiotics, for the control of Gram-positive problematic bacteria which are outlined in the following sections. Progress in this area has been thoroughly discussed in a number of indepth reviews [82-85]. In relation to Gram-negative bacteria, the presence of the outer cell membrane prevents access to the peptidoglycan by exogenously-applied lysins. For this reason the main focus of the following section is on the potential of endolysins which target Gram-positive pathogens. It is worth noting however, that methods enabling Gram-negative lysins to permeate the outer membrane are actively being pursued [86-90].

3.1. Endolysin structure & function

A broad range of recombinant endolysins targeting pathogenic bacteria have been characterised to date. Endolysins encoded by phages infecting Gram-positive hosts generally have a modular architecture containing a peptidoglycan hydrolase domain, usually located at the N-terminus and quite often a cell wall binding domain (CBD) at the C-terminus. Two prominent exceptions to this rule are staphylococcal and mycobacterial phage endolysins which commonly contain three domains. Staphylococcal endolysins generally contain two peptidoglycan hydrolysing domains (most commonly an amidase and an endopeptidase) and a CBD domain [91]. The mycobacterial phage endolysins generally contain a CBD, a peptidoglycan hydrolysing domain and a putative peptidase which may also cleave the peptidoglycan [92]. Due to the complex structure of the mycobacterial cell wall the lysis mechanism demonstrates a complementary level of complexity. In addition to involving a holin and endolysin it also requires lysin B which targets mycolic acid and chaperones to direct the endolysin to the peptidoglycan [92].The lytic domains of endolysins can be classified into five types based on where they cleave the peptidoglycan structure, as seen in Fig. 2.

The function of the CBD in endolysins is two-fold. It targets the lytic domain to its substrate and it keeps it secured there after lysis so that it is not available to lyse nearby cells which could act as a host for phage replication. The lysins which have been characterised for Gram-negative bacteria are generally single-domain globular enzymes which do not contain a CBD [84]. In the case of Gram-negative endolysins, the absence of a CBD could be explained by the fact that released endolysins cannot harm potential phage host cells as is the case with Gram-positive lysins. However, there are exceptions to this single domain rule. A recent investigation by Oliveira et al. gives a comprehensive account of the the structure of lysins targeting both Gram postive and Gram-negative endolysins and reports putative binding domains in several Gram-negative endolysins [91]. It is noteworthy that studies have been performed which demonstrate that removal of the CBD can result in altered bacteriolytic activity. In some cases the activity was enhanced [93, 94] while in others it was reduced [95-97]. Such findings demonstrate the potential to alter the character of an endolysin via protein engineering. This topic will be discussed in more depth later in this chapter. In order to gain better understanding of the mechanism of action used by endolysins, attempts have been made to elucidate the protein structure by X-ray crystallography. Several single-domain, globular phage lysins and some individual lytic domains from modular lysins have been successfully crystalised [84]. However, to date, only three

Fig. 2 The structure of Staphylococcus aureus peptidoglycan with the cleavage site of the five main types of enzymatic domain found in endolysins: 1.) N-acetyl-β-D-muramidase (lysozymes) 2.) lytic transglycosylase 3.) N-acetyl-β-D-glucosaminidase 4.) N-acetylmuramoyl-L-alanine amidases 5.) Endopeptidase. NAM: N-acetylmuramic acid. NAG: N-acetylglucosamine.


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complete modular endolysins have been crystalised, namely, Clp-1 [98], PlyPSA [96] and PlyC [99]. The inherent difficulty in crystallising whole endolysins with modular structure has been attributed to the flexible linker region between domains [84]. The PlyC lysin presents an interesting case as it is the only reported endolysin encoded by more than one gene. Crystal structure analysis revealed that the functional PlyC endolysin is composed of a PlyCA molecule in association with a ring-shaped assembly of eight PlyCB subunits [99]. Other methods which have been used to investigate the structure of phage lysins include nuclear magnetic resonance [100], in silico modelling based on homologous sequences [101, 102] and various biophysical techniques such as circular dichroism, small angle X-ray scattering data and sedimentation velocity experiments [103].

3.2. Specificity of endolysins

One of the advantages of using phage lysins over antibiotics is their targeted specificity, which greatly reduces the risk of dysbiosis. Both the enzymatic and CBD domain can contribute to the overall specificity of an endolysin. There is a high level of variability in peptidoglycan chemotypes in Gram-positive genus [104]. By targeting a bond in the peptidoglycan which is highly genus- or species-specific, the catalytic domain has a high level of discrimination in terms of target bacteria. The binding specificity of a CBD can be investigated by fusing the CBD domain with indicator proteins such as GFP. Its target site is commonly found to be genus-specific, as has been demonstrated for the SH3b domain of certain staphylococcal phage endolysins [105], but in some cases it can have specificity down to the serovar level, as has been reported for Listeria monocytogenes phage endolysins [106]. Because of their high specificity, CBD’s fused with indicator proteins have significant potential as tools for rapid detection of bacterial pathogens.

3.3. Bacterial endolysin resistance

The prevalence of antibiotic-resistant bacteria is a serious threat to the modern healthcare system. One significant advantage of lysins is that, unlike antibiotics and whole phages there have been no reports of development of bacterial resistance. Repeated exposure of bacteria to low levels of phage lysin, both on agar and in liquid culture, did not result in development of resistant strains [107]. In the same study, mutagenesis was also employed to increase the chance of finding lysin resistant strains, but none were detected. However, using similar methods to induce resistance to the antibiotics novobiocin and streptomycin, resistant mutants were readily identified. When strains were exposed to the same mutagenesis strategy, roughly 1,000-fold and 10,000-fold increases, in novobiocin and streptomycin resistance occured, respectively. Thus, the likelihood of bacteria developing endolysin resistance appears to be very low.

3.4. In vivo trials & immunogenicity

In 2001, the group of Fischetti [15] reported the first successful in vivo endolysin trial, which involved prophylaxis and treatment of upper respiratory streptococcal infection in mice, using streptococcal lysin PlyC. Several publications since then have investigated the in vivo efficacy of phage lysins against a wide range of pathogens such as, S. aureus (including MRSA) [108-110], B. anthracis [111, 112], S. pneumoniae [113-118] and group B streptococci (GBS) [94, 119]. These studies have demonstrated that phage lysins are capable of treating systemic and mucosal infections. The latter is a very significant result, as mucosal membranes are a reservoir for many pathogens, including antibiotic resistant strains and currently prophylactic treatment against such infections are limited to polysporin and mupirocin ointments [82]. In addition phage endolysins have been shown to act synergistically with other bacteriolytic enzymes and antibiotics in vivo [114, 120]. A few studies have investigated the immunogenicity of endolysins and how this can effect the efficacy of these enzymes in control of systemic and mucosal infections [116, 121, 122]. In general, the immunogenicity studies undertaken have demonstrated that phage lysins do induce antibody production. However, in the case of Cpl-1, it was observed that antibodies only moderately reduced the efficacy of lysin treatment in vivo [116]. In addition, it was found that cytokine concentration was increased in mice treated with a continuous intravenous infusion of Cpl-1 phage lysin compared to untreated mice [115]. However, in a separate study involving the same enzyme administered at 12h intervals, a reduced cytokine concentration was recorded in lysin-treated compared to untreated mice [118]. This demonstrates the importance of choosing the correct dosage and timing for treatment. Another clinically relevant point about phage lysins is that they have proven to be effective in preventing and eliminating bacterial biofilms [123, 124] which generally prove refactory to removal by the immune system or antibiotics.

3.5. Engineered lysins

The modular structure of lysins allows for domain swapping by protein engineering and has been employed to produce chimeric lysins with altered host spectra and improved lytic activity [125,126]. It has also been demonstrated that site-directed or random mutagenesis may be used to improve lysin activity [94] or to make lysins more thermostable [127]. The production of recombinant full length endolysins is often hindered by low levels of solubility. With this in mind chimeric lysins have been produced which have improved solubility over the parental molecule [128, 129]. In addition


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by modifying lysins with polyethylene glycol [130] or the Fc region of IgG [131] the half-life of these molecules could be extended to improve the efficacy of these molecules in treating systemic infection.

4. Conclusion

Phages and phage-encoded lysins present alternative, non-toxic methods of specifically controlling pathogenic bacteria alone or in combination with currently used antibacterial agents. Antibiotics are no longer sufficient for the control of many infections and the requirement for novel approaches is clearly evident. There is a considerable amount of data available which demonstrates the efficacy of phage-based antimicrobials for treatment and prophylaxis of bacterial infections. However, there remain several hurdles which must be overcome before they become either a complementary or a first-line defence against bacterial pathogens. From a regulatory point of view, the classification of phage-based products for therapeutic application requires clarification and may require adaptation of current medical product regulatory frameworks. While the results of the clinical trials described in this chapter are very promising, there is an urgent need for additional well-designed, double-blinded, placebo-based, large scale human clinical trials. There is also a need for in-depth characterisation of the functional role of as-yet unassigned open reading frames encoded in the genomes of therapeutic phages; research which could well result in novel phage-derived products. With regard to endolysins, the transition from animal to human clinical trials will require further knowledge on their pharmacokinetic and pharmacodynamic nature and also their immunogenic properties. Furthermore, better understanding of the interactions between phage and their bacterial host can help identify novel targets for phage-based drug development [132].

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