Bacteriophages as anti-infective agents: recent developments and regulatory challenges

Abstract

The biennial meeting on ‘Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine’, held in London, UK, on 20 January 2012, and chaired by George Salmond (University of Cambridge, UK) hosted over 50 participants representing 13 countries. The highly multidisciplinary meeting covered a diverse range of topics, reflecting the current expansion of interest in this field, including the use of bacteriophages as the source of biochemical reagents for molecular biology, bacteriophages for the treatment of human and animal diseases, bacteriophage-based diagnostics and therapeutic delivery technologies and necessity for, and regulatory challenges associated with, robust clinical trials of phage-based therapeutics. This report focuses on a number of presentations from the meeting relating to cutting-edge research on bacteriophages as anti-infective agents.

Keywords::

• bacteriophage
• biofilm
• Campylobacter jejuni biofilm
• clinical trial
• Clostridium difficile
• phage delivery

Bacteriophages are the most abundant biological entities on earth, and it is estimated that phage predation reduces the global bacterial population by half every 48 h. Bacteriophages also drive adaptive evolution in their bacterial hosts both through predator–prey interaction dynamics and via adaptive impacts of lysogeny. Despite predating the discovery of antibiotics by several decades, bacteriophage therapy was largely supplanted by antibiotics and their use in Western medicine declined. However, bacteriophages have several characteristics that make them potentially attractive next-generation therapeutic agents, with several benefits over conventional antibiotics. Phages are highly specific and, therefore, do not disturb normal flora; replicate in their host, facilitating effective treatment by delivery of a low-phage dose; are amplified at the site of infection; and are capable of rapid adaptation to combat emergence of bacterial resistance. Bacteriophages, therefore, hold significant promise as a therapeutic approach in the face of widespread antibiotic resistance. In addition, bacteriophages are sources of biochemical reagents, which are of value in modern molecular biology and as anti-infective chemotherapeutics in their own right.

Bacteriophages in anti-infective therapy

Martha Clokie (University of Leicester, UK) presented recent work on the nature, isolation and exploitation of phages for pathogenic anaerobes, in particular Clostridium difficile. C. difficile (of which there are over 350 ribotypes) is a major causative organism in antibiotic-associated diarrhea and pseudomembranous colitis. Outbreaks of C. difficile infections are common in industrialized nations and are associated with significant morbidity and mortality. Furthermore, such outbreaks impose a significant financial burden on healthcare providers, yet remain difficult to rapidly diagnose and treat effectively. The work of Clokie and colleagues has revealed that temperate phage carriage among infected individuals is common and may contribute to virulence [1]. Her group has spearheaded the search for lytic C. difficile phages, which may have diagnostic and therapeutic value, and has led to the establishment of a panel of 20 lytic bacteriophages, which target clinically relevant, dominant ribotypes of C. difficile commonly associated with infection outbreaks in UK hospitals. This phage library has been extensively characterized according to their morphology, genome sequence, infection parameters and host range using a number of in vitro and in vivo methods. These bacteriophages are likely to be of significant therapeutic potential in the management of such outbreaks and, importantly, this work has pioneered novel methods for isolation of phages targeting anaerobic pathogens.

Ian Connerton (University of Nottingham, UK) highlighted the potential of bacteriophages as alternatives to conventional antimicrobial approaches in the control of food-borne pathogens, in particular Campylobacter species, including Campylobacter jejuni, a leading cause of food-borne illness globally. Bacteriophages have the potential to be used to reduce the high incidence of Campylobacter carriage in UK poultry (up to 80%) [2] and thus reduce their entry into the human food chain. In addition, Campylobacter bacteriophages could prove potentially useful agents not just for therapy but also in bio-sanitation, whereby phages may be directly applied to food (carcasses or poultry meat) and inanimate surfaces in processing and packaging facilities (e.g., surface-adhered biofilms) and reduce the number of food-borne pathogens in poultry products. Biofilms in engineered water systems, such as drinker lines in poultry farms, are widely recognized as a potential reservoir for pathogens, including enteric pathogens such as Campylobacter spp. A number of bacteriophages isolated and characterized by Connerton and coworkers have been shown to be effective in the control of C. jejuni and to disperse C. jejuni biofilms in vitro. For example, Camplyobacter-specific virulent phages CP8 and CP30 significantly (1- to 3-log reductions) reduced C. jejuni biofilms in vitro [3,4]. The use of bacteriophage in control of food-borne pathogens would be subject to regulation in the EU under directive 89/107EEC (Food Additives and Processing Aids), which bans the use of any chemical or substance in food preparation or processing unless approved by the EU. This poses a particular regulatory challenge relating to the use of bacteriophages for decontamination of poultry products, as they could potentially be classified as both an additive and a processing aid.

Marine Henry (Institut Pasteur, France) described recent investigations into the utility of environmental bacteriophages isolated (from sewage in the Paris area, France) as potential therapeutic agents for the treatment of Pseudomonas aeruginosa infections. Using a mouse model of pulmonary infections (as previously described [5]) and employing a bioluminescent strain of P. aeruginosa (PAK) to record real-time imaging of infection progression, the efficacy of eight different P. aeruginosa bacteriophage treatments was established and correlated to in vitro efficacies. Using this model, a group from Institut Pasteur has demonstrated that bacteriophages isolated from the environment are active against both PAK and CHA P. aeruginosa strains, and are both preventative and curative in this model of pulmonary infection [6]. Phages from this panel (PAK-P1-5) were particularly effective, producing more than 70% survival rates 8 days postinfection. Importantly, this study provides clear evidence that light quantification 8 h postinfection (6 h after phage treatment) could be used as a rapid screening tool for the prediction of bacteriophage therapeutic potential in vivo.

Another potential application for bacteriophages and their products in anti-infective therapy, namely the use of biopolymer-degrading enzymes as tools for biofilm disruption, was the subject of a presentation by Jakub Barylski (Adam Mickiewicz University, Poland). The isolation of an unknown bacteriophage and host (16 sRNA gene sequencing indicates significant similarity to Bacillus pumilis) from lake Goreckie (Poland) led to the identification of two novel enzymes speculated to be involved in biopolymer degradation, poly-γ-glutamate and a polysaccharide lyase (similar to pectin lyase-like proteins described previously from various bacilli and their phages). These enzymes are likely to be involved in initial degradation of the host mucoid capsule and may be useful in disruption of the exopolysaccharide biofilm matrix. Such enzymes may augment the effectiveness of conventional antimicrobial agents against bacteria in the biofilm mode of growth.

The ability of bacteria to survive phage infection was the subject of an exciting presentation by Tim Blower (University of Cambridge, UK). Bacteria have evolved a number of mechanisms to resist phage infection. Phage abortive infection systems promote cell death (altruistic suicide) in infected cells and thus limit phage replication within the bacterial population. The phage abortive infection system from Erwinia carotovora subspecies atroseptica, ToxIN, represents a novel class of protein–RNA toxin–antitoxin (TA) pair, consisting of a protein toxin and a specific RNA antitoxin [7]. Homolog of this new class of TA system are widespread in various enteric bacteria and mediate abortive infection of multiple phages. In response to stimuli (e.g., phage infection), the antitoxin component is degraded and the toxin elaborates its effects [8]. Although much remains unknown about the exact biological roles of TA systems, the antiphage activity of these systems will be a fundamentally important consideration in phage therapy.

Brendan Gilmore (Queen’s University Belfast, UK) discussed the potential utility of bacteriophages in the treatment of biofilm-mediated medical device infections [9]. The ultimate success of bacteriophage therapeutics for the treatment of a broad spectrum of bacterial infections will depend on the development of suitable delivery technologies and advanced formulations [10]. The stability of T4 bacteriophage in commonly used medical device polymers was described, highlighting the challenges in maintaining phage stability and viability in the formulation of bacteriophages into polymeric systems. The use of microneedle arrays, minimally invasive drug delivery systems, which provide a conduit for therapeutic agents across the stratum corneum [11], to the blood vessels of the papillary dermis, was then examined. Recent work has demonstrated for the first time that the use of a novel prototype polycarbonate hollow microneedle array facilitated the transdermal delivery of a significant T4 phage dose both in vitro (10⁶ PFU/ml) and in vivo (~10³ PFU/ml) in a rat model. Although this delivery technology may provide a useful platform for the transdermal delivery of bacteriophages to the systemic circulation, many challenges remain in the maintenance of phage stability in polymeric systems and in controlling the rate of release of these agents via the transdermal route, for anti-infective therapies.

Finally, the regulatory challenges associated with realizing the potential of bacteriophage therapy were the subject of a presentation by David Harper (AmpliPhi Biosciences Corporation, UK). Bacteriophage therapeutics enjoyed relatively brief exposure in Western medicine before their use was supplanted by conventional antibiotics. Lack of evidence of clinical effectiveness, spurious, unsubstantiated claims of efficacy and, of course, the demonstrated effectiveness of antibiotics themselves resulted in a steep decline in use. Currently, there are no therapeutic phage products approved (or in Phase III trials) for human or veterinary use in the EU or USA, due in part to a dearth of robust clinical trial data of sufficient quality to satisfy Western regulatory bodies, such as the US FDA or European Medicines Agency, regarding the safety and potential efficacy of such therapeutic agents. A number of companies have been actively involved in the Phase I and II clinical trials of bacteriophage preparations for human and animal bacterial infections (e.g., see [12]). Such clinical trial data will be required in order to gain approval for widespread use of phage therapeutics and to accelerate bacteriophage therapy from the bench to the bedside.

Summary

The future of bacteriophage research in fundamental biology, genomics and translational science will be significant drivers of discovery and innovation in diverse fields from diagnostics and other biotechnological applications to therapeutic applications in humans, plants and animals. Several challenges remain in realizing the potential of bacteriophages in these areas but the outcomes, both in fundamental research and translational applications, have the potential to be highly significant.

This meeting was organized by Euroscicon [101]. The next meeting on ‘Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine’, will take place on 22 January 2014 [102].

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.