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A R T I C L E

At a recent conference on infection control, hosted by microbiology expert Oxoid, Professor Geoff Hanlon described bacteriophages and explained the role they could play in treating bacterial infection. Professor Hanlons talk prompted many questions from the audience and became the hot topic of the conference. Here, he expands on a fascinating subject.

Bacteriophage therapy
A once and future solution?
Scientists and clinicians currently working in medical microbiology will have spent most of their working lives attempting to address the issue of antibiotic resistance. It is not a new phenomenon and yet it is an issue that seems to have reached a certain defining moment in time. This is partly because the level of bacterial resistance has reached alarming proportions, but also because the subject has attracted profound media attention and, at the same time, the pharmaceutical industry has backed away from research and development of new antibiotics. So, as the problem marches relentlessly onwards, we do not have the assurance that new drugs for the treatment of multiresistant infection lie just around the corner. The expectant public (and, consequently, politicians) are therefore looking for answers and there is clearly a need to explore alternative avenues to reduce our reliance on antibiotics. Here, I will focus on the possibilities offered by the use of bacteriophages to treat bacterial infections. It is outside the scope of this article to discuss the subject of antibiotic resistance in detail. We are all aware of the extent to which certain bacteria have acquired multiple resistance to antibiotics, and, while media attention has focused on methicillin-resistant Staphylococcus aureus (MRSA), it could be argued that the problems posed by Acinetobacter baumanii and Clostridium difficile are far more acute. It has been suggested that one of the reasons for the emergence of resistance in the West is inappropriate prescribing (partly driven by public demand). Steps are now being taken to address this but antibiotic resistance is a worldwide problem and while we may slow the spread the trend will not be reversed. In the past, the pharmaceutical industry has provided a constant stream of new classes of antibiotics to help stem the tide of resistance development. However, the US Food and Drug Administration (FDA) recently showed that of the nine antibacterial agents approved by it since 1998 only two had novel mechanisms of action. While me too drugs have their value, the battle against antibiotic resistance can only make major strides with the discovery of new chemical classes of drugs. In its study, the FDA looked at the 15 largest pharmaceutical companies to see what products were currently in development. Of 303 new molecular entities currently undergoing research and development (R&D), only five were new antibacterials and none had novel mechanisms of action. Given the long lead-in times for new drugs we cannot expect any new antibiotics in the near future. The reasons for this loss of interest in antibiotic research are entirely understandable. The cost of bringing a new chemical entity to the market place has been estimated to be somewhere between $400 and $800 million, and the process takes about eight years, from phase I clinical trials to product launch. When a new antibiotic is launched, the medical profession quite rightly adopts a cautious approach and often keeps the drug in reserve. Coupled with the fact that, by definition, antibiotics cure the disease they are treating, the pharmaceutical industry has limited opportunities for recouping its R&D outlay before resistance almost inevitably occurs, or patents expire.

Fig 1. Bacteriophages exhibit a variety of different morphological forms.

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Each bacteriophage will only attack one species or even a single strain of bacterium
Figure 3 illustrates the life cycle of a typical lytic bacteriophage. The virus attaches to the bacterial cell via specific receptor sites and then, after making a small hole in the cell wall, injects its DNA into the cell. The viral genome then takes over the metabolic machinery of the bacterium, redirecting its metabolic processes to the manufacture of new virus components. These components are then assembled into complete virions which are released when the cell bursts. In excess of 100 new virus particles may be liberated from a single bacterial cell and each is able to go on to infect a new bacterial cell. The process of phage replication will therefore continue until all the susceptible bacterial cells have been killed. Lysogenic phages are viruses that do not immediately enter a lytic cycle, but instead they integrate their DNA into the host cell DNA. Thus, a permanent association is formed between the phage and the bacterium. When the bacterial DNA replicates, the phage DNA replicates at the same time and so each daughter cell will contain the viral DNA (known as prophage). The prophage directs the synthesis of repressor proteins, blocking its own genes and also those of closely related viruses. Therefore, the cell benefits in some way by this type of immunity to infection from other phages. A prophage sometimes escapes regulation by the repressor and its DNA is cut free,

Fig 2. Typical structure of a bacteriophage.

No wonder then that companies are diverting their resources to the development of products for the long-term treatment of the burgeoning elderly population, such as antihypertensives, lipid-lowering drugs, antidepressants and antidementia drugs. In order to bring industry back onboard, changes are necessary at the highest level, perhaps to provide tax incentives for antibiotic research, to extend the patent life for new antibiotics, or possibly to introduce fasttrack drug approval procedures for new antibacterial agents.

Alternatives to antibiotics

Although much has been said about the end of the antibiotic era, it is fairly certain that these agents will be with us for some time to come and will continue to be the frontline approach for the treatment of bacterial infections. However, we need to take the pressure off them and reduce our reliance on them as the only option available. To this end, a variety of alternative strategies are being explored and some, including photodynamic disinfection, essential oil therapy and treatment with bacteriophages, are showing promise. The remainder of this article will examine the potential of bacteriophage therapy as a possible adjunct to antibiotic treatment of bacterial infections.

morphological types contained within 12 families and their genome may comprise single- or double-stranded DNA (ss-DNA, ds-DNA) or RNA (Fig 1). Of course, they have co-evolved with bacteria over the last 3-4 billion years and so have developed similar abilities to adapt to changing environments. The typical structure of a bacteriophage is shown in Figure 2. The head (or capsid) is a protein shell often of icosahedral shape, and this contains the viral genome. The collar is a contractile structure to which tail fibres are connected. These fibres have receptors at their tips that recognise attachment sites on the bacterial cell wall.

Characteristics of bacteriophages

Bacteriophages (phages) are viruses that only kill bacteria. Indeed, they are highly specific and each phage will only attack one species or even a single strain of bacterium. They are the most abundant life form on earth, outnumbering bacteria by a factor of 10, and have been isolated from many different habitats. There are a variety of different

Fig 3. Life cycle of a typical lytic bacteriophage.

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allowing it to embark on a lytic cycle. However, mistakes are often made when the prophage DNA is cut free and bacterial genes may be incorporated in the viral DNA and then transferred to the next viral host cell. This process is known as transduction and is responsible for the horizontal transference of genes from one bacterial cell to another. Lysogenic phages are not useful for therapy and will not be considered further.

Patients have travelled from the West to Tblisi in Georgia in order to obtain phage therapy not available elsewhere'
due mainly to the fact that those working in the area had little or no understanding of basic phage biology. In 1931 the Council on Pharmacy and Chemistry of the American Medical Association published a damning report which cast doubt on the value of phage therapy. It was at about this time that antibiotics came to the fore and phage therapy was quietly forgotten, at least in the West. However, phage therapy proliferated in the USSR and other Eastern bloc countries such as Poland. The failure of phage therapy in those early days was almost inevitable and was the result of high expectations on the part of the public and poor knowledge of the science behind phage replication. Indeed, it was thought at one time that only a single type of bacteriophage attacked all bacterial cells. The exquisite specificity of phages was not appreciated, nor the difference between lytic and lysogenic viruses. The methods used to prepare the phages were crude and often patients were dosed with products containing no viable phage but large amounts of endotoxin. Patients were also poorly diagnosed and this led to treatment with inappropriate phage products. This situation has, of course, changed and we now know a great deal about the biology of bacteriophages, which have been instrumental in improving our understanding of molecular genetics and basic cell biology. Methods for their isolation and purification are highly refined and preparations can now meet the exacting standards required of pharmaceutical products. These facts, together with our current knowledge of effective clinical trials design, means that we are well placed to revisit some past work and evaluate the true potential of phage therapy. While the West began its love affair with antibiotics the USSR embraced phage therapy, which became part of normal healthcare. The Eliava Institute grew to become a research establishment and a major manufacturer of phage products. At its peak, it employed 1200 people and tons of phage products were manufactured daily and exported throughout the USSR, being used both for therapy and prophylaxis. A major user of phage products was the military, which routinely included these preparations in first aid kits. Therefore, there is a wealth of expertise in the former USSR on the application of bacteriophages in the treatment of bacterial infections. However, much of the work has been published in rather obscure journals, often written in Georgian or Ukrainian, and hence is rather inaccessible. In addition, the quality of the scientific work would not meet the rigorous standards applied in the West, particularly in areas such as clinical trials. The Eliava Institute currently produces a range of phage preparations in a variety of pharmaceutical forms. They can be administered topically, orally, rectally, by inhalation or by injection. The purified phage may be a single clone active against one bacterial species or a mixture of phages that can be used against a broad range of pathogens. An example of a monoclonal preparation is a single staphylococcal bacteriophage active against 8095% S. aureus strains, including MRSA. This product can be used for local and generalised infections, sepsis in the newborn, osteomyelitis, pneumonia etc. A phage mixture that can be used for the treatment and prophylaxis of purulent wound infections is a preparation called Pyophage. This contains bacteriophages active against staphylococci, streptococci, Pseudomonas aeruginosa, Proteus species and Escherichia coli. This is used in surgery (both pre- and post-operatively), burn wounds, osteomyelitis, skin infections and eye and ear infections. For the treatment of gastrointestinal infections there is an 11-component mixture active against six different Salmonella species, and a 17component cocktail effective against a broad range of intestinal pathogens. The advantages afforded by bacteriophage over conventional antibiotic treatment are as follows: They kill only harmful pathogens and the normal gut microflora are not affected. They are effective against antibioticresistant bacteria. As they act in a completely different way to antibiotics, the presence of resistance determinants is irrelevant. The pharmacokinetics of therapy is quite different from that of antibiotics. After administration, the number of bacteriophages increases and continues to do so until all the susceptible bacteria are dead. Often there is no need to give a repeat dose. Clinical experience suggests there are few side effects or allergic reactions. We have grown up surrounded by bacteriophages and so we do not react to them. They are cheap and easy to produce. As bacteriophages are abundant in the environment, it is a relatively simple task to obtain and purify new ones when necessary.

History of bacteriophages

The concept of using phages in antibacterial therapy is not new. In 1896 Ernest Hankin observed that the Ganges and Jumna rivers in India seemed to possess antibacterial properties, and he surmised that this might in some way be responsible for the reduced number of cases of gastrointestinal infection, particularly cholera, in those villages close to the river. It was not until 1915 that Frederick Twort, an English army officer, suggested that this antibacterial activity might be due to a virus. Twort did not pursue his ideas and it was left to Felix dHerelle to continue this work. He named the viruses bacteriophages (bacteria eaters) and quickly realised their potential for the treatment of bacterial infections. In 1919 he was working in Paris, treating patients with dysentery, and his first patient was a 15-year-old boy. DHerelle obtained the phages from contaminated stool samples and purified them. To test that they were safe he took a dose of the phage preparation himself. The following day, having suffered no ill effects, he gave the boy a single draft of the preparation. The boy recovered rapidly and dHerelle went on to treat other cases in the ward. Needless to say, this generated considerable interest, coming as it did a decade before the discovery of penicillin, and in an age when contracting a bacterial infection was highly likely to lead to death. About this time, dHerelle began working with Giorgi Eliava, who was from Tblisi in Georgia in the then USSR. Eliava was convinced of the importance of phage therapy and decided to set up a research institute in his home country. The Eliava Institute became, and remains today, a world centre for research into bacteriophage therapy. Unfortunately, Eliava fell foul of the authorities, was arrested and executed in 1937 as an enemy of the people. There is a suggestion that his friendship with the wife of the local KGB chief may have led to his undoing. Following his initial success, dHerelle went on to study the potential of phage therapy in a range of bacterial diseases and treated patients with typhoid and paratyphoid fevers, cholera, wound infections and urinary tract infections. Other researchers became interested and pharmaceutical companies started manufacturing bacteriophage preparations. There was a huge expansion of research papers in the area but unfortunately there were also many failures, which were

Bacteriophage preparations

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Table 1. Comparison of the results of phage treatmentand antibiotc treatment.

TREATMENT Salmonella phage NO OF PATIENTS 130 PARAMETERS OF RECOVERY Improvement of general status Normalisation of stool Elimination of pathogen Improvement of general status Normalisation of stool Elimination of pathogen 10 20 20 2 8 10 18 12 10 10 16 14 2 30 31 15 3 40 54 46 DAYS OF TREATMENT 4 30 33 41 5-8 20 8 20 12 10 4 4 >13

Antibiotics only

50

Clinical studies

During the Second World War, phages were used by the troops of the USSR for the treatment of wound infections, particularly gangrene. Studies showed that the mortality in phage-treated soldiers was less than 20%, whereas those in the control groups had mortality rates in excess of 50%. A major trial was conducted in the 1960s on the prophylactic use of phages against dysentery in children in Georgia. A town was divided in two along the main street and children on one side were given phages while those on the other were given a placebo. A total of 30,769 children between the ages of 6 months and seven years were included in the trial: 17,044 were given Shigella phage orally once every seven days, while 13,725 received placebo in place of the phage. Both groups were visited once a week and faecal samples from those children with gastrointestinal disorders were examined microbiologically. The trial lasted for 109 days. Outcome was based on clinical diagnosis and it was found that the incidence of dysentery was 3.8 times higher in the placebo group than in the phage treated group. Phage therapy was also used widely in Poland and a large amount of clinical data was summarised recently. Over a period of time during the 1980s a total of 550 patients (aged from one week to 86 years) were treated at 10 different hospitals. Of these patients, 518 had previously been treated unsuccessfully with antibiotics. Their conditions ranged from wound infections, respiratory tract infections, peritonitis and bacteraemia, and the pathogens included staphylococci, Klebsiella spp., Pseudomonas spp., E. coli and Salmonella spp. The phages were obtained from a culture collection of bacteriophages and treatment involved 10 mL suspension by mouth, half an hour before meals (after gastric acid neutralisation), or topically using phagesoaked dressings. The success rate, defined by complete recovery and negative cultures,

Knowledge of phase biology underpins an understanding of their mode of action in vivo

was reported as 75100% (94% overall) depending upon the pathogen. A clinical study was published comparing the effect of bacteriophages and antibiotics in the treatment of salmonella infections. Using both symptomatic and microbiological criteria, the phage-treated group generally responded more quickly than the antibiotic group and the infections resolved more quickly (Table 1). Unfortunately, although this provides interesting information on the potential of phage therapy, the trial was not well designed and vital information was lacking. There have been examples of patients from the West travelling to Tblisi in order to obtain phage therapy not available elsewhere. One celebrated example was Fred Bledsoe from the USA who contracted an MRSA infection in his foot after stepping on a nail. Despite 10 weeks intravenous antibiotic treatment, he was told the condition was incurable and amputation of the leg was scheduled. Relatives heard of the work being conducted in Georgia and they arranged for him to travel to the Eliava Institute for treatment. For two weeks he was given phage solutions and powders on the infected wounds and tests after three weeks revealed the bacteria had been eradicated and the wounds healed. He has now completely recovered. A number of scientists in the West have been impressed by the results from the former USSR but were aware that anecdotal evidence and poorly conducted experiments would not convince the regulatory authorities to allow its use here. Thus, it

was necessary to begin to assemble a substantial body of hard scientific evidence proving the value of this form of therapy. In the early 1980s, Smith and Huggins conducted a series of studies on the bacteriophage treatment of animals artificially infected with a pathogenic strain of E. coli. When a group of mice were injected intramuscularly with a dose of 106 colony forming units of the bacterial pathogen they all died. However, concurrent treatment with 104 phage specifically lytic against this strain of E. coli resulted in survival of all the animals. Interestingly, the phages used in the experiment were selective for the K1 capsule antigen on the bacterial host; thus, any resistant bacterial strains that emerged had no capsule and were therefore much less virulent. A parallel study compared the efficacy of phages in this system with a range of antibiotics. The only one that gave comparable results was streptomycin, and that required multiple dosing. Tetracycline, ampicillin, chloramphenicol and trimethoprim/sulphafurazole all gave inferior results. Further studies on diarrhoeal disease in calves reinforced these laboratory studies. More recently, Soothill carried out a series of experiments using bacteriophages to prevent graft rejection in burn wounds. A guinea-pig model was used in which a wound was artificially infected with either P. aeruginosa or Acinetobacter baumanii. He showed that appropriate phage treatment would prevent graft rejection even when as few as 100 phages were used against an inoculum of 108 bacteria.

Issues to be considered

If a population of bacterial cells is infected with a dose of a single clone of bacteriophage then it is very likely that some cells within that population will be resistant to the phage. This resistance can arise as a result of alteration in the phage surface receptor site, inhibition of phage DNA penetration, modified restriction endonucleases resulting in degradation of phage DNA, or inhibition of viral intracellular development. Thus, it is essential to use promiscuous phages with a broad host range, or, alternatively, employ mixtures to cover all likely variants within a population. If a resistant strain of bacteria should evolve then it is relatively easy to isolate further lytic phages from the environment. Unfortunately, this approach is not likely to find favour with regulatory authorities. The use of highly characterised bacteriophages from a culture collection obviously has its advantages, as the spectrum of activity and virulence will be well defined. Moreover, administration of a bacteriophage with defined characteristics should avoid the inadvertent use of lysogenic phages or those with transducing potential, particularly if they have the ability to transfer virulence genes.

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More information is required on the interaction of phages with the immune system. There is no specific evidence that they generate an immune response but work is required to confirm the effect of multiple dosing. Similarly, clearance by nonspecific defences is not well understood, although Merrill and co-workers have isolated bacteriophage with the property of prolonged circulation in the body. At present there are at least 16 companies worldwide devoted to bacteriophage technology with a view to exploiting phage therapy in the near future. There are still issues of patentability to address, together with the difficulties of overcoming regulatory hurdles.

Time to renew your HPC registration

Biomedical scientists are about to renew their registration with the Health Professions Council (HPC). Here, UK Registration Manager Claire Harkin provides an update on how the process will work. When do biomedical scientists need to renew their registration? Biomedical scientists renew their registration every two years; their registration period runs from the 1 December to 30 November. How will biomedical scientists know when to renew? The HPC has written to every biomedical scientist on the register to let them know that they need to renew their registration. They should have received a renewal form in September. What payment options do registrants have? The renewal fee is 120 (less if an individual has recently registered and is receiving the reduced rate) for two years. Registrants can pay by cheque or by postal/money order, in which case the amount must be paid in full. Registrants can spread the cost of registration over the two-year registration cycle by paying by Direct Debit. The HPC will deduct 30 (less if receiving the reduced rate) over the two-year registration cycle. Notification of the Direct Debit instalment dates will have been included in the letter accompanying the renewal form. If biomedical scientists already have a Direct Debit in place, do they need to do anything else? Yes, it is vital that they read all the information provided by the HPC. Even if a registrant has an existing Direct Debit they must still sign the professional declaration and return it to the HPC before the end of November in order to remain on the register. What if people cannot remember how they usually pay? The HPC will have indicated on the renewal form if the registrant has a current Direct Debit in place. If they do and have not changed their banking details, they are only required to sign the declaration. What happens if people do not receive their renewal form? A renewal notice has been sent to everyone on the register; however, if they have moved and forgotten to notify the HPC, or they think their form may have been mislaid, they should contact the UK Registration Department, either by phone (0845 3004 472 [lo-call, if calling from within the UK]) or by email (registration @hpc-uk.org), and the HPC will issue them with a duplicate copy of the renewal form. Alternatively, go online and complete the online request form at www.hpc-uk.org. How will people know if they have renewed successfully? The register will be updated as soon as renewal and payment have been processed, and the date displayed next to a registrant's name on the online register will be updated to show 1 December 2005 30 November 2007. Will biomedical scientists receive a certificate? Yes, and it will be valid for two years. In addition to a certificate, registrants will receive a credit card-sized card on which will be a unique number. This is designed to combat identity fraud. Registrants should keep the card in a safe place, as the HPC will require it to be used in the future. Is CPD linked to re-registration? No. The Council has taken the decision to suspend CPD requirements for a year. CPD will be linked to reregistration from August 2006, and the first audit will take place in August 2008. This means that although biomedical scientists will have to record their CPD from next year, their first audit will be in November 2009.

In summary

Bacteriophages are viruses capable of killing bacteria, including strains that are resistant to multiple antibiotics, in a highly specific manner. Abundance in the environment makes it a relatively simple task to isolate phages against any given pathogen and these can be characterised using a series of known protocols. The timescale and costs for the development of a new phage for therapy will be a fraction of those for a new antibiotic. There is an extensive clinical provenance for the use of bacteriophages in the treatment of bacterial infections. They have been used for the past 80 years in Eastern bloc countries, with many favourable reports of clinical benefits and few incidents of adverse reactions. This is accompanied by a scientific rationale to their clinical use where knowledge of phage biology underpins an understanding of their mode of action in vivo. This is in comparison to alternative therapies such as homeopathy and acupuncture where, even if we feel they are of significant clinical benefit, their use is hard to rationalise on a scientific basis. It is important to learn from mistakes made in the past. Early phage therapy was dogged by the combination of high expectations and poor performance. If it is to become a useful adjunct to antibiotic therapy for bacterial infections then its use must be introduced in a careful and rational way, supported by a firm underpinning of good science.
Geoff Hanlon is professor of pharmaceutical microbiology in the School of Pharmacy and Biomolecular Sciences at the University of Brighton. This article is based on a talk given at the Oxoid Infection Control Seminar, held in Stratford upon Avon in May.

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