Bacteriophages offer opportunities for safely managing bacterial infections
Alexander Sulakvelidze
Alexander ("Sandro") Sulakvelidze is an Associate Professor of Molecular Genetics and Microbiology at the University of Florida College of Medicine, and Chief Scientist of Intralytix, Inc., a Maryland- based biotechnology company focused on developing and commercializing various bacteriophagebased products.
Summary
· Efforts are needed to identify novel classes of antibacterial agents, including environmentally friendly agents that kill only specific pathogens without harming beneficial flora, and lytic bacteriophages may provide one such modality.
· Bacteriophages were discovered nearly a century ago and very quickly were tested clinically as potential therapeutic agents against bacterial pathogens.
· Reduced financial resources, scientific and regulatory hurdles, and general unfamiliarity with phages impede their development as antibacterial agents.
· Bacteriophages offer complementary approaches to conventional antibiotics and other antimicrobial agents, and they can be used in various applications ranging from food safety to human therapeutics.
Bacteriophages, viruses that kill bacteria, are likely the oldest and most ubiquitous organisms on Earth. They date back 3 billion years, and their numbers range from 1030-1032. In their predator-prey relationship with bacteria, phages play a key role in maintaining balances in every ecosystem where bacteria exist.
Bacteriophages were discovered independently by Frederick Twort in 1915 and Felix d'Herelle in 1917. Their efforts make a fascinating story about different scientific approaches involving two dramatically different personalities. d'Herelle, with help from his wife, coined the term "bacteriophage" by combining bacteria with phagein, which is Greek for devour. Phage discovery came well before antibiotics were developed, adding to the interest that they generated within the worldwide scientific community. For example, 21 reports concerning phages were published during 1920 alone.
By 1919, d'Herelle and his collaborators at the Hoˆ pital des Enfants-Malades in Paris, France, began using bacteriophages therapeutically. In this first, small-scale clinical trial, they treated four young children suffering from bacterial dysentery. Each patient was administered one dose of d'Herelle's anti-dysentery phage preparation, and all began to recover within 24 hours. Before treating the children, d'Herelle, chief pediatrician Victor- Henri Hutinel, and several interns ingested phage samples to prove the treatment would be safe in what might be considered to be the first "phase I safety clinical trial" of a phage preparation. The results of that study were not immediately published.
The first publication reporting the efficacy of phages in treating an infectious disease of humans was by Richard Bruynoghe and Joseph Maisin during 1921, who used bacteriophages successfully to treat staphylococcal skin infections. Their publication was followed by hundreds of other reports from investigators using bacteriophages to treat bacterial infections in humans and other animals. Phage therapy also caught the attention of scientists at pharmaceutical companies, including Eli Lilly and Company, E.R. Squibb & Sons, and Swan- Myers/Abbott Laboratories, who produced phage preparations for clinical applications. Separately, physicians in the Russian and German Armies used phages to treat soldiers.
However, the results of those clinical applications were sometimes controversial and, with the advent of antibiotics that seemed to work like "magic bullets," interest in phage therapy started to decline in the West during the 1940s and 1950s. Despite controversy, phage therapy continued to be used in the Soviet Union and in some Eastern European countries. Moreover, with development of antibiotics on the decline amid the emergence of multidrug-resistant bacterial pathogens, interest in developing novel antibacterial agents has rekindled. This interest includes revisiting the practical applications of bacteriophages.
Modes of Action and Prevalence of Bacteriophages
Bacteriophages fall into two main groups: lytic (or virulent) and temperate phages. The key difference between the two involves their  replication cycles. Lytic phages replicate inside specific bacterial hosts and their progeny phages are released by lysis (Fig. 1). Temperate phages may integrate their DNA into their host genome by the process called lysogenization. Lysogenized bacteria may replicate for many generations or they may undergo lysogenic induction, during which the integrated phage DNA excises from the bacterial chromosome (Fig. 2).
Typically, only lytic phages are used during phage therapy, primarily for two reasons. First, lytic phages are much more potent killers of their targeted host bacteria than are temperate phages, making them more effective as therapeutic agents. Second, after their infected host bacteria undergo lysogenic induction, temperate phages may transfer fragments of host bacterial DNA into other bacterial species through phage-mediated transduction. Thus, if those fragments contain toxin-encoding or antibiotic resistance-mediating genes, they could produce new virulent strains. Because lytic phages are incapable of transduction, their use provides an added safeguard.
Bacteriophages are the most ubiquitous microorganisms on Earth. For example, (i) the total number is estimated to be 1030-1032 phage particles or plaque-forming units (PFU); (ii) within the United States, each person sheds approximately 3 x 109PFU of coliphages per day; (iii) 1 ml of nonpolluted water often contains as many as 2 x 108 PFU of phages; (iv) phages are harbored in dental plaque and saliva as well as in the intestinal tract; (v) they are present in animal feed; and (vi) we regularly consume phages in drinking water and in virtually all nonprocessed foods.
Safety of Bacteriophages and Regulatory Approvals
Using lytic bacteriophages to treat bacterial infections in human patients and to improve food safety are two of the safest, "green" antibacterial applications now available. Phages are highly specific-active against only a specific bacterial species, strain, or subgroup of strains-and cannot infect eukaryotic cells. Moreover, their ubiquity in the environment means we are exposed to them routinely, further certifying their safety. In the 90 years during which phages have been used to treat human infections, their use has led to no reported serious side effects. Phages have been administered in several ways, including: (i) orally, in tablet or liquid formulations (105-1011 PFU/dose), (ii) rectally, (iii) topically to the skin, eye, ear, and nasal mucosa, (iv) as aerosols or intrapleural injections, and, albeit less frequently, (v) intravenously. The few reported minor side effects such as mild fever likely arose from poorly purified preparations containing lipopolysaccharide (LPS) endotoxins from gramnegative bacteria used to propagate the phages and, when phages were administered intravenously, also from the rapid release of endotoxin when phages lyse large numbers of targeted gram-negative pathogens.
Despite this safety record, the U.S. Food and Drug Administration (FDA) did not have guidelines for reviewing bacteriophage preparations until recently, and those guidelines are not yet fully finalized. Earlier FDA-approved clinical uses in the 1970s, 1980s, and 1990s involved phage phi X174, which was administered (usually intravenously) for nontherapeutic purposes to patients with Down syndrome, Wiskott-Aldrich syndrome, HIV infections, and other immunodeficiencies. FDA also conducted a safety review of bacteriophages in 1973 after phages were found in several vaccines. Agency officials concluded that the vaccines were safe and allowed their continued use.
Without guidelines, obtaining regulatory approvals for bacteriophages tended to be challenging and lengthy. For instance, it took 4 years to approve the first food safety-related phage product, ListShieldTM, a phage cocktail that targets Listeria monocytogenes contaminants in meat and poultry products. After ListShieldTM was cleared by the FDA in 2006, reviews of similar food safety applications appear to be progressing reasonably.
Meanwhile, in 2008, FDA officials approved the first phage phase I clinical trial evaluating phage. In that case, the investigators used a cocktail containing eight phages targeting Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, and the trial included 42 patients with venous leg ulcers. The trial results fully supported the safety of the complex, multiphage preparation in the infected patients. Patients are being recruited for at least two other phage therapy trials, according to the website www.clinicaltrials.gov.
That first U.S. trial also alleviated a concern among those developing phage therapies in humans that FDA officials might insist on singlephage preparations instead of cocktails. The long-term efficacy of single-phage preparations is questionable, and early commercial failures could hinder development of phage therapy in the United States. Notably, most phage therapy trials in the former Soviet Union and Eastern Europe have involved multivalent phage cocktails, according to published reports. Challenges in Developing Phage Therapies
Although several factors make bacteriophages attractive, they are not "magic bullets" and they might not work in certain settings. Thus, understanding phage properties is critical for designing therapeutic interventions. Paradoxically, one key limitation involves the specificity of phages, which is considered one of their strengths. However, that specificity also means that the identity of the targeted pathogen must be precisely determined before phage therapy begins. Recent advances in rapid diagnostics could help to address this challenge.
There are other issues to address, including the potential in vivo elimination of bacteriophages, bacteriophage-neutralizing antibodies, and phage-resistant mutants. So far, these problems have not materialized. Moreover, they can be circumvented. For example, in animal tests, bacteriophages persist long enough to sustain their therapeutic effects; also, if necessary, several doses of bacteriophages can be administered during the illness, much like antibiotic therapy where a single dose of an antibiotic is seldom used. Although phage-resistant mutants are anticipated, they have not affected phage therapy trials so far. Furthermore, when such mutants emerge, new phages with full potency can replace those that no longer work. From a regulatory standpoint, this practice would be akin to the annual updating of influenza vaccines.
Another nontechnical obstacle is potentially more challenging. For one reason or another some experts continue to question the scientific validity of phage therapy-a skepticism that traces to the 1920s when the nature of phage was the subject of fierce debate. Indeed, many scientists questioned d'Herelle's studies, and some skeptics believed that phages were enzymes rather than viruses.
The efficacy of phage therapy was controversial from the outset, and several early studies yielded negative results in part because of their inappropriate use of phages. For example, phages were sometimes used to treat viral diseases or in other cases where the disease agent was unknown. Some skeptics set aside phage therapy, discounting it for being a "Soviet" approach while raising criticisms that perhaps stemmed from political and ideological sources. However, the emergence of multi-antibiotic-resistant pathogens caused that attitude to begin to change, and phage therapy has been increasingly re-evaluated in the West during the last 5-10 years.
Future of Phage Therapy
Educating the general public about phage as therapeutic agents looms as another major challenge. During 2006, shortly after FDA approved the first phage-based preparation for food safety, ensuing open-forum Internet discussions illustrated considerable confusion regarding this technology. For instance, somemembers of the public worried that food producers were using phages to "sneak a price increase on their products" (the rationale being along the lines that phages added to meat products will leave less meat per pound and, therefore, effectively increase the cost of meat for customers). Others worried that the phages were somehow "trained" like guard dogs to be "sicced on" L. monocytogenes contaminating foods. While these examples may be amusing to microbiologists, they also indicate how little the general public knows about bacteriophages. This ignorance, in turn, could leave them reluctant to eat phage-treated foods, even though those foods typically contain billions of phages naturally. Members of grant-funding and regulatory agencies also display uneasiness with using bacteriophages for food safety and human therapeutics. Although regulatory officials are learning more about bacteriophages, regulatory reviews are still hindered by a lack of in-depth understanding and, in some cases, guidelines for reviewing phage-based products.
The relative shortage of applied phage research in academic laboratories also interferes with general acceptance of phage therapy. Some funding agencies preferentially support basic research. Thus, some scientists consider applied approaches such as phage therapy as being too low tech to deserve grant support. Even programs designed to fund research with a high potential for rapid commercialization, such as the federal Small Business Innovation Research (SBIR) program, share that problem. One way to overcome that obstacle is to select reviewers who will objectively evaluate phage therapy research proposals. In addition, National Institutes of Health support for phage clinical trials would further increase awareness of their therapeutic potential.
Lytic phages are not "magic bullets" for overcoming diseases caused by multidrug-resistant bacterial pathogens. However, phages do offer an alternative modality to help prevent and treat bacterial infections. Lytic bacteriophages also provide a means for specifically targeting and killing problem bacteria without damaging beneficial bacterial flora. Although phages are potentially as effective as antibiotics in killing sensitive target bacteria, the phage act differently in terms of mechanism and the means by which bacteria develop resistance.
More than 70 years ago, George Dryer, Chairman of the Sir William Dunn School of Pathology at Oxford University, suppressed further research on penicillin after realizing that the active substance was not a bacteriophage-then considered the most powerful antibacterial agent. After several years, Dryer's successor Howard Florey reinstated the penicillin research in the same department at Oxford, leading to the historic Lancet paper "Penicillin as a chemotherapeutic agent"-and to the birth of the antibiotic era. We hope that the situation will not repeat itself with bacteriophages and that history will remind us not to ignore something that is very promising but different from what we use today.
SUGGESTED READING
Boyd, F. 2005. Bacteriophages and bacterial virulence, p. 223-265. In E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: Biology and Applications, 1st ed. CRC Press, Boca Raton, Fla.
Chain, E., H. W. Florey, A. D. Gardner, N. G. Heatley, M. A. Jennings, J. Orr-Ewing, and A. G. Sanders. 1940. Penicillin as chemotherapeutic agent. Lancet 2:226.
Duckworth, D. H. 1976. "Who discovered bacteriophage?" Bacteriol Rev. 40:793-802.
Friedman, M., and G. W. Friedland. 1998. Alexander Fleming and antibiotics, p. 168-191. In Medicine's 10 greatest discoveries. Yale University Press, New Haven.
Kutter, E., and A. Sulakvelidze. 2005. Bacteriophages: biology and application. CRC Press, Boca Raton, Fla. Projan, S. 2004. Phage-inspired antibiotics? Nature Biotechnol. 22:167-168.
Schoolnik, G. K., W. C. Summers, and J. D. Watson. 2004. Phage offer a real alternative. Nature Biotechnol. 22:505-506.
Sulakvelidze, A., and G. Pasternack. 2010. Industrial and regulatory issues in bacteriophage applications in food production and processing, p. 297 - 326. In P. M. Sabour and M. W. Griffiths (ed.), Bacteriophages in the control of food- and waterborne pathogens. ASM Press, Washington, D.C.
Summers, W. C. 1999. Fe´lix d'Herelle and the origins of molecular biology. Yale Univ Press, New Haven. Thiel, K. 2004. Old dogma, new tricks-21st Century phage therapy. Nature Biotechnol. 22:31-36.
Wolcott, R., D. Rhoads, M. Kuskowski, L. Ward, and A. Sulakvelidze. 2009. Bacteriophage therapy of venous leg ulcers in humans: results of a Phase I safety trial. J. Wound Care 18:237-243.
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