Posted: 03/08/2012
The Bacteriophage: Too Good to Be True?
What if we had an agent that could kill bacteria without contributing to the development of antimicrobial resistance? What if this agent were also low cost, easy to develop, and harmless to patients?
Would you believe that such an agent has been known to medical science since the early 20th century? It's called a bacteriophage, essentially a virus that kills bacteria. A bacteriophage (or "phage") attaches itself to a specific bacterial host cell and destroys it by internal replication and bacterial lysis. Bacteriophages are not antibiotics -- but that is the beauty of these viruses. Because they aren't chemical antibiotics, their use can avoid most of the disadvantages of the infection-fighting drugs we have available today.
Discovery and Rediscovery
Predating the discovery of antibiotics, the existence of bacteriophages was suspected in 1896, when antimicrobial activity against Vibrio cholerae was observed in the waters of the Ganges and Jumna rivers in India.[1] In the early 20th century, scientists around the world were isolating and studying these "agents of transmissible bacterial lysis"[2] for their ability to eradicate undesirable bacteria -- not only in humans, but in animals and plants, as well.[3]
According to bacteriophage scientist Dr. Alexander Sulakvelidze, "during the early 20th century, the remarkable antibacterial activity of bacteriophages prompted some physicians and veterinarians to use lytic phages to treat various bacterial diseases of humans and domesticated livestock, respectively, but their use gradually declined in the West after antibiotics became widely available." Thus, instead of becoming the long-awaited key weapon in the fight against infectious diseases, bacteriophage research gave way to the new miracle of modern medicine -- antibiotics. However, phage research never stopped in some parts of the world, particularly in Eastern Europe and the former Soviet Union.[4]
Resurrected interest in bacteriophages was concurrent with an emerging global crisis of drug-resistant pathogens and a corresponding decline in the development of effective new antibiotics. Fear of returning to the preantibiotic era has spurred a renewal of research efforts to bring bacteriophage technology to the current infectious disease crisis.
Bacteriophages: Abundant and Diverse
Bacteriophages are found everywhere that bacteria proliferate, making them the most widely distributed biological entity in the world.[5] We ingest bacteriophages daily in water and unprocessed foods, harboring them in our saliva, dental plaque, and gastrointestinal tract.[6]Second only to their abundance is the diversity of bacteriophages.[7] More than 100 million phage species are believed to exist.[8] From an evolutionary perspective, bacteriophages help regulate bacterial populations. Over millions of years, bacteriophages have coevolved with bacteria, adapting to and thwarting the antiviral tactics of the bacterial population, to thrive and become expert natural predators.[7, 9] Predation by bacteriophages destroys an estimated one-half of the bacterial population worldwide every 48 hours.[10] Phages tend to be highly specific, targeting only certain species of bacteria.[11]
Bacteriophages: Lethal by Design
Looking like a cross between a robotic insect and a lunar landing device, the structure of a common (nonfilamentous) bacteriophage includes a head, a genome, a tail sheath, tail plug, and tail fibers (Figures 1a and 1b).
- Head: a polyhedral capsid composed of coat protein that protects the nucleic acid genome in its core
- Genome: nucleic acid codes for enzymes and proteins necessary to replicate itself. Can be made of DNA or RNA, and can be linear or circular, single or double-stranded
- Tail sheath: encloses a channel through which the genome travels from head to bacterial host
- Tail plug: distal portion of the tail, which penetrates the cell wall and membrane of the host bacterium, allowing the genome to be injected into the host
- Tail fibers: attach the phage to the surface of the host bacterium
Figure 1a. Diagram showing the structural components of a common phage. Image courtesy ofGraham Colm, Wikimedia Commons. 1b. Electron microscopy of a phage. Image courtesy of Hans-Wolfgang Ackermann,Wikimedia Commons.
Phages infect and kill only bacteria. They have no effect on human, animal, or plant cells, making them essentially harmless to all but their bacterial targets.
A Medicine That Keeps Growing
A most fascinating and useful ability of phages is to replicate themselves with the aid of host bacteria. For this reason, a single "dose" of bacteriophage could theoretically be adequate to quell an infection. The initial bacteriophage needn't be replenished, as with antibiotic therapy, for the bactericidal effect to persist (although in practice, multidose regimens are required for optimally effective phage therapy).
Like other viruses, phages are cellular parasites (Figure 2). They carry the DNA necessary to replicate themselves, but cannot do so without a host.[12]
Figure 2. Electron micrograph of bacteriophages attached to a bacterial host. These viruses are the size and shape of coliphage T1. Image courtesy ofGraham Colm, Wikimedia Commons.
The first order of business is for the phage to recognize the bacterium it is able to infect. Lytic bacteriophages display surface ligands that are specific for receptors on the surface of their bacterial targets.[13] They subsequently have 2 methods of reproducing within these host cells -- the lytic cycle and the lysogenic cycle.
Lytic cycle. Used by lytic (or "virulent") type of phage, the lytic cycle is a cascade of events involving several structural and regulatory genes that disrupt bacterial metabolism and cause the bacterium to lyse.[8] In this mechanism of destruction, the virion attaches itself to a bacterium and injects its DNA into the host cell. The phage genome is replicated, and phage particles (progeny) are formed and assembled into new phages. These progeny multiply, and with the aid of lytic proteins eventually lyse the cell to make their escape. In the space of about 30 minutes, hundreds of identical, new phages burst into the environment in search of new hosts.
Lysogenic cycle. In this mechanism, characteristic of the "temperate" bacteriophage, the phage integrates its genome into the host genome without immediately transcribing it to make new phage particles. The phage genome can exist indefinitely in this prophage state. If the DNA is damaged, the lytic cycle is induced, and new phage particles are produced, which can then lyse the host bacterial cell. This property renders lysogenic phages less useful for killing bacteria because of the potential for chronic habitation within the bacterium converting the phage-sensitive bacterium into an insensitive one and possibly encoding bacterial virulence factors that are later released.[14,15]
Bacteriophages as Antimicrobials
Attempts to treat infections with bacteriophages began before the discovery of penicillin.[16] The first therapeutic use of bacteriophages is described in the treatment of dysentery.[3] Even though bacteriophage therapy wasn't always successful, it was believed to be safe and without risk for serious adverse reactions, and this knowledge encouraged continued bacteriophage research and experimentation.[8]
Phages are still being used in some countries to treat bacterial infections that fail to respond to conventional antibiotics. The most immediate and obvious clinical use for bacteriophages today is as antimicrobials, particularly for infections that are no longer susceptible to currently available agents, or to provide safer alternatives to some of the older, toxic agents that clinicians have been forced to use.
Interest in bacteriophages for both prophylaxis and treatment of multidrug-resistant bacterial strains is growing.[4] Because the mechanisms of resistance of antibiotics and phages are completely different, current pathogens that are highly resistant to antibiotics will still be susceptible to bacteriophages, providing a new avenue for attack.[8] Overcoming phage resistance is unlikely to be problematic, given the plethora of available bacteriophages.[17]
Phage Therapy vs Conventional Antibiotic Therapy
Dr. Sulakvelidze pinpoints the essential difference between phages and traditional antimicrobials. "The mechanisms by which antibiotics and lytic phages kill bacteria, and the mechanisms of bacterial resistance to antibiotics and phages, are fundamentally different from one another. Lytic phages can killbacteria that cannot be killed by currently available antibiotics, and the use of phages in various settings (including for improving food safety) does not create selective pressure for antibiotic-resistant strains to emerge," explains Dr. Sulakvelidze. "Lytic phages are very effective in killing their targeted host bacteria. However, in contrast to antibiotics, they are very specific -- phages will lyse related strains or a subgroup of strains (usually within the same bacterial species or within closely related bacterial species), but they will not lyse strains of other, unrelated bacteria."
On the surface, phages have numerous advantages over chemical antibiotics (Table). Phages do have limitations, as well, which may ultimately determine how they will be most effectively used in clinical therapeutics.[8]
Table. Brief Look at Antibiotics and Bacteriophages
| Some are bacteriostatic, inhibiting growth rather than killing bacteria, allowing development of resistance | Bactericidal -- target bacteria no longer viable | Phage virulence and other properties can vary, making those with poor killing ability or undesirable properties (such as virulence factor carriage) less effective therapeutic agents |
| Toxic to other human cells, causing an array of adverse effects, and harmful to commensal bacterial flora | Nontoxic except to targets; no serious adverse effects, and minimal disruption of desirable bacterial flora | Although phages don't kill other cells, bacterial endotoxins released in the process of lysis can harm other cells or stimulate systemic responses |
| Systemic antibiotics are metabolized and excreted -- require redosing for continued effect | Phage titers keep rising. In theory, phages exhibit self-perpetuating dosing -- killing continues following initial dose as long as targets are present | For many reasons (clearance of phages from the mammalian organism, their attachment to the cell debris of already lysed bacteria), multiple dosing regimens are often required to maintain killing effect of phages. Defined dosages of bacteriophages to maintain effective bacteriophage density could be more complicated |
| Many act on more than one type of bacteria, species, or strain. Broad spectrum activity allows treatment before pathogens are identified | Narrow host range -- very specific target is necessary for action | If bacteria are unknown, empiric treatment of infection is impossible. Rapid test methods are needed. Could require combinations (cocktails) of different bacteriophages that will require an unorthodox drug development and approval process |
| Hindered significantly by the presence of biofilms | Demonstrated ability to overcome some biofilms and prevent some biofilm formation | Biofilms made of more than one type of bacteria can complicate bacterial availability to phages. Biofilms may also be able to trap phages |
Data from Loc-Carrillo C et al,[14] Kutateladze M, et al,[18] and Abedon SA.[19]
Bacteriophage Therapy: Lingering Issues and Stumbling Blocks
Phage therapy has already been successfully integrated into the agricultural,
food-processing, and fishery industries.[17] We know little, however, about how bacteriophages behave in the human body, so phage research for human applications will require more carefully controlled and conducted studies of safety and efficacy.[17]
A few of the lingering doubts about phage therapy in humans, although not considered insurmountable, include:[13,17]
- Antibody response. Intravenous administration of phages or use after repeated phage treatments could stimulate the release of bacteriophage-neutralizing antibodies;
- Rapid uptake and inactivation of phages by the spleen; and
- Contamination of therapeutic phage preparations with endotoxin from bacterial debris.
Although bacteriophages have a long record of safety, the possibility that bacteriophages exert an immunomodulatory effect on humans has been raised, and has not been ruled out. If bacteriophages are found to significantly induce immunosuppressive activity, the risk for other infections could increase, a situation more dangerous in patients with cancer and other immunocompromised hosts. The bulk of evidence to date suggests that bacteriophages are efficacious and safe in individuals with impaired immunity.[20]
The answer to some of these issues could be to avoid using intact (whole) phage therapy, which may turn out to be impractical.[13] The solution could be using products derived from phages.
Bacteriophage Engineering Possibilities
In the approach to using bacteriophages as the antimicrobials most widely employed to date, whole phage particles would be used to lyse specific bacteria that are susceptible to them. But phage engineers aren't stopping there, and the therapeutic potential of engineered phages and phage products appears promising.
Capitalizing on some of the phage's extraordinary abilities, enhancing bacteriophages with new abilities, or combining bacteriophage elements with conventional antibiotics could result in new infection-fighting tools. For example, creating bacteriophages with biofilm-degrading enzymes would enable the phage to break through the extracellular polymeric substance layers that protect bacterial colonies.[21] Antibiotic-enhancing phages can be coupled with antibiotics in a new antimicrobial strategy -- using bacteriophages as antibiotic adjuvants.[22]
In addition to using whole, intact phages, phage lysins show promise as therapeutic agents. These lysins -- enzymes able to rapidly cleave essential bonds in the bacterial cell wall to permit the release of a phage's progeny -- have been used to kill bacteria on mucosal surfaces and infected tissues, sources of infection that are particularly tricky to eradicate.[23]
Phage lysins show promise in one area where traditional antibiotics have failed: the control of pathogenic colonization on mucous membranes.[23] Fear of increasing antibiotic resistance precludes the use of antibiotics to destroy the reservoirs of disease-causing bacteria living in the respiratory tract, gastrointestinal tract, or urogenital tract of many institutionalized patients. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to gram-positive bacteria, could safely and efficiently destroy these pathogens and greatly reduce the burden of disease.[9] Furthermore, phage lysins can be developed with broad lytic activity able to eradicate numerous gram-positive pathogens.[24]
Alternatively, bacteriophages could be engineered to lack endolysin enzymatic activity, rendering them incapable of cell lysis. These nonreplicating, "endolysin-deficient" phages remain lethal to the cells they infect, but with little or no release of progeny phages, thereby avoiding potential problems associated with rapid and excessive release of dangerous endotoxins from the targeted bacteria.[25] One advantage of such an approach would be the ability to provide phage treatment in a defined dosage.
Biotechnical Applications for Phages
The therapeutic use of phages or phage components analogous to an antibiotic to fight infection is by no means the only potential application for phages in infectious disease. The biotechnology applications of phages are as exciting as they are limitless, and include:[26]
- Phage display: for the synthesis of polypeptides with novel characteristics;
- Phage typing: typing of bacterial strains and identification of pathogenic bacteria or detection of antibody-antigen binding;
- Gene delivery vehicle: phages can target specific cell types for delivery of genes to those cells;
- Vaccine delivery: phages can carry vaccine antigens expressed on their surfaces; and
- Biocontrol: phage-mediated biocontrol of plant pathogens.
Bacteriophages: Old Ideas Reborn
Years of clinical experience with phage therapy, a plethora of animal studies, and an improved understanding of bacteriophage biology should bolster the chances of successful reintroduction of bacteriophage therapy into the treatment of modern infectious diseases.[16]
Could bacteriophages entirely replace antibiotics? Probably not in the foreseeable future, but scientists hope that bacteriophages can complement our more conventional antibiotics in the treatment of the multidrug-resistant bacterial infections that are threatening to return us to the preantibiotic era.[26] Bacteriophages come from the preantibiotic era, but, ironically, their "novelty" could hamper widespread acceptance.[8] In the days of personalized medicine, what could be better than a weapon that attacks only your target?
References
- Abedon ST, Thomas-Abedon C, Thomas A, Mazure H. Bacteriophage prehistory: is or is not Hankin, 1896, a phage reference? Bacteriophage. 2011;1:174-178.
- Duckworth DH. "Who discovered bacteriophage?". Bacteriol Rev. 1976;40:793-802. Abstract
- Sulakvelidze A, Morris JG Jr. Bacteriophages as therapeutic agents. Ann Med. 2001;33:507-509. Abstract
- Górski A, Borysowski J, Miêdzybrodzki R, Weber-D¹browska B. Bacteriophages in medicine. In: Mc Grath S, van Sinderen D, eds. Bacteriophage: Genetics and Molecular Biology. Norfolk, England: Caister Academic Press; 2007.
- McAuliffe O, Ross RP, Fitzgerald GF. The new phage biology: from genomics to applications. In Mc Grath S, van Sinderen D, eds. Bacteriophage: Genetics and Molecular Biology. Norfolk, England: Caister Academic Press; 2007.
- Sulakvelidze A. Safety by nature: potential bacteriophage applications. http://www.microbemagazine.org/index.php/01-2011-home/3192-safety-by-nature-potential-bacteriophage-applications/. Microbe Magazine, American Society for Microbiology. Published January 2011. Accessed January 2, 2012.
- Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nat Rev Microbiol. 2010;8:317-327. Abstract
- Sulakvelidze A. The challenges of bacteriophage therapy. Pharmacy. 2011;10:14-18.
- Fenton M, Ross P, McAuliffe O, O'Mahony J, Coffey A. Recombinant bacteriophage lysins as antibacterials. Bioeng Bugs. 2010;1:9-16.
- Deresinski S. Bacteriophage therapy: exploiting smaller fleas. Clin Infect Dis. 2009;48:1096-1101. Abstract
- Monroe D. Looking for chinks in the armor of bacterial biofilms. PLoS Biol. 2007;5:e307.
- Mader SS. Viruses, bacteria, and archaea. In: Mader SS, ed. Biology. 10th ed. New York, NY: McGraw-Hill; 2009.
- Fischette VA. Phage antibacterials make a comeback. Nature Biotechnol. 2001;19:734-735.
- 14.Loc-Carrillo C, Abedon ST. Pros and cons of phage therapy. Bacteriophage. 2011;1:111-114.
- Wagner PL, Waldor MK. Bacteriophage control of bacterial virulence. Infect Immun. 2002;70:3985-3993. Abstract
- Hanlon GW. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Int J Antimicrob Agents. 2007;30:118-128. Abstract
- Inal JM. Phage therapy: a reappraisal of bacteriophages as antibiotics. Arch Immunol Ther Exp (Warsz). 2003;51:237-244. Abstract
- Kutateladze M, Adamia R. Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol. 2010;28:591-595. Abstract
- Abedon SA. Bacteriophages and biofilms. In: Bailey WC, ed. Biofilms: Formation, Development and Properties. Hauppauge, NY: Nova Science Publishers; 2011.
- Borysowski J, Górski A. Is phage therapy acceptable in the immunocompromised host? Int J Infect Dis. 2008;12:466-471.
- Lu TK, Collins JJ. Dispersing biofilms with engineered enzymatic bacteriophage. Proc Natl Acad Sci USA. 2007;104:11197-11202. Abstract
- Lu TK, Collins JJ. Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy. Proc Natl Acad Sci USA. 2009;106:4629-4634. Abstract
- Fischetti VA. Exploiting what phage have evolved to control gram-positive pathogens. Bacteriophage. 2011;1:188-194.
- Yoong P, Schuch R, Nelson D, Fischetti VA. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J Bacteriol. 2004;186:4808-4812. Abstract
- Paul VD, Sundarrajan S, Rajagopalan SS, et al. Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection. BMC Microbiol. 2011;31:195.
- Haq IU, Chaudhry WN, Akhtar MN, Andleeb S, Qadri I. Bacteriophages and their implications on future biotechnology: a review. Virol J. 2012;9:9.
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