DEVELOPMENT OF ENHANCED BACTERIOPHAGE AS ANTIBACTERIAL AGENT

National Institute Of Mental Health

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Abstract

Increased prevalence of multidrug resistant bacterial pathogens, such as enterococci resistant to vancomycin (VRE), motivated us to enhance the therapeutic efficacy of bacterial viruses or bacteriophages (phages). Previous studies had demonstrated that phage administered to animal was rapidly removed by the host defense systems, particularly the organs of the reticuloendothelial system (RES). To reduce phage elimination by the host defense system, we developed a serial passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of phage for a number of different species of bacteria. We also demonstrated that these long-circulating phage mutants have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Additional factors known to have impeded therapeutic antibacterial applications of phage included: the failure to recognize the relatively narrow host range of phages and the presence of toxins in unpurified phage. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. In these efforts we have isolated bacteriophage with activity against enterococci resistant to vancomycin (VRE). The emergence of VRE poses a problem for patients with immune deficiency related illnesses (including those who have been immune suppressed for organ transplantation). From a clinical point of view, there are currently few therapeutic agents commercially available with established efficacy for patients infected by VRE. We tested the ability of phage to rescue mice infected with lethal doses of a strain of VRE isolated from a human infection. Preliminary studies demonstrated a dose response for the phage titers used to rescue VRE infected mice. Experiments were also performed to determine whether phage could be used to rescue mice late in the course of a VRE illness. Although the mouse experiments do not completely mimic the course of human illness with VRE, primarily because we administered a larger lethal dose of bacteria than most humans are confronted with early in their disease. Despite this difference we were encouraged by the fact that we were able to rescue significant numbers of mice late in the infectious cycle.Two additional concerns have confronted those interested in the use of phage as an antibacterial agent. The first, has been a belief that bacteria will quickly develop resistance to phage. However, in a comparative study of mice given potentially lethal intramuscular or intracereberal injections of bacteria, a single intramuscular dose of phage was more effective than multiple intramuscular injections of: tetracycline, ampicillin, choramphenicaol or trimethoprim plus sulphafuraxole. The authors of this study, Smith and Huggins, noted that: ‘the therapeutic success of phage was due to its high in vivo activity and the failure of phage-resistant mutants to proliferate during treatment’. Levin and Bull repeated this study with similar results and they concluded that the exponential growth of the phage also reduced the appearance of resistant stains of bacteria. The second concern, is that the FDA would never approve the use of a virus to treat bacterial infections. In this regard it should be noted that a phage, phiX174, has been approved as a marker for assessing the immune response in AIDs and other immune deficient patients. In this application phiX174 is injected intravenously and serum is monitored for the presence of phage and antibody production against the phage. Preparations of this product have been shown to be safe for human use as a biological product. The guidelines that have been used to determine this can be easily applied to our preparations of VRE phage. In addition, the FDA has known for over a decade that phage has often been found to be a contaminant in human vaccine preparations. When phage contamination of the vaccines was brought to the attention of the FDA, they suggested that phage have no direct harmful effect on humans and they obtained an executive order permitting the use of phage contaminated vaccines. Despite the general belief that phage are harmless for humans, some types of phages can be indirectly harmful, due to their ability to transfer bacterial virulence factors, including toxin genes. For example, diphtheria toxin is a phage-encoded protein. With modern genomic sequencing techniques, however, it should be possible to identify such genes and eliminate them for these applications. In addition to our success in selecting phage for their ability to remain for longer periods in the mammalian circulatory system we recognize that the same scheme could be used to select phage for other similar tasks. In fact, phage have recently been selected for their ability to bind to the endothelium in the kidney and brain. It has been suggested that such phage could be used as vehicles to more accurately deliver pharmaceutical agents. Such applications of phage, particularly for gene therapy may prove to be more efficient than those employing mammalian viruses. Much of the research in this project to-date has been done in collaboration with a CRADA partner. This CRADA partner is currently working with the FDA to develop clinical trails for VRE phage antibacterial therapies. The Laboratory has taken the initiative to establish the Phage-Tech Interest Group (PhTIG), and is involved in the establishment of a WWW site http://www.nih.gov/sigs/phtig/ - multidrug resistant, antibacterial agents, bacteriophages,enterococci resistant to vancomycin (VRE), immune deficiency