Increases in the prevalence of multidrug resistant bacterial pathogens such as enterococci resistant to vancomycin (VRE) have produced a need for alternatives to antibiotic therapies. We have been engaged in studies to enhance the therapeutic efficacy of bacterial viruses or bacteriophages (phages). Most phages are very specific to the species or strain of bacteria. Although this specificity may limit the usefulness of phage therapy, it may be clinically useful, because such phages can be targeted to the specific infectious bacteria and will not affect the normal flora of the body. However in certain situations such as in urinary tract infections with strains of E. coli. it would be useful to have a single phage that could affect more than one pathogenic strain to address this problem. We characterized E. coli phages that are specific for certain pathogenic K antigen strains. In this group of phages, host range is determined by presence of specific hydrolytic tail proteins. We discovered that if a phage contains genes for more than one specific hydrolytic tail protein, such phage are polyvalent in their capacity to infect more than one strain of bacteria, expressing an outer capsule that can serve as a substrate for that hydrolytic enzyme found on the phage tail. This discovery allows us to develop additional phage with extended host ranges. Much of the phage work in the lab focuses on genomics, evolution, and ecology. Last year, in collaboration with I. Molineux of the Univ. of TX and S. Adhya of the NCI, we completed the genome sequences of phages K1-5 and SP6 (Scholl et al., 2004. J. Mol. Biol. 335(5):1151-71.). Another project involving the role of bacterial capsules as a defense against phages has recently been completed, and showed how lytic phages contribute to the evolution of bacterial pathogens by providing pressure for bacteria to produce and evolve different protective capsules. This work was presented at the ASM phage summit in August 2004 and a manuscript ?The E. coli E1 capsule is a barrier to Bacteriophage T7? (Scholl, D., Adhya, S. and Merril, C.) has been submitted to Appl. Environ. Microbiol. A book chapter ?Polysaccharide-degrading phages? (Scholl, D., and Merril, C.) is also currently in press. We have also isolated and characterized a novel phage, M59-2, which is specific for E. coli strains that overproduce colanic acid, an important component in biofilm formation. We have found that M59-2 encodes a tail protein that degrades colonic acid. This phage and its colanic acid hydrolase are potential tools for control of biofilms and we are conducting experiments to examine this. This work was also presented as both a lecture and posters at the August 2004 ASM phage summit, where Dr. Merril and Dr Scholl were invited speakers. We have completed a draft sequence of the genome of M59-2 and a manuscript ?M59-2, A novel phage specific for colanic acid-producing strains of E. coli.? (Scholl, Adhya, McKinstry, and Merril) is in preparation. We are working on the second year of the 3 yr. NIAID biodefense proposal for intramural biodefense research that was funded to study bacteriophage therapy for Y. pestis (plague) and to develop phage based detection methods for plague. A patent regarding this work was filed in June 2004 titled ?Reporter plasmid phage packaging system for detection of bacteria? (Merril, Scholl, Adhya, Edgar, McKinstry) Two researchers continue to work on this project in collaboration with Dr. Adhya in the NCI. Given the importance of rapidly determining whether a particular phage will be effective as an antibacterial therapeutic agent, we are currently developing phage that express reporter genes. Prior to this, determination was performed by plaque assays that can take from 12 hrs to several days to develop. We are currently developing methods for incorporating reporter genes, such as b-galactosidase or luciferase genes into phaages that may be of clinical use as antibacterial agents. An invention report has been submitted. Proposals to explain clinical failures of phage therapy include the interactions of phage with the immune system. However, in experiments with germ free mice, with no detectable adaptive immune system antibodies to lambda phage, phage titers in the circulatory system of mice were found to decrease exponentially by more than 109pfu within 48 hours of intraperitoneal (i.p.), intravenous or oral phage administration. Based on these observations, lambda phage mutants were selected, using a serial passage technique, with a 13,000 to 16,000 fold greater capacity to remain in the Balb/C mouse circulatory system 24 hours after i.p. injection. These """"""""long-circulating lambda phage (Argo phage) found to have a mutation(s) including one in the lambda capsid Eprotein that results in the change of a glutamic acid to a lysine at residue 158. In the current experiments we demonstrate, that this specific lambda mutation at residue 158 of the lambda capsid E protein is sufficient to confer the full """"""""long-circulating"""""""" phenotype to wild type lambda phage. This Argo phage, created by maker rescue, with the single E158K substitution and its isogenic parental wild type strain will provide guidance for the development of more effective antibacterial therapeutic phage strains and they may be useful in studies of the innate immune system. This work was presented this year at the ASM Phage meeting and a manuscript will be submitted for publication. Other factors known to have impeded therapeutic antibacterial applications of phage include: 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 immune suppressed from 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. Our studies demonstrated a dose response for the phage titers used to rescue VRE infected mice even late into the course of a VRE illness. A manuscript concerning this work was published. Two additional concerns have confronted interest in the use of phage as an antibacterial agent: 1) The belief that bacteria will quickly develop resistance to phage. In a comparative study, by Smith and Huggins, 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 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. 2) The FDA would never approve the use of a virus to treat bacterial infections. 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.
