US008003323B2

(12) United States Patent (10) Patent No.: US 8,003,323 B2 Morris et al. (45) Date of Patent: *Aug. 23, 2011

(54) REDUCTION IN BACTERIAL FOREIGN PATENT DOCUMENTS COLONIZATION BY ADMINISTERING DE 3714699 1, 1988 BACTERIOPHAGE COMPOSITIONS DE 19828,596 2, 1999 EP O 29O 295 11, 1988 EP O 414 304 2, 1991 (75) Inventors: J. Glenn Morris, Baltimore, MD (US); EP O 510907 10, 1992 Alexander Sulakvelidze, Towson, MD GB 2 253859 9, 1992 (US); Zemphira Alavidze, Tbilisi (GE): JP 62-123104 6, 1987 WO WO90, 13631 11, 1990 Gary R. Pasternack, Baltimore, MD WO WO95/27043 10, 1995 (US); Torrey C. Brown, Severna Park, WO WO 97.39.111 10, 1997 MD (US) WO WOOOf 69269 11, 2000 (73) Assignee: Intralytix, Inc., Baltimore, MD (US) OTHER PUBLICATIONS Bolder et al. The effect of flavophospholipol (Flavomycin) and (*) Notice: Subject to any disclaimer, the term of this Salinomycin Sodium (Sacox) on the excretion of Clostridium patent is extended or adjusted under 35 perfiringens, Salmonella enteritidis, and Campylobacter jejuni in U.S.C. 154(b) by 0 days. broilers after experimental infection. Poult Sci. Dec. This patent is Subject to a terminal dis 1999;78(12): 1681-9.* claimer. Smith et al. Factors Influencing the Survival and Multiplication of Bacteriophages in Calves and in Their Environment. Journal of Gen Appl. No.: 12/264,582 eral Microbiology 133 (1987), 1127-1135.* (21) Adamia, Revaz S., et al., “The Virulent Bacteriophage IRA of Sal monella typhimurium: Cloning of Phage Genes which are Potentially (22) Filed: Nov. 4, 2008 Lethal for the Host Cell”, J Basic Microbiol., vol. 30, p. 707-716, 1990. (65) Prior Publication Data Alavidze, A., et al., “Isolationof Specific Lytic Phages Multidrug US 2009/0053144A1 Feb. 26, 2009 Resistant Pseudomonas aeruginosa'. The American Society for Microbiology, vol. 99, p. 447, 1999. Alavidze, Z. et al., “Isolation of Specific Lytic Phases against Related U.S. Application Data Multidrug Resistant Pseudomonas aeraiginosa, American Society for Microbiology, Final Program, 1999. (63) Continuation of application No. 107724.900, filed on Barrow, et al., “Salmonellosis—Prospects for Microbiological Con Dec. 2, 2003, now Pat. No. 7,459.272, which is a trol in Poultry”, Avian Pathology, vol. 18, p. 557-561, 1989. continuation of application No. 09/757,685, filed on Barrow, et. al., Use of Lytic Bacteriophage for Control of Experimen Jan. 11, 2001, now abandoned. tal Escherichia coli Septicemia and Meningitis in Chickens and Calves. Clinical and Diagnostic Laboratory Immunology 5(3): 294 (60) Provisional application No. 60/175.377, filed on Jan. 298. 1998. Barrow, P. A., “Bacteriophages Mediating Somatic 11, 2000, provisional application No. 60/175,415, Antigenic Conversion in Salmonella cholerae-suis: their Isolation filed on Jan. 11, 2000, provisional application No. from Sewage and Other Salmonella Serotypes Processing the 60/175,416, filed on Jan. 11, 2000, provisional Somatic 6 Antigen”, Journal of General Microbiology, vol. 132, p. application No. 60/205.240, filed on May 19, 2000. 835-837, 1986. (51) Int. C. (Continued) CI2O I/68 (2006.01) Primary Examiner — Michele Joike A6 IK35/00 (2006.01) (74) Attorney, Agent, or Firm — Hunton & Williams LLP (52) U.S. Cl...... 435/6; 424/93.1 (58) Field of Classification Search ...... None (57) ABSTRACT See application file for complete search history. The present invention provides a method for reducing the risk of bacterial infection or sepsis in a susceptible patient by (56) References Cited treating the Susceptible patient with a pharmaceutical com U.S. PATENT DOCUMENTS position containing bacteriophage of one or more strains 4,375,734 A 3, 1983 Kozloff et al. which produce lytic infections in pathogenic bacteria. Pref 4,891,210 A 1, 1990 Norris erably, treatment of the patient reduces the level of coloniza 4,957,686 A 9, 1990 Norris tion with pathogenic bacteria Susceptible to the bacteriophage 5,132,221 A 7, 1992 Ward et al. by at least one log. In a typical embodiment, the Susceptible 5,612, 182 A 3, 1997 Pearson et al. patient is an immunocompromised patient selected from the 5,641,464 A 6/1997 Briggs, III et al. group consisting of leukemia patients, lymphoma patients, 5,660,812 A 8, 1997 Merrill et al. 5,688,501 A 11/1997 Merrilet al. carcinoma patients, sarcoma patients, allogeneic transplant 5,766,892 A 6, 1998 Merrill et al. patients, congenital or acquired immunodeficiency patients, 5,811,093 A 9, 1998 Merrill et al. cystic fibrosis patients, and AIDS patients. In a preferred 6,015,816 A 1/2000 Kostyniak et al. mode, the patients treated by this method are colonized with 6,056,954 A 5, 2000 Fischetti et al. 6,121,036 A 9, 2000 Ghanbari et al. the pathogenic bacteria Subject to infection by said bacte 6,322,783 B1 1 1/2001 Takahashi riophage. 6,942,858 B1* 9/2005 Ghanbari et al...... 424,936 2005/0260171 A1 11/2005 Ghanbari et al. 35 Claims, No Drawings US 8,003,323 B2 Page 2

OTHER PUBLICATIONS Slopek, et al., “Results of Bacteriophage Treatment of Suppurative Barrow, Paul A., et al., “Bacteriophage Therpay and Prophylaxis: Bacterial Infections; IV. Evaluation of the Results Obtained in 370 Rediscovery and Renewed Assessment of Potential”. Trends in cases”. Arch. Immunol. Therapiae Exper, vol. 33, p. 219-240, 1985. Microbiology, vol. 5, p. 268-271, 1997. Slopek, et al., “Results of Bacteriophage Treatment of Suppurative Berchieri, A., Jr., et al., The Activity in the Chicken Alimentary Tract Bacterial Infections; V. Evaluation of the Results Obtained I Chil of Bacteriophages Lytic for Salmonella typhimurium, Res. dren'. Arch. Immunol. Therapiae Exper, vol. 33, p. 241-260, 1985. Microbiol., vol. 142, p. 541-549, 1991. Slopek, et al., “Results of Bacteriophage Treatment of Suppurative Bogovazova, G. G. et al., “Immunobiological Properties & Thera Bacterial Infections: VI. Analysis of Treatment of Suppurative peutic Effectiveness of Preparations from Klebsiella Bacterioph Staphylococcal Infections'. Arch. Immunol. Therapiae Exper... vol. ages”, Zh. Mikrobiol. Epidemiol. Immunobiol., vol. 3, p. 30-33, 33, p. 261-275, 1985. 1992. Slopek, et al., “Results of Bacteriophage Treatment of Suppurative Carlton, R., Phage Therapy: Past History and Future Prospects Bacterial Infections in the Years 1981-1986', Arch. Immunol. Archivum Immunologiae et Therapiae Experimentalis 47: 267-274, Therapiae Exper, vol. 35, p. 569-584, 1987. 1999. Slopek, et al., Results of Bacteriophage Treatment of Suppurative Gachechiladze, K. K., et al., “Host-Controlled Modification and Bacterial Infections; II. Detailed Evaluation of the Results, Arch. Restriction as a Criterion of Evaluating the Therapeutical Potential of Immunol. Therapiae Exper, vol. 31, p. 293-327, 1981. Pseudomonas Phage'. J. Basic Microbiol., vol. 31, p. 101-106, 1991. Slopek, S., et al., “Results of Bacteriophage Treatment of Suppura Karin E. Byers et al., “Disinfection of Hospital Rooms Contaminated tive Bacterial Infections VI. Analysis of Treatment of Supparative with Vancomyci-Resistant Enterococcus faecum”, col. 4, p.261-264. Staphylococcal Infections'. Database Biosis 'Online, Biosciences Apr. 1988. Information Service, 1985. Kudva, Indira T., et al., "Biocontrol of Escherichia coli O157 with Soothill, J. S., “Bacteriophage Prevents Destruction of Skin Graft by O157-Specific Bacteriophages'. Applied and Environmental Pseudomonas aeruginosa'. Bums, vol. 20, p. 209-211, 1994. Microbiology, vol. 65, p. 3767-3773, 1999. Soothill, J. S., et al., “The Efficacy of Phages in the Prevention of the Lenski, Richard E., “Dynamics of Interactions Between Bacteria and Destruction of PigSkin invitro by Pseudomonas aerarginosa”. Med. Virulent Bacteriphage'. Adv. Microb. Ecol., vol. 10, p. 1-44, 1988. Sci. Res., vol. 16, p. 1287-1288, 1988. Levin, Bruce R. et al., “Phage Therapy Revisited: The Population Soothill, J.S., "Treatment of Experimental Infections of Mice with Biology of a Bacterial Infection and its Treatment with Bacterioph Bacteriophages”. J. Med. Microbiol., vol. 37, p. 258-261, 1992. age and Antibiotics”. The American Naturalist, vol. 147, p. 881-898, Williams Smith, H., et al., “Effectiveness of Phages in Treating 1996. Experimental Escherichia coil Diarrhoea in Calves, Piglets and Ochs, Hans D., “Immunologic Responses to Bacteriophage (bX 174 Lambs”, Journal of General Microbiology, vol. 129, p. 2659-2675, in Immunodeficiency Diseases”. The Journal of Clinical Investiga 1983. tion, vol. 50, p. 2559-2567, 1971. Williams Smith, H., et al., “Effectiveness of Phages in Treating Risi, et al. Prevention of Infection in the Immunocompromised host, Experimental Escherichia coli Diarrhoea in Calves by Means of American Jouranl Infect. Control 26: 594-604, 1998. Bacteriophages”. Journal of General Microbiology, vol. 133, p. Slopek, et al., “Results of Bacteriophage Treatment of Suppurative 1111-1126, 1987. Bacterial Infections; I. General Evaluation of the Results’. Arch. Williams Smith, H., et al., “Successful Treatment of Experimental Immunol. Therapiae Exper, vol. 31, p. 267-291, 1983. Escherichia coli Infections in Mice Using Phage: Its General Supe Slopek, et al., “Results of Bacteriophage Treatment of Suppurative riority Over Antibiotics”,Journal of General Microbiology, vol. 128, Bacterial Infections; III. Detailed Evaluation of the Results Obtained p. 307-318, 1982. in Further 150 cases”. Ach. Immunol. Therapiae Exper, vol. 32, p. 317-335, 1984. * cited by examiner US 8,003,323 B2 1. 2 REDUCTION IN BACTERIAL trations.” Arch. Intern: Med., 126: 255-9). However, during COLONIZATION BY ADMINISTERING the 1980's enterococcal strains with high levels of aminogly BACTERIOPHAGE COMPOSITIONS coside resistance and resistance to penicillin, mediated both by a plasmid-encoded B-lactamase and by changes in peni RELATED APPLICATIONS cillin binding proteins, appeared (Mederski-Samoraj, et al. (1983), “High level resistance to gentamicin in clinical iso This application is a Continuation of U.S. patent applica lates of enterococci.” J. Infect. Dis., 147:751-7: Uttley, et al. tion Ser. No. 107724,900, filed on Dec. 2, 2003, now U.S. Pat. (1988), “Vancomycin resistant enterococci.” Lancet i:57-8). No. 7,459.272, which is a Continuation of U.S. patent appli In 1988 the first VRE isolates were identified (Leclercq, et al. cation Ser. No. 09/757,685, filed on Jan. 11, 2001, now aban 10 (1988), “Plasmid mediated resistance to Vancomycin and doned which claims priority to U.S. Provisional Patent Appli teicoplanin in Enterococcus faecium." N Engl. J. Med., 319. cation Nos. 60/175,377, 60/175,415, and 60/175,416 all of 157-61). Such organisms, called VRE because of resistance which were filed on Jan. 11, 2000, and U.S. Provisional to Vancomycin, are also resistant to the penicillin-aminogly Application No. 60/205.240, filed on May 19, 2000. Each of roside combination. VRE includes strains of several different the foregoing applications are incorporated herein by refer 15 enterococcal species with clinically significant VRE infec ence in their entirety. tions caused by Enterococcus faecium and Enterococcus faecalis. BACKGROUND OF THE INVENTION Enterococci can cause a variety of infections including wound infection, endocarditis, urinary tract infection and 1. Field of the Invention bacteremia. After Staphylococcus aureus and coagulase The present invention is directed to the field of medical negative staphylococci, enterococci are the most common treatment and prevention of infections diseases; in particular, cause of nosocomial bacteremia. Among immunocompro use of therapeutic compositions containing bacteriophage to mised patients, intestinal colonization with VRE frequently reduce or eliminate colonization with potentially pathogenic precedes, and serves as a risk factor for, Subsequent VRE bacteria (including bacterial strains resistant to many or most 25 bacteremia (Edmond, et al. (1995), “Vancomycin resistant commonly used antimicrobial agents), thereby reducing the Enterococcus faecium bacteremia: Risk factors for infec risk of Subsequent disease occurrence. tion.” Clin. Inf Dis., 20:1126-33; Tornieporth, N. G., R. B. 2. Description of Related Art Roberts, J. John, A. Hafner, and L. W. Riley, 1996, “Risk Vancomycin-Resistant Enterococcus factors associated with Vancomycin-resistant Enterococcus Over the last ten years there has been an emergence of 30 faecium infection or colonization in 145 matched case bacterial pathogens, which demonstrate resistance to many, if patients and control patients.” Clin. Infect. Dis., 23:767-72.I. not all antimicrobial agents. This is particularly relevant in the By using pulse field gel electrophoresis as a molecular typing institutional environment where nosocomial pathogens are tool investigators at the University of Maryland at Baltimore under selective pressure due to extensive antimicrobial usage. and the Baltimore VA Medical Center have shown VRE A particular problem in this regard has been Vancomycin 35 strains causing bacteremia in cancer patients are almost resistant enterococci (VRE), which are not treatable with always identical to those which colonize the patients gas standard classes of antibiotics. Despite the recent release of trointestinal tract (Roghmann MC, Qaiyumi S. Johnson JA, two drugs to which VRE are susceptible (quinupristin/dalfo Schwalbe R, Morris J G (1997), “Recurrent vancomycin pristin and linezolid (Plouffe J. F. Emerging therapies for resistant Enterococcus faecium bacteremia in a leukemia serious gram-positive bacterial infections: A focus on lin 40 patient who was persistently colonized with vancomycin-re eZolid. Clin Infect dis 2000 Suppl 4:S144-9), these microor sistant enterococci for two years. Clin Infect Dis 24:514-5). ganisms remain an important cause of morbidity and mortal The risk of acquiring VRE increases significantly when there ity in immunocompromised patients. is a high rate of VRE colonization among patients on a hos Enterococci are grain positive facultatively anaerobic pital ward or unit (i.e., when there is high “colonization cocci found in a variety of environmental sources including 45 pressure'). In one study in the Netherlands, colonization soil, food and water. They are also a common colonizing pressure was the most important variable affecting acquisi bacterial species in the human intestinal tract (i.e., the intes tion of VRE among patients in an intensive care unit (Bonten tinal tract serves as a reservoir for the microorganism). MJ, et al., “The role of “colonization pressure' in the spread Although the taxonomy of enterococci has not been finalized, of Vancomycin-resistant enterococci: an important infection it is generally accepted that the genus consists of 19 species. 50 control variable.” Arch Intern Med 1998:25:1127-32). Use of Antibiotic management of serious enterococcal infections antibiotics has been clearly shown to increase the density, or has always been difficult due to the intrinsic resistance of the level of colonization, in an individual patient (Donskey CJ et organisms to most antimicrobial agents Arden, R. C., and B. al, “Effects of antibiotic therapy on the density of Vancomy E. Murray, 1994, "Enterococcus: Antimicrobial resistance.” cin-resistant enterococci in the stool of colonized patients.” N In: Principles and Practice of Infectious Diseases Update, 55 EnglJMed 2000:343:1925-32): this, in turn, would appear to volume 2, number 4 (February, 1994). New York: Churchill increase the risk of Subsequent infection, and the risk of Livingstone, Inc. 15 pps. Landman, D., and J. M. Quale, transmission of the organism to other patients. 1997, “Management of infections due to resistant entero Multi-Drug Resistant Staphylococcus aureus (MDRSA) cocci: a review of therapeutic options. J. Antimicrob. S. aureus is responsible for a variety of diseases ranging Chemother, 40:161-70; Moellering, R. C., 1998, “Vancomy 60 from minor skin infections to life-threatening systemic infec cin-resistant enterococci.” Clin. Infect. Dis. 26:1196-9. In tions, including endocarditis and sepsis Lowy, F. D., 1998, the 1970s enterococcal infections were treated with the syn “Staphylococcus aureus infections.” N. Engl. J. Med., 8:520 ergistic combination of a cell wall active agent Such as peni 532. It is a common cause of community- and nosocomially cillin and are aminoglycoside (Moellering, et al. (1971), acquired septicemia (e.g., of approximately 2 million infec "Synergy of penicillin and gentamicin against enterococci.” 65 tions nosocomially acquired annually in the United States, J. Infect. Dis., 124:S207-9: Standiford, et al. (1970), “Antibi approximately 260,000 are associated with S. aureus Emori, otic synergism of enterococci: relation to inhibitory concen T. G., and R. P. Gaynes, 1993, 'An overview of nosocomial US 8,003,323 B2 3 4 infections, including the role of the microbiology laboratory.” coccus aureus bacteremia. Arch Intern Med 2000: 160:1001 Clin. Microbiol. Rev., 4:428-442). Also, approximately 20% 4). As seen with VRE, the rate of colonization of persons with of the human population is stably colonized with S. aureus, MDRSA on a unit (the colonization pressure) significantly and up to 50% of the population is transiently colonized, with increases the risk of acquisition of MDRSA for other patients diabetics, intravenous drug users, patients on dialysis, and on the unit (Merrer J. etal, “Colonization pressure' and risk patients with ADS having the highest rates of S. aureus colo of acquisition of methicillin-resistant Staphylococcus aureus nization Tenover, F.C., and R. P. Gaynes, 2000, “The epide in a medical intensive care unit.” Infect Control Hosp Epide miology of Staphylococcus infections. p. 414-421. In: V. A. miol 2000: 21:718-23). Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Multi-Drug Resistant Pseudomonas aeruginosa Rood (ed), Gram-positive pathogens, American Society for 10 Pseudomonas aeruginosa is a highly virulent gram-nega Microbiology, Washington, D.C.. The organism is respon tive bacterial species that is responsible for bacteremia, sible for approximately one-half of all skin and connective wound infections, pneumonia, and urinary tract infections. tissue infections, including folliculitis, cellulitis, furuncules, Increasing problems with multi-antibiotic resistance in and pyomyositis, and is one of the most common causes of Pseudomonas has been noted in hospitals, with particular Surgical site infections. The mortality rate for S. aureus sep 15 concern focusing on Strains which are generally designated as ticemia ranges from 11 to 48% Mortara, L. A., and A. S. “Imipenem-resistant Pseudomonas', reflecting the last major Bayer, 1993, “Staphylococcus aureus bacteremia and antimicrobial agent to which they have become resistant. endocarditis. New diagnostic and therapeutic concepts.” Many of these strains are resistant to all major antibiotic Infect. Dis. Clin. North. Am., 1:53-68). classes, presenting Substantive difficulties in management of Methicillin was one of the first synthetic antibiotics devel infected patients. oped to treat penicillin-resistant staphylococcal infections. As seen with other Gram-negative microorganisms, However, the prevalence of methicillin-resistant S. aureus Pseudomonas strains often emerge as the primary colonizing strains or “MRSA (which also are resistant to oxacillin and flora of the posterior pharynx during hospitalization. Strains nafcillin) has drastically increased in the United States and present in the posterior pharynx, in turn, are more likely to be abroad Panlilio, A. L., D. H. Culver, R. P. Gaynes, S. Ban 25 aspirated into the lungs, and cause pneumonia. In this setting erjee, T. S. Henderson, J. S. Tolson, and W. J. Martone, 1992, colonization with multi-drug resistant Pseudomonas repre “Methicillin-resistant Staphylococcus aureus in U.S. hospi sents a potentially serious risk factor for development of tals, 1975-1991.' Infect. Control Hosp. Epidemiol., 10:582 multi-drug resistant Pseudomonas pneumonia. 586. For example, according to the National Nosocomial Bacteriophage Infections Surveillance System National Nosocomial Infec 30 Bacteriophage has been used therapeutically for much of tions Surveillance (NNIS) report, data summary from Octo this century. Bacteriophage, which derive their name from the ber 1986-April 1996, issued May 1996, “A report from the Greek word “phago” meaning “to eat” or “bacteria eaters', National Nosocomial Infections Surveillance (NNIS) Sys were independently discovered by Twort and independently tem. Am. J. Infect. Control. 5:380-388), approximately 29% by D'Herelle in the first part of the twentieth century. Early of 50,574S. aureus nosocomial infections from 1987 to 1997 35 enthusiasm led to their use as both prophylaxis and therapy were resistant to the B-lactam antibiotics (e.g., oxacillin, for diseases caused by bacteria. However the results from nafcillin, methicillin), and the percent of MRSA strains early studies to evaluate bacteriophage as antimicrobial among U.S. hospitals reached approximately 40% by the end agents were variable due to the uncontrolled study design and of the same period. At the University of Maryland Medical the inability to standardize reagents. Later in well designed Center, >50% of all S. aureus blood isolates are now methi 40 and controlled studies it was concluded that bacteriophage cillin resistant. were not useful as antimicrobial agents (Pyle, N.J. (1936), J. In this setting, there is great concern about the possible Bacteriol., 12:245-61; Colvin, M. G. (1932), J. Infect Dis. emergence of methicillin-resistant/multi-drug resistant S. 51:17-29; Boyd et al. (1944), Trans R. Soc. Trop. Med. Hg., aureus strains which are Vancomycin resistant—and which 37:243-62). would be essentially untreatable. Although overt resistance to 45 This initial failure of phage as antibacterial agents may Vancomycin has not yet been documented in clinical isolates, have been due to the failure to select for phage that demon there have been several reports of clinical infections with S. strated high in vitro lytic activity prior to in vivo use. For aureus strains having intermediate resistance to Vancomycin example, the phage employed may have had little or no activ (MICs=8 g/ml), which suggests that untreatable staphylo ity against the target pathogen, were used against bacteria that coccal infections may not be too far away Tenover, F.C., and 50 were resistant due to lysogenization or the phage itself might R. P. Gaynes. 2000. Given the virulence of S. aureus, the be lysogenic for the target bacterium (Barrow, et al. (1997), emergence of Such untreatable strains would be devastating “Bacteriophage therapy and prophylaxis: rediscovery and and have a major impact on the way in which medicine is renewed assessment of potential.” Trends in Microbiology, practiced in this country. 5:268-71). However, with a better understanding of the Staphylococcal species, including MDRSA, are common 55 phage-bacterium interaction and of bacterial virulence fac colonizers of the human nose; in one community-based study, tors, it was possible to conduct studies which demonstrated 35% of children and 28% of their guardians had nasal Sta the in vivo anti-bacterial activity of the bacteriophage phylococcus aureus colonization (Shopsin B, et al., “Preva (Asheshov, et al. (1937), Lancet, 1:319-20: Ward, W. E. lence of methicillin-resistant and methicillin-susceptible Sta (1943), J. Infect. Dis..., 72:172-6: Lowbury, et al. (1953), J. phylococcus aureus in the community.” J Infect Dis 2000; 60 Gen. Microbiol., 9:524-35). In the U.S. during the 1940’s Eli 182:359-62.). Persons who are nasally colonized with MRSA Lilly commercially manufactured six phage products for have an increased risk of developing serious systemic infec human use including preparations targeted towards staphylo tions with this microorganism, and, in particular, colonization cocci, streptococci and other respiratory pathogens. or prior infection with MDRSA significantly increases the With the advent of antibiotics, the therapeutic use of phage risk of subsequent bacteremia with MDRSA (Roghmann M 65 gradually fell out of favor in the U.S. and Western Europe and C, “Predicting methicillin resistance and the effect of inad little subsequent research was conducted. However, in the equate empiric therapy on Survival in patients with Staphyllo 1970s and 1980s there were reports of bacteriophage US 8,003,323 B2 5 6 therapy continuing to be utilized in Eastern Europe, most guinea pigs (See, e.g., Smith. H. W., and M. B. Huggins notably in Poland and the former Soviet Union. “Successful treatment of experimental Escherichia coli Phage therapy has been used in the former Soviet Union infections in mice using phages: its general Superiority over and Eastern Europe for over half a century, with research and antibiotics'J. Gen. Microbial., 128:307-318 (1982); Smith, production centered at the Eliava Institute of Bacteriophage H.W., and M. B. Huggins “Effectiveness of phages in treating in Tbilisi, in what is now the Republic of Georgia. The inter experimental E. coli diarrhea in calves, piglets and lambs' J. national literature contains several hundred reports on phage Gen. Microbiol., 129:2659-2675 (1983); Smith, H. W. and R. therapy, with the majority of the publications coming from B. Huggins “The control of experimental E. coli diarrhea in calves by means of bacteriophage'. J. Gen. Microbial., 133: researchers in the former Soviet Union and eastern European 1111-1126 (1987); Smith, H. W., R. B. Huggins and K. M. countries. To give but a few examples, phages have been 10 Shaw "Factors influencing the survival and multiplication of reported to be effective in treating (i) skin and blood infec bacteriophages in calves and in their environment J. Gen. tions caused by Pseudomonas, Staphylococcus, Klebsiella, Microbial., 133:1127-1135 (1987)). These trials measured Proteus, and E. coli Cislo, M., M. Dabrowski, B. Weber objective criteria Such as Survival rates. Efficacy against Sta Dabrowska, and A. Woyton, 1987. “Bacteriophage treatment phylococcus, Pseudomonas and Acinetobacter infections of suppurative skin infections. 35(2):175-183: Slopek, S., I. 15 were observed. These studies are described in more detail Durlakowa, B. Weber-Dabrowska, A. Kucharewicz below. Krukowska, M. Dabrowski, and R. Bisikiewicz, 1983, One U.S. study concentrated on improving bioavailability “Results of bacteriophage treatment of suppurative bacterial of phage in live animals (Merril, C. R. B. Biswas, R. Carlton, infections. I. General evaluation of the results.” Archivum. N. C. Jensen, G. J. Greed, S. Zulo, S. Adhya “Long-circulat Immunol. Therapiae Experimental, 31:267-291; Slopek, S., ing bacteriophage as antibacterial agents' Proc. Natl. Acad. B. Weber-Dabrowska, M. Dabrowski, and A. Kucharewicz Sci. USA, 93:3188-3192 (1996)). Reports from the U.S. relat Krukowska, 1987, “Results of bacteriophage treatment of ing to bacteriophage administration for diagnostic purposes suppurative bacterial infections in the years 1981-1986, have indicated phage have been safely administered to 35:569-83), (ii) staphylococcal lung and pleural infections humans in order to monitor humoral immune response in Meladze, G. D., M. G. Mebuke, N. S. Chkhetia, N. I. 25 adenosine deaminase deficient patients (Ochs, et al. (1992), Kiknadze, G. G. Koguashvili, I. I. Timoshuk, N. G. Lari Antibody responses to bacteriophage phi X174 in patients with adenosine deaminase deficiency.” Blood, 80: 1163-71) onova, and G. K. Vasadze, 1982, “The efficacy of Staphylo and for analyzing the importance of cell associated molecules coccal bacteriophage in treatment of purulent diseases of in modulating the immune response in humans (Ochs, et al. lungs and pleura. Grudnaya Khirurgia, 1:53-56 (in Russian, (1993), “Regulation of antibody responses: the role of Summary in English), (iii) Paeruginosa infections in cystic 30 complement acra adhesion molecules. Clin. Immunol. fibrosis patients Shabalova, I. A. N. I. Karpanov, V. N. Kry Immunopathol., 67:S33-40). lov, T. O. Sharibjanova, and V. Z. Akhverdijan. “Pseudomo Additionally, Polish, Georgian, and Russian papers nas aeruginosa bacteriophage in treatment of P aeruginosa describe experiments where phage was administered sys infection in cystic fibrosis patients.’’ abstr. 443. In Proceed temically, topically or orally to treat a wide variety of antimi ings of IX international cystic fibrosis congress, Dublin, Ire 35 crobial resistant pathogens (See, e.g., Shabalova, I. A. N. I. land, (iv) neonatal sepsis Pavlenishvili, I., and T. Tserts Karpanov, V. N. Krylov, T.O., Sharibjanova, and V. Z. Akh vadze. 1985. “Bacteriophage therapy and enterosorbtion in Verdijan. “Pseudomonas aeruginosa bacteriophage in treat treatment of sepsis of newbornes caused by gram-negative ment of P aeruginosa infection in cystic fibrosis patients.” bacteria. In abstracts, p. 104, Prenatal and Neonatal Infec Abstr. 443. In Proceedings of IX International Cystic Fibrosis tions, Toronto, Canada, and (v) Surgical wound infections 40 Congress, Dublin, Ireland; Slopek, S. I. Durlakowa, B. Peremitina, L. D., E. A. Berillo, and A. G. Khvoles, 1981, Weber-Dabrowska, A. Kucharewicz-Krukowska, M. Dab "Experience in the therapeutic use of bacteriophage prepara rowski, and R Bisikiewicz. 1983. “Results of bacteriophage tions in supportive surgical infections.” Zh. Mikrobiol. Epi treatment of suppurative bacterial infections. I. General demiol. Immunobiol. 9:109-110 (in Russian). Several evaluation of the results.” Archivum, Immunol. Therapiae reviews of the therapeutic use of phages were published dur 45 Experimental, 31:267-291; Slopek, S., B. Weber-Dabrowska, ing the 1930s-40s Eaton, M.D., and S. Bayne-Jones, 1934, M. Dabrowski, and A. Kucharewicz-Krukowska. 1987. “Bacteriophage therapy: review of the principles and results “Results of bacteriophage treatment of suppurative bacterial infections in the years 1981-1986, Archivum Immunol. of the use of bacteriophage in the treatment of infections. J. Therapiae Experimental, 35:569-83. Am. Med. Assoc., p. 103: Krueger, A. P., and E. J. Scribner, Infections treated with bacteriophage included osteomy 1941, “The bacteriophage: its nature and its therapeutic use.” 50 elitis, sepsis, empyema, gastroenteritis, Suppurative wound J. Am. Med. Assoc., p. 116 and recently Barrow, P.A., and J. infection, pneumonia and dermatitis. Pathogens involved S. Soothill, 1997, “Bacteriophage therapy and propylaxis— included Staphylococci, Streptococci, Klebsiella, Shigella, rediscovery and renewed assessment of potential.” Trends in Salmonella, Pseudomonas, Proteus and Escherichia. These Microbiol., 5(7):268-271; Lederberg, J., 1996, “Smaller articles reported a range of Success rates for phage therapy fleas... ad infinitum: therapeutic bacteriophage.” Proc. Natl. 55 between 80-95% with only rare reversible allergic or gas Acad. Sci. USA, 93:3167-3168. In a recent paper published trointestinal side effects. These results indicate that bacte in the Journal of Infection (Alisky, J. K. Iczkowski, A. Rapo riophage may be a useful adjunct in the fight against bacterial port, and N. Troitsky, 1998, “Bacteriophages show promiseas diseases. However, this literature does not describe, in any antimicrobial agents. J. Infect., 36:5-15), the authors way anticipate, or otherwise Suggest the use of bacteriophage reviewed Medline citations (published during 1966-1996) of 60 to modify the composition of colonizing bacterial flora in the therapeutic use of phages in humans. There were twenty humans, thereby reducing the risk of Subsequent develop seven papers from Britain, the U.S.A., Poland and the Soviet ment of active infections. Union, and they found that the overall reported success rate for phage therapy was in the range of 80-95%. SUMMARY OF THE INVENTION These are several British studies describing controlled tri 65 als of bacteriophage raised against specific pathogens in According to one embodiment of the present invention, a experimentally infected animal models such as mice and lytic bacteriophage which infects Enterococcus, wherein no US 8,003,323 B2 7 8 more than 30% of the Enterococcus strains in a collection of In another embodiment, this invention provides a method more than 100 genetically diverse Vancomycin resistant for reducing the incidence of infection by selected bacteria in Enterococcus (VRE) strains are resistant to infection by said a medical facility by administering to patients who are admit bacteriophage is disclosed. The bacteriophage may produces ted to said medical facility a bacteriophage preparation which lytic infection in at least 200 genetically diverse VRE isolates. reduces the colonization level by the selected bacteria in The bacteriophage preparation may be administered orally to patients at risk for infection by the selected bacteria. In a patients who are colonized with VRE who are admitted to a typical embodiment, the patients at risk for infection are medical facility. These patients may include, inter alia, leu selected from the group consisting of leukemia patients, lym kemia patients, lymphoma patient, transplant patients and phoma patients, carcinoma patients, sarcoma patients, allo 10 geneic transplant patients, congenital or acquired immuno AIDS patients. The bacteriophage may also be administered deficiency patients, cystic fibrosis patients, and ADS patients. to all patients admitted at the medical facility. Administration In another embodiment, the bacteriophage preparation is of this bacteriophage will reduce or eliminate colonization administered to Substantially all patients admitted to said with VRE, thereby reducing the risk that these patients will medical facility. In a preferred embodiment, the bacterioph Subsequently develop serious systemic infections with this 15 age preparation is administered to Substantially all patients highly resistant microorganism. Reduction or elimination of colonized with the selected bacteria who are admitted to said colonization will also decrease the VRE “colonization pres medical facility. In another preferred embodiment, the sure' in the hospital or specific units of the hospital, thereby selected bacteria is VRE, MDRSA, or multi-drug resistant reducing the risk that VRE will be acquired by persons who Pseudomonas. currently are neither colonized or infected with the pathogen. According to another embodiment of the present invention, In one embodiment, the present invention provides a a bacteriophage preparation which reduces the number of method for reducing the risk of bacterial infection or sepsis in VRE in experimentally infected mice by at least 1 log is a Susceptible patient by treating the Susceptible patient with a disclosed. pharmaceutical composition containing bacteriophage of one According to another embodiment of the present invention, or more strains which produce lytic infections in pathogenic 25 a lytic bacteriophage which infects Staphylococcus aureus, bacteria. Preferably, treatment of the patient reduces the level wherein no more than 30% of the Staphylococcal strains in a of colonization with pathogenic bacteria Susceptible to the collection of more than 100 genetically diverse multi-drug bacteriophage by at least one log. In a typical embodiment, resistant Staphylococcus aureus (MDRSA) Strains are resis the Susceptible patient is an immunocompromised patient tant to infection by said bacteriophage is disclosed. The bac selected from the group consisting of leukemia patients, lym 30 teriophage may produces lytic infection in at least 200 geneti phoma patients, carcinoma patients, sarcoma patients, allo cally diverse MDRSA isolates. The bacteriophage geneic transplant patients, congenital or acquired immuno preparation may be administered via nasal spray to individu deficiency patients, cystic fibrosis patients, and AIDS als who are nasally colonized with MDRSA, particularly to a patients. In a preferred mode, the patients treated by this Subpopulation made up of all Such individuals who are admit method are colonized with the pathogenic bacteria Subject to 35 ted to a medical facility. The bacteriophage may also be infection by said bacteriophage. administered to all patients admitted at the medical facility. In a preferred embodiment of this invention, the risk of Administration of this bacteriophage will reduce or eliminate infection is reduced by administering a composition compris colonization with MDRSA, thereby reducing the risk that ing bacteriophage which produce lytic infections in patho these patients will Subsequently develop serious systemic genic bacteria selected from Vancomycin-resistant enterococ 40 infections with this highly resistant microorganism. Reduc cus (VRE), pneumococcal species, methicillin-resistant tion or elimination of colonization will also decrease the Staphylococcus aureus, multi-drug resistant Staphylococcus MDRSA “colonization pressure' in the hospital or specific aureus (MDRSA), multi-drug resistant Pseudomonas spe units of the hospital, thereby reducing the risk that MDRSA cies, Neisseria sp., Hemophilus sp., Proteus sp., Klebsiella will be acquired by persons who currently are neither colo sp. and Esherichia coli. Preferably, the pathogenic bacteria 45 nized or infected with the pathogen. are selected from VRE, MDSA, and multi-drug resistant According to one embodiment of the present invention, a Pseudomonas. In a preferred embodiment of this invention, lytic bacteriophage which infects Pseudomonas aeruginosa, the bacteriophage composition is in a form selected from a wherein no more than 30% of the Pseudomonas strains in a parenteral composition, an oral tablet, capsule or liquid, a collection of more than 100 genetically diverse multi-antibi nasal aerosol, a throat wash, a toothpaste, and a topical oint 50 otic resistant Pseudomonas aeruginosa strains are resistant to ment. Preferably, the pharmaceutical composition contains a infection by said bacteriophage is disclosed. The bacterioph plurality of bacteriophage strains. More preferably, the phar age may produces lytic infection in at least 200 genetically maceutical composition contains bacteriophage strains diverse isolates. The bacteriophage preparation may be which produce lytic infections in pathogenic bacteria of a administered by mouth wash or gargle to individuals who are plurality of bacterial strains or bacteriophage strains which 55 colonized with multi-drug resistant Pseudomonas aerugi produce lytic infections in pathogenic bacteria of a plurality nosa, particularly to a subpopulation made up of all Such of bacterial species. individuals who are admitted to a medical facility. The bac In a preferred embodiment, the present invention provides teriophage may also be administered to all patients admitted a method for reducing the risk of bacterial infection or sepsis at the medical facility. Administration of this bacteriophage in a patient having a wound selected from an ulcer, a lacera 60 will reduce or eliminate colonization with multi-drug resis tion, a deep penetrating wound and a Surgical wound by tant Pseudomonas Strains, thereby reducing the risk that these treating the patient with a pharmaceutical composition con patients will Subsequently develop serious systemic infec taining bacteriophage of one or more strains which produce tions with this highly resistant microorganism. This may also lytic infections in pathogenic bacteria capable of infecting be expected to reduce the “colonization pressure of this these wounds. Preferably, the composition is a topical oint 65 microorganism, thereby reducing the risk that it will be ment, an irrigation Solution or a component of a wound dress acquired by persons who are currently not colonized or 1ng. infected. US 8,003,323 B2 10 DETAILED DESCRIPTION OF THE The general rule is that patients first become colonized by EMBODIMENTS pathogenic bacteria present in their immediate environment before developing illness due to those bacteria. Serious VRE Bacteriophage technology can be of value in managing a infections, including septicemia, usually are preceded by large variety of bacterial infections because: (i) bacterioph intestinal colonization with the infecting organisms; there ages are highly specific and very effective in lysing targeted fore, the risk of septicemia is likely to be decreased by reduc pathogenic bacteria, (ii) bacteriophages are absolutely spe ing colonization prior to periods when patients are severely cific for prokaryotes, and do not affect humans or animals, neutropenic or otherwise immunosuppressed (i.e., reducing (iii) bacteriophages are safe, as underscored by their exten intestinal colonization may also reduce the risk of blood 10 stream invasion). The present inventors have discovered that sive clinical use in Eastern Europe and the former Soviet certain strains of bacteriophage are particularly effective at Union, and the commercial sale of phages in the 1940s in the lysing VRE. By administering these VRE-active bacterioph United States, (iv) phage preparations can rapidly be modi age to persons colonized with VRE, it is possible to substan fied to combat the emergence of newly arising bacterial tially reduce or even eliminate VRE from the colonized per threats, and (V) phage production is seen to be cost-effective 15 son. Thus, the present invention provides strains of phage for large-scale applications in a variety of medical settings. Of which are particularly effective against VRE, methods for particular relevance, bacteriophage will not kill non-patho obtaining additional Strains of VRE-active phage, methods genic, “normal flora' bacteria, thereby retaining the “coloni for treating patients colonized with VRE by administering Zation resistance' of reservoirs Such as the human intestinal VRE-active phage, and methods of reducing nosicomial tract, the nose, and the posterior pharynx. Accordingly, the infection rate by administering VRE-active phage in Vivo, ex present invention envisions using lytic phages (in combina Vivo, or both, to selected locations, areas, objects and/or per tion with antibiotics or alone) to prophylactically ortherapeu SOS. tically eliminate various bacteria capable of causing diseases Analogous approaches using bacteriophage targeted to of the gastrointestinal, genitourinary, and respiratory tracts, other pathogenic bacteria are also contemplated by this inven and skin, oral cavity, and bloodstream. In accordance with 25 tion. S. aureus phage preparations can reduce contamination this invention, therapeutic phages can be administered in a of skin and wounds with S. aureus, which in turn may prevent number of ways, in various formulations, including: (i) orally, the development of serious Surgical site infections and septi in tablets or liquids, (ii) locally, in tampons, rinses or creams, cemia. Phage active against Pseudomonas species can be (iii) aerosols, and (iv) intravenously. used to reduce colonization that threatens to develop into One benefit of bacteriophage therapy when compared to 30 pneumonia in immunocompromised patients or in individu antibiotic therapy relates to the relative specificity of the two als Suffering from cystic fibrosis. therapeutic modalities. Bacteriophage are specific for par Isolation of Bacteriophage ticular bacterial strains or species, while antibiotics typically The present inventors have isolated several lytic phages are broadly effective against a large multiplicity of bacterial active against genetically diverse (as assessed by pulsed field species or genera. It is well known that normal individuals are 35 gel electrophoresis and/or arbitrary pruned polymerase chain colonized with innocuous bacteria, and this colonization may reaction or other nucleic acid amplification techniques)VRE be beneficial to the colonized individual (see U.S. Pat. No. strains. In vitro susceptibility tests involving 234VRE strains 6,132,710, incorporated herein by reference). Antibiotic (184 E. faecium, 41 E. faecalis and 6 E. gallinarium isolated therapy can severely alter colonization or even eliminate ben from patients at the University of Maryland and the Baltimore eficial colonization completely. This may have adverse 40 VA Medical Center, and 3 E. faecium ATCC strains), resulted effects, such as the outgrowth of opportunistic species such as in the Intralytix phage collection being able to cumulatively Clostridium difficile, which then leads to an antibiotic-asso lyse all VRE strains in the collection, with one particular ciated colitis. In contrast, bacteriophage therapy specifically phage being able to lyse 95% of VRE strains. Furthermore affects the bacterial strains that are sensitive or susceptible to mice whose gastrointestinal tract was colonized with VRE lytic infection by the particular bacteriophage in the thera 45 under selective pressure of antibiotic administration, were peutic composition, but leaves other (innocuous or beneficial) orogastrically administered VRE-active phages, which bacteria unaffected. Thus, bacteriophage therapy is prefer resulted in a 1 to 3 log reduction of VRE gastrointestinal able for prophylactic treatment where alteration of normal colonization compared to a control group of animals not microflora should be minimized. given phage. This occurred within a 48 to 72 hour time frame. In a preferred mode of this invention, phage technology is 50 No side effects due to the phage were observed. focused on two important human pathogens. VRE and Bacteriophage strains may be isolated by analogous pro MDRSA, and the value of VRE- and MDRSA-specific lytic cedures to those used to isolate the VRE-active strains phages in different settings: (i) oral administration of phages described herein. Suitable bacteriophage may be isolated for prophylaxis against septicemia, (ii) local application of from any sample containing bacteriophage, which typically phages for prophylaxis/treatment of skin and wound infec 55 are found in association with their host bacteria. Thus, any tions, (iii) intravenous administration of phages fortherapy of source that might be expected to contain VRE is suitable for septicemia, and (iv) the use of aerosolized phages against use as a source of VRE-active bacteriophage. Such samples respiratory pathogens. include fecal, urine, or sputum samples from patients, par VRE infection has become a particularly serious problem ticularly patients undergoing acute or prophylactic antibiotic among immunocompromised and/or seriously ill patients in 60 therapy, patients in intensive care units or immunocompro intensive care units, cancer centers and organ transplant units. mised patients. Such patients may include but are not limited Since VRE are resistant to all currently used antimicrobials, to burn patients, trauma patients, patients receiving bone mar alternate approaches to reducing or eliminating VRE gas row and/or organ transplants, cancer patients, patients with trointestinal colonization in immunocompromised patients congenital or acquired immunodeficiency diseases, dialysis must be found in order to reduce the prevalence of VRE 65 patients, liver disease patients, and patients with acute or bacteremia. Oral administration of lytic bacteriophage active chronic renal failure. Body fluids including ascites, pleural against VRE is one such approach. effusions, joint effusions, abscess fluids, and material US 8,003,323 B2 11 12 obtained from wounds. While humans are the primary reser cific host, or based upon the amino acid sequences or anti voir for VRE, the organism also can be readily found in the genic properties of Such proteins. immediate environment of infected/colonized patients such Quantities of broad-spectrum VRE-active bacteriophage as bedrails, bed sheets, furniture, etc. (Bodnar, U. R. et al needed fortherapeutic uses described below may be produced (1996). “Use of in house studies of molecular epidemiology by culture on a suitable host strain in the manner described and full species identification of controlling spread of Vanco above for enrichment culture. When performing an enrich mycin resistant Enterococcus faecalis isolates'. J. Clin. ment culture to produce bacteriophage for therapeutic use, a Microbiol., 34:2129-32: Bonten, M.J. M. etal (1996), “Epi host strain is selected based on its ability to give a maximum demiology of colonization of patients and the environment yield of phage, as determined in pilot experiments with sev 10 eral different host VRE strains. If two or more host strains with Vancomycin resistant enterococci.” Lancet, 348: 1615 give similar yield, the strain most sensitive to antibiotics is 19: Noskin, G. A. (1995), “Recovery of Vancomycin resistant selected. enterococcion fingertips and environmental Surfaces.” Infect. The techniques described herein for isolation of VRE Control Hosp. Epidemiol., 16: 577-81). Consequently, monophages are applicable to isolation of bacteriophages that samples for bacteriophage isolation may also be obtained 15 are lytic for other pathogenic bacteria. It is within the skill in from nonpatient sources, including sewage, especially sew the art to substitute host strains of other bacteria in the meth age streams near intensive care units or other hospital venues, ods described herein in order to isolate phage specific for or by Swabinhospital areas associated with risk ofnosicomial those bacteria. Starting the phage isolation process with infection, such as intensive care units. Other Suitable Sam samples selected from environments that also contain bacte pling sites include nursing homes, rest homes, military bar ria of the host species will accelerate the process. racks, dormitories, classrooms, and medical waste facilities. Patient Population Phages also can be isolated from rivers and lakes, wells, water Any patient who is at risk for colonization with VRE or tables, as well as other water sources (including salt water). who has proven VRE colonization is a candidate for treatment Preferred sampling sites include water sources near likely according to the method of this invention. Intestinal coloni sites of contamination listed above. 25 zation with VRE is relatively common in institutionalized Suitable methods for isolating pure bacteriophage strains patients undergoing antimicrobial therapy. In studies con from a bacteriophage-containing sample are well known, and ducted in 1993-94, 17-19% of a random sample of all patients such methods may be adapted by the skilled artisan in view of at the University of Maryland Hospital were colonized with the guidance provided herein. Isolation of VRE-active bacte VRE (Morris, et al. (1995), “Enterococci resistant to multiple riophage from Suitable samples typically proceeds by mixing 30 antimicrobial agents including Vancomycin.” Ann. Int. Med., the sample with nutrient broth, inoculating the broth with a 123:250-9), while in an identical study conducted in 1996 this host bacterial strain, and incubating to enrich the mixture with increased to 23.8%. Once colonized with VRE, a patient may bacteriophage that can infect the host strain. An Enterococcus remain colonized for life; however once off antimicrobial sp. strain will be used as the host strain, preferably a VRE therapy, VRE colonization may drop to levels not detectable strain. After the incubation for enrichment, the mixture is 35 in routine stool culture. Colonized persons though who also filtered to remove bacterial leaving lytic bacteriophage in the Subsequently become immunocompromised are at risk for filtrate. Serial dilutions of the filtrate are plated on a lawn of developing bacteremia (Edmond, et al., 1995; Tornieporth, et VRE, and VRE-active phage infect and lyse neighboring all (1996), “Risk factors associated with Vancomycin resistant bacteria. However the agar limits the physical spread of the Enterococcus faecium colonization or infection in 145 phage throughout the plate, resulting in Small visibly clear 40 matched case patients and control patients. Clin. Infect. Dis., areas called plaques on the plate where bacteriophage has 23:767-72). destroyed VRE within the confluent lawn of VRE growth. VRE infection is a particularly serious problem among Since one plaque with a distinct morphology represents one immunocompromised and/or seriously ill patients in cancer phage particle that replicated in VRE within that area of the centers, intensive care units, and organ transplant centers. In bacterial lawn, the purity of a bacteriophage preparation can 45 case control studies VRE has been linked to antimicrobial use be ensured by removing the material in that plaque with a and severity of illness (as measured by APACHE score) pasteur pipette (a "plaque pick) and using this material as the (Handwerger, et al. (1993), “Nosocomial outbreak due to inoculum for further growth cycles of the phage. The bacte Enterococcus faecium, highly resistant to Vancomycin, peni riophage produced in Such cycles represent a single strain or cillin and gentamicin.” Clin. Infect. Dis., 16:750-5; Monte "monophage. The purity of phage preparation (including 50 calvo, et al. (1996), “Bloodstream infections with Vancomy confirmation that it is a monophage and not a polyvalent cin resistant enterococci.” Arch. Intern. Med., 156:1458–62; phage preparation) is assessed by a combination of electron Papanicolaou, et al. (1996), “Nosocomial infections with microscopy, SDS-PAGE, DNA restriction digest and analyti Vancomycin-resistant Enterococcus faecium in liver trans cal ultracentrifugation. In addition, each phage is uniquely plant patients: Risk factors for acquisition and mortality.” identified by its DNA restriction digest profile, protein com 55 Clan. Infect. Dis., 23:760-6: Roghmann, et al., (1997), position, and/or genome sequence. “Recurrent Vancomycin resistant Enterococcus faecium bac Individual VRE-active bacteriophage strains (i.e., teremia in a leukemic patient who was persistently colonized monophages) are propagated as described for enrichment with Vancomycin resistant enterococci for two years.” Clin. culture above, and then tested for activity against multiple Infect. Dis., 24: 514-5). Investigators at the University of VRE strains to select broad-spectrum VRE-active bacte 60 Maryland at Baltimore and the Baltimore VA Medical Center riophage. Efforts are made to select phages that (i) are lytic, have demonstrated by pulse field electrophoresis that VRE (ii) are specific to enterococci, (iii) lyse more than 70% of the strains causing bacteremia in cancer patients are almost VRE strains in our VRE strain collection, and/or (iv) lyse always identical to those that colonize the patient's gas VRE strains resistant to other VRE phages previously iden trointestinal tract. tified. It is also possible to select appropriate phages based 65 Three categories of immunocompromised patients Sub upon the sequences of DNA or RNA encoding proteins jected to prolonged antimicrobial administration in a institu involved in the binding and/or entry of phage into their spe tionalized setting and who would be susceptible to VRE gas US 8,003,323 B2 13 14 trointestinal colonization are: 1) leukemia (30,200 patients Phages also may be incorporated in a tablet or capsule which per year in the U.S.) and lymphoma patients (64,000 patients will enable transfer of phages through the stomach with no per year in the U.S.), 2) transplant patients (20,961 per year in reduction of phage viability due to gastric acidity, and release the U.S.), and 3) AIDS patients (66.659 patients per year in of fully active phages in the Small intestine. The frequency of the U.S.). The total number of patients in the immunocom dosing will vary depending on how well the phage is tolerated promised category is 181,800 per year in the U.S. Pfundstein, by the patient and how effective a single versus multiple dose et al., found that the typical rate of enterococcal gastrointes is at reducing VRE gastrointestinal colonization. tinal colonization among renal and pancreas transplant The dose of VRE-active bacteriophage and duration of patients receiving antibiotics in an institutional setting was therapy for a particular patient can be determined by the 34% (38/102) with 4 (11%) of these isolates being VRE 10 skilled clinician using standard pharmacological approaches (Pfundstein, et al. (1999), “A randomized trial of surgical in view of the above factors. The response to treatment may be antimicrobial prophylaxis with and without Vancomycin in monitored by, analysis of blood or body fluid levels of VRE, organ transplant patients.” Clin. Transplant., 13:245-52). orVRElevels in relevant tissues or monitoring disease state in Therefore the rate of gastrointestinal colonization by VRE in the patient. The skilled clinician will adjust the dose and this immunocompromised population would be 15 duration of therapy based on the response to treatment 0.34x0.11=0.04 or 4% of the total patient population. One revealed by these measurements. can therefore estimate VRE gastrointestinal, colonization to One of the major concerns about the use of phages in be 181,800x0.04=7272 patients per year. clinical settings is the possible development of bacterial resis Formulation and Therapy tance against them. However, as with antimicrobial resis According to this invention, VRE-active bacteriophage are tance, the development of resistance to phages takes time. The preferably formulated in pharmaceutical compositions con Successful use of phages in clinical settings will require con taining the bacteriophage and a pharmaceutically acceptable tinual monitoring for the development of resistance, and, carrier, and can be stored as a concentrated aqueous solution when resistance appears, the Substitution of other phages to or lyophilized powder preparation. Bacteriophage may be which the bacterial mutants are not resistant. In general, formulated for oral administration by resuspending purified 25 phage preparations may be constructed by mixing several phage preparation in aqueous medium, Such as deionized separately grown and well-characterized lytic monophages, water, mineral water, 5% sucrose solution, glycerol, dextran, in order to (i) achieve the desired, broad target activity of the polyethylene glycol, Sorbitol, or such other formulations that phage preparation, (ii) ensure that the preparation has stable maintain phage viability, and are non-toxic to humans. The lytic properties, and (iii) minimize the development of resis pharmaceutical composition may contain other components 30 tance against the preparation. So long as the other components do not reduce the effective The development of neutralizing antibodies against a spe ness (ineffectivity) of the bacteriophage so much that the cific phage also is possible, especially after parenteral admin therapy is negated. Pharmaceutically acceptable carriers are istration (it is less of a concern when phages are administered well known, and one skilled in the pharmaceutical art can orally and/or locally). However, the development of neutral easily select carriers suitable for particular routes of admin 35 izing antibodies may not pose a significant obstacle in the istration (Remington's Pharmaceutical Sciences, Mack Pub proposed clinical settings, because the kinetics of phage lishing Co., Easton, Pa., 1985). action is much faster than is the host production of neutraliz The pharmaceutical compositions containing VRE-active ing antibodies. For VRE for example, phages will be used for bacteriophage may be administered by parenteral (Subcuta just a few days, sufficient to reduce VRE colonization during neously, intramuscularly, intravenously, intraperitoneally, 40 the time period when immunocompromised patients are most intrapleurally, intravesicularly or intrathecally), topical, oral, susceptible to the development of potentially fatal VRE sep rectal, inhalation, ocular, otic, or nasal route, as necessitated ticemia, but not long enough for phage-neutralizing antibod by choice of drug and disease. ies to develop. If the development of antiphage antibodies is Injection of specific lytic phages directly into the blood a problem, several strategies can be used to address this issue. stream can eliminate or significantly reduce the number of 45 For example, different phages having the same spectrum of targeted bacteria in the blood. If, after either oral or local activity (but a different antigenic profile) may be adminis administration, phages get into the bloodstream in Sufficient tered at different times during the course of therapy. On a numbers to eliminate bacteria from the bloodstream, septice more Sophisticated level, therapeutic phages may be geneti mia may be treated by administering phages orally (or cally engineered which will have abroadlytic range and/or be locally). If the phages do not get into the bloodstream in 50 less immunogenic in humans and animals. sufficient numbers to eliminate bacteria from the blood Environmental Therapy stream, the utility of direct i.v. injection of phages for treating In the 1980's a number of British studies were conducted septic infections can be used to treat bloodstream infections which demonstrated the efficacy of bacteriophage prophy caused by VRE and other pathogenic bacteria, and can pro laxis and therapy in mice and farm animal models. These vide an urgently needed means for dealing with currently 55 studies were significant because the titers of the phage prepa untreatable septicemic infections. rations administered were significantly less than the bacterial Dose and duration of therapy will depend on a variety of inoculum indicating in Vivo bacteriophage multiplication. factors, including the patient age, patient weight, and toler For example, Smith et al (Smith, et al. (1982), “Successful ance of the phage. Bacteriophage may be administered to treatment of experimental Escherichia coli infections in mice patients in need of the therapy provided by this invention by 60 using phage: its general Superiority over antibiotics. J. Gen oral administration. Based on previous human experience in Microbiol., 128:307-1825) found intramuscular inoculation Europe, a dose of phage between 107 and 10' PFU will be of mice with 10 CFU of E. coli with K1 capsule killed 10/10 Suitable in most instances. The phage may be administered mice. However when mice were simultaneously intramuscu orally in, for example, mineral water, optionally with 2.0 larly inoculated with 10 PFU of phage, at a separate site, grams of sodium bicarbonate added to reduce stomach acid 65 10/10 mice survived. Smith and coworkers demonstrated that ity. Alternatively, sodium bicarbonate may be administered administration of a mixture of two phage resulted in high separately to the patient just prior to dosing with the phage. levels of protection of calves with diarrhea induced by E. coli US 8,003,323 B2 15 16 with K 88 or K99 fimbriae (Smith, et al. (1983), “Effective "Strain phage Staphylococci applicable for prophylaxis and ness of phages in treating experimental Escherichia coli diar therapy of poultry Staphylococcus.” Soviet patent N1389287. rhea in calves, piglets and lambs. J. Gen. Microbiol., 129: Dec. 15, 1987). 2659-75; Smith, et al. (1987), “The control of experimental In the future, application of VRE phage to the environment Escherichia coli diarrhea in calves by means of bacterioph of farm animals such as chickens or cattle maybe necessary to age.” J. Gen. Microbiol., 133:1111-26: Smith, et al. (1987), reduce VRE in this setting if VRE become prevalent in such “Factors influencing the survival and multiplication of bacte environments and Such animal VRE are capable, upon being riophages in calves and in their environment.” J. Gen. Micro consumed in contaminated food, of transiently colonizing the biol., 133:1127-35). If the phage was administered before or 10 human gastrointestinal tract long enough to transferantibiotic at the same time as E. coli no deaths occurred and complete resistance gene transposons to normal gut flora (Latta, S. protection was attained. Control animals developed watery (1999) “Debate heats up over antibiotic-resistant foodborne diarrhea and died within 2 to 5 days. If phage administration bacteria.” The Scientist 13: (14) 4-5). was delayed until the onset of diarrhea, protection was not Alternatively, colonization in the farm animals may be complete although the severity of infection was greatly 15 reduced and no deaths were observed. Berchieri, et al., found reduced by administering bacteriophage to the animals (or that fewer chicks orally infected with 10 PFU of Salmonella treating the animals environment) using phage that produce typhimurium died when 10 PFU of Salmonella specific lytic infections in target bacteria which colonize the animals. phage was orally administered soon after initiation of the Such unwanted colonization may be a particular problem bacterial infection (Berchieri, et al. (1991), “The activity in with intensive agricultural techniques, such as those used for the chicken alimentary tract of bacteriophages lytic for Sal chickens or Veal calves. Target organisms include Salmonella monella typhimurium.Res. Microbiol. 142:541-49). They sp. and E. coli O157:H7. also found that the phage was readily spread between the Bacteriophage Cocktails different infected birds. This invention also contemplates phage cocktails which Environmental applications of phage in health care insti 25 may be custom tailored to the pathogens that are prevalent in tutions could lie most useful for equipment Such as endo a certain situation. Typically, pathogenic bacteria would be Scopes and environments such as ICUs which maybe poten initially isolated from a particular source (e.g., a patient or tial sources of nosocomial infection due to pathogens such as location contaminated with VRE) and susceptibility testing of VRE but which may be difficult or impossible to disinfect. the pathogens to various bacteriophage Strains would be per Phage would be particularly useful in treating equipment or 30 environments inhabited by bacterial genera Such as formed, analogous to antimicrobial Susceptibility testing. Pseudomonas which may become resistant to commonly Once each pathogen's phage susceptibility profile is deter used disinfectants. In the Soviet Union there has been a report mined, the appropriate phage cocktail can beformulated from that application of phage to the hospital environment has phage strains to which the pathogens are Susceptible and resulted in killing targeted bacteria Such as Staphylococci and 35 administered to the patient. Since phage would often be used Pseudomonas within 48-72 hours. Phage persisted in the in institutional settings where pathogens are resistant to many environment as long as there were target bacteria present and antimicrobial agents, phage cocktails would often consist of upon elimination of target bacteria, phage became undetect phage lytic for the most prevalent institutional pathogens able in 6-8 days (Alavidze, et al., 1988, “Use of specific which, in addition to enterococci, are Staphylococcus aureus, bacteriophage in the prophylaxis of intrahospital infections 40 Staphylococcus epidermidis, E. coli and Pseudomonas caused by P. aeruginosa. in Abstracts. All-Soviet Union aeruginosa. Also since enterococci are often involved in conference “Modern biology at the service of public health’. polymicrobial infections along with other gastrointestinal Kiev, Ukraine). commensals, such as in pelvic wound infections, the Phage compositions used to disinfect inanimate objects or approach of therapeutically using cocktails of phage lytic the environment may be sprayed, painted, or poured, onto 45 against different bacterial species would be most appropriate. Such objects or Surfaces inadueous solutions withphage titers Since phage cocktails would be constructed of phage against ranging between 107-10' PFU/ml. Alternatively, phage may institutional pathogens, isolation of Such phage would be be applied by aerosolizing agents that might include dry most Successful from the sewage of Such institutions. Typi dispersants which would facilitate distribution of the phage cally, the phage cocktail will include one or more VRE-active into the environment. Such agents may also be included in the 50 spray if compatible with phage viability and nontoxic in bacteriophage according to this invention. nature. Finally, objects may be immersed in a solution con It may be appropriate to use certain phage cocktails in taining phage. The optimal numbers and timing of applica agricultural settings where there are certain human pathogens tions of phage compositions remains to be determined and Such as Salmonella and Campylobacter inherent to poultry or would be predicated by the exact usage of Such products. 55 livestock and which contaminate the environment of Such Since phage are normally widely present in the environ animals on an ongoing basis. The result is a continuing source ment and are found even in food or drugs, there is minimal of infection by Such pathogens. safety concern with regard to applying phage preparations to Bacteriophage cocktails may be applied contemporane the environment. ously—that is, they may be applied at the same time (e.g., in As reported above, Smith and Huggins in England found 60 that E. coli induced diarrhea in calves could be prevented by the same application), or may be applied in separate applica simply spraying the litter in the calf rooms with an aqueous tions spaced in time Such that they are effective at the same phage preparation or even by keeping the calves in uncleaned time. The bacteriophage may be applied as a single applica rooms previously occupied by calves whose E. coli infections tion, periodic applications, or as a continuous application. had been treated with phage. There is also data from the 65 Other bacteria within the contemplation of the present Soviet Union indicating the efficacy of phage to rid chicken invention include, inter alia, Campylobacter, E. coli O157: houses of Staphylococci (Ponomarchuk, et al., (1987), H7, and Listeria, and Staphylococcus. US 8,003,323 B2 17 18 EXAMPLES using specific primers selected from published gene sequences (Goering, R. V. and the Molecular Epidemiologi Example 1 cal Study Group (1994) “Guidelines for evaluating pulsed field restriction fragment patterns in the epidemiological Obtaining VRE Isolates analysis of nosocomial infections.” (Abstract) Third Interna tional Meeting of Bacterial Epidemiological Markers; Cam Isolation of VRE bridge England). VRE were isolated by standard methods from patients in the Surgical intensive care and intermediate care units of the Example 2 University of Maryland Medical Center in Baltimore. Tryp 10 ticase Soy Agar supplemented with 5% sheep blood (BBL, Isolation of VRE Phage Cockeysville Md.) was used to isolate enterococci from urine, wounds and sterile body fluids. VRE were isolated from stool 500 ml of raw sewage from the University of Maryland is specimens on Colistin Nalidixic Acid (CNA) agar (Difco mixed with 100 ml of 10 times concentrated LB broth (Difco labs, Detroit, Mich.) supplemented with defibrinated sheep 15 Laboratories). This sewage-broth mixture is inoculated with a blood (5%), Vancomycin (10 g/ml) and amphotericin (1 18-24 hour LB broth culture (1 ml) of a VRE strain and g/ml). See Facklam, R. R., and D. F. Sahm. 1995. Enterococ incubated at 37° C. for 24 hours to enrich the mixture for cus. In: Manual of Clinical Microbiology, 6' edition, Ameri bacteriophage which can infect the VRE strain added. After can Society for Microbiology, Washington, D.C., pp. 508 incubation, the mixture is centrifuged at 5000 g for 15 min 312. utes to eliminate matter which may interfere with subsequent Identification of VRE filtration. The supernatant is filtered through a 0.45 m Milli Enterococci were identified by esculin hydrolysis and pore filter. Filtrate is assayed using the Streak Plate Method growth in 6.5% NaCl at 45° C. Identification to the species and/or Appelman Tube Turbidity Test to detect lytic activity level was done using conventional testing as indicated in against different strains of VRE. Facklam and Collins (Facklam, et al. (1989), “Identification 25 Method for Testing Phage Against VRE Isolates of Enterococcus species isolated from human infections by a Three methods are employed: Plaque Assay; Streak Plate conventional method test scheme. J. Clin. Microbiol., Method; and Tube Turbidity Method, and the procedures for 27:731-4). each follow. Antimicrobial Susceptibility Testing of VRE Plaque Assay: Antimicrobial Susceptibilities to amplicillin, Vancomycin, 30 A 18-24 hour nutrient broth culture of the VRE strain (0.1 streptomycin, and gentamicin were determined using the E nil) to be tested for susceptibility to infection and dilutions of test quantitative minimum inhibitory concentration proce a VRE phage preparation (1.0 ml) are mixed and then added dure (AB Biodisk, Solna Sweden). Quality control stains of to 4.5 ml 0.7% molten agar in nutrient broth at 45° C. This E. faecium (ATCC 29212, 51299) were used to ensure mixture is completely poured into a petri dish containing 25 potency of each antimicrobial agent tested. With exception of 35 ml of nutrient broth solidified with 2% agar. During overnight Vancomycin, Susceptibility interpretations from the National incubation at 37° C. VRE grow in the agar and form a con Committee for Clinical Laboratory Standards were adhered fluent lawn with some VRE cells being infected with phage. to (National Committee for Clinical Laboratory Procedures These phages replicate and lyse the initially infected cells and (1993), “Methods for Dilution Antimicrobial Susceptibility Subsequently infect and lyse neighboring bacteria. However Tests for Bacteria that Grow Aerobically.” 3rd Edition. 40 the agar limits the physical spread of the phage throughout the National Committee for Clinical Laboratory Standards Vill plate, resulting in Small visibly clear areas called plaques on anova Pa.; National Committee for Clinical Laboratory Stan the plate where bacteriophage has destroyed VRE within the dards (1993), “Performance Standards for Antimicrobial confluent lawn of VRE growth. Disk Susceptibility Tests' 5th Edition, National Committee The number of plaques formed from a given Volume of a for Clinical Laboratory Standards, Villanova Pa.). A VRE 45 given dilution of bacteriophage preparation is a reflection of isolate was defined as one which had a minimum inhibitory the titer of the bacteriophage preparation. Also, since one concentration to Vancomycin of at least 16 g/ml. plaque with a distinct morphology represents one phage par Defining Genetically Distinct VRE Strains ticle that replicated in VRE in that area of the bacterial lawn, Distinct VRE isolates were characterized as such by con the purity of a bacteriophage preparation can be ensured by tour-clamped homogeneous electric field electrophoresis 50 removing the material in that plaque with a pasteur pipette (a after digestion of chromosomal DNA with SmaI (Verma, P. et “plaque pick) and using this material as the inoculum for al. (1994) “Epidemiologic characterization of Vancomycin further growth cycles of the phage. On this basis, doing fur resistant enterococci recovered from a University Hospital ther plaque assays on preparations of phage grown from this (Abstract). In; Abstracts of the 94th General Meeting of the plaque pick, one would expect all plaques to have a single American Society for Microbiology, Las Vegas Nev.; Dean, et 55 appearance or plaque morphology which is the same as the al. (1994) “Vancomycin resistant enterococci (VRE) of the plaque picked, a further indication of purity. Therefore this VanB genotype demonstrating glycoprotein (G) resistance technique can not only be used to test bacteriophage potency inducible by Vancomycin (V) or teicoplanin (T). In; Abstracts but also bacteriophage purity. of the 94th General Meeting of the American Society for Streak Plate Method: Microbiology, Las Vegas Nev.). Electrophoretic studies were 60 Eighteen hour LB broth cultures of the different entero also performed using Apal digestion for VRE strains which cocci strains to be tested are grown at 37° C. (resulting in differed only by 1-3 bands after initial analysis (Donabedian, approximately 10 CFU/ml) and a loopful of each culture is S. M. etal (1992) “Molecular typing of ampicillin-resistant, streaked across a nutrient agar plate in a single line. This non-beta lactamase producing Enterococcus faecium isolates results in each plate having a number of different VRE from diverse geographic areas.” J. Clin. Microbiol. 30: 2757 65 streaked across it in single straight lines of growth. Single 61). The Vancomycin-resistant genotype (VanA. VanB or drops of phage filtrates to be tested are applied to the steaks of VanC) was defined by polymerase chain reaction analysis each VRE growth, and the plate is incubated 6 hours at 37°C., US 8,003,323 B2 19 20 at which time the steaks of the different VRE strains are (Pharmacia Biotech Piscataway N.J.) and a 0-1 M NaCl gra examined for the ability of phage to form clear areas devoid of dient in the start buffer. Phage tends to be eluted from the bacterial growth, indicating lysis of that particularVRE strain column between 150-170 mM NaCl with each fraction being by that particular phage. assessed for the presence of phage by standard plaque assay The VRE host range for a given phage filtrate can be 5 technique. Fractions collected and assayed are pooled if the ascertained by which VRE streaks it is capable of causing a phage titer by the plaque assay is no greater than 3 logs lower clear area devoid of growth and which strains of VRE the than the phage preparation put onto the column (e.g., 10" phage is incapable of doing this. PFU/ml is put onto the column therefore pool only those Appelman Tube Turbidity Test (from Adams, M. H. 1959. fractions with titers>107 PFU/ml). Pooled fractions are tested Bacteriophages. Interscience Publ. New York N.Y.): 10 for endotoxin by the Limulus Amebocyte Lysate Assay (Bio 18 hour LB broth cultures of different VRE Strains are Whittaker Inc Walkersville Md.). Pools demonstrating, >50 prepared. 0.1 ml of phage filtrate or a dilution thereof is added EU/ml of endotoxin are passed through a Affi-prep poly to 4.5 ml of VRE broth cultures and incubated at 37° C. for 4 myxin support column (Bio-Rad Labs, Hercules, Calif.) to hours (monophages) or 4-18 hours (polyvalent phages). remove residual endotoxin. Phage free VRE broth cultures are used as controls. Broth 15 The phage pool is buffer exchanged against 100 mM cultures which are normally turbid due tobacterial growth are ammonium bicarbonate using size exclusion with Sephadex examined for the ability of the phage to lyse the VRE strain as G-25 chromatography (Pharmacia Biotech). 1 ml aliquots of indicated by the clearing of the culture turbidity. the purified phage are freeze dried in the presence of gelatin The host range of a given phage can be ascertained by and stored at room temperature. The purity of the phage which VRE broth cultures the phage is capable of clearing and preparation is assessed by a combination of electron micros which broth cultures it cannot induce clearing. copy, SDS-PAGE, DNA restriction digest and analytical Example 3 ultracentrifugation. A Phage Strain is Active Against Over 200 VRE 25 Example 5 Isolates Determination of a Protective Dose of Bacteriophage A collection of 234VRE isolates, 187, E. faecium of which 3 strains are from ATCC, 41 E. faecalis strains, and 6 E. Establishment of Sustained VRE Colonizationina Animal gallinarium strains as well as 6 E. faecium strains which are 30 Model Vancomycin sensitive were tested for susceptibility of infec CD-1 mice are pretreated for seven days with 0.1 mg/ml of tion by 7 monophages isolated as described in Example 2. gentamicin and 0.5 mg/ml of Streptomycin in drinking water Susceptibility of infection was determined by the 3 tech to reduce their normal intestinal flora. VRE are then admin niques described. The majority of VRE strains in this collec istered to the mice, who have fasted for 6 hours, by consump tion were isolated from patients at the University of Maryland 35 tion of one food pellet inoculated with 10 CFU of VRE. VRE and Baltimore VA Medical Centers as indicated in Example 1. intestinal colonization is confirmed in mice by standard Such VRE isolates were determined to be distinct and geneti colony counts of >10 CFUVRE/gram of feces on CNA agar cally diverse by pulsed field gel electrophoresis typing. Of the containing 10 g/ml of Vancomycin, 1 g/ml of amphotericin B 7 monophages. VRE/E2 and VRE/E3 have a relatively narrow and 10 g/ml of gentamicin. The colonization procedure is host range compared to other VRE phages, but are able to 40 infect the small proportion of VRE strains which were resis considered successful if there is consistent shedding of >10 tant to other phages collected. A phage cocktail containing the CFU of VRE per gram of feces for 5-7 days after consumption above 7 VRE monophages lysed 95% of the VRE strains in of the spiked food pellet. VRE colonization may persist for 4 the collection. weeks by this method. Mice are given drinking water con 45 taining the above mixture of antibiotics throughout the dura Example 4 tion of the experiment. Use of a In Vivo Mouse Model to Demonstrate Efficacy of Producing Bacteriophage-Containing Compositions Lytic Bacteriophage in Reducing VRE Gastrointestinal Colo nization 0.1 ml amounts of a 18-24 LB broth culture' of a strain of 50 Twenty-four hours after detecting>10 CFUVRE/grain of VRE, which has been previously selected on the basis of feces, mice were administered VRE phage (by having them being able to produce a maximum yield of bacteriophage are consume one food pellet inoculated with 10 PFU of VRE). mixed with 1.0 ml of a VRE monophage filtrate and then Control groups consisted of (1) non-VRE-colonized mice mixed with 4.5 ml of 0.7% moltenagar in nutrient brothat 450 sham dosed (no phage in dose), (2) VRE-colonized mice C. This mixture is completely poured into a petri dish con- 55 which are sham dosed, and (3) non-VRE-colonized mice taining 25 ml of nutrient broth solidified with 2% agar. After dosed with phage. Five mice were used in each group. overnight incubation at 37°C., the softtopagar layer with the The efficacy of phage treatment to reduce VRE gastrointes phage is recovered by gently scraping it off the plate, and this tinal colonization was determined by quantitating VRE, on a recovered layer is mixed with a small volume of broth (1 ml daily basis, in weighed fecal samples from the mice in the per plate harvested). This Suspension is 60 different groups. In addition, at the end of the experiment, LB broth culture contains Bacto LB Broth. Miller (Luria-Bertani, dehydrated) mice were sacrificed and the number of VRE and phage in reconstituted according to instructions by Difco Laboratories, Detroit, Mich. centrifuged at 5,000-6,000 g for 20 minutes at 4°C. and the phage containing their liver, spleen, and blood determined. If administration of Supernatant is carefully removed. The Supernatant is filtered through a 0.45 m phage reduced VRE gastrointestinal colonization/overall filter and centrifuged at 30,000 g for 2-3 hours at 4°C. load in mice by at least 1 log as compared to the control The phage containing pellet is Suspended in 1-5 ml of 65 groups within 48-98 hours after phage administration, then phosphate buffer and is further purified by ion exchange this dose of the particular phage was deemed efficacious. chromatography using a Q resource ion exchange column More preferably, colonization was reduced by at least 3 logs. US 8,003,323 B2 21 22 Example 6 related fields are intended to be within the scope of the inven tion, which is limited only by the appended claims. Reduction of Colonization in Humans All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled The primary objective of this study is to (i) determine the in the art to which this invention pertains. All publications and efficacy of a candidate phage preparation in transiently elimi patent applications are herein incorporated by reference to the nating/reducing VRE colonization in humans, and (ii) further same extent as if each individual publication or patent appli assess the kinetics of turnover and the safety of the phages in cation was specifically and individually indicated to be incor immunocompromised patients, who are at greatest risk for porated by reference. 10 The invention claimed is: VRE infections. The study is a double-blinded, placebo-con 1. A method for reducing risk of bacterial infection in an trolled trial of oral phage administration in hospitalized animal colonized with bacteria comprising treating an animal patients colonized with VRE. with a composition containing bacteriophage of one or more VRE-colonized patients are enrolled in the study. The strains which produce lytic infections in said bacteria, said patients are randomized to receive VRE-specific phages or a 15 animal having been colonized by said bacteria for at least 24 placebo. Stool samples are collected immediately before hours, but not infected with said bacteria, wherein said treat administration of the phages or placebo and 1, 2 and 3 days ment of the animal reduces the level of colonization with after administration of the phages or placebo; for patients bacteria Susceptible to the bacteriophage by at least one log, who remain hospitalized, additional stool samples may be and wherein said composition is administered intravesicu obtained 7 and 10 days after phage/placebo administration. larly, topically, orally, rectally, ocularly, otically, nasally, or The amount of VRE and VRE-specific phages in the stools is via inhalation. quantitated, and data is recorded on patient diagnosis, level of 2. The method of claim 1, wherein the bacteria are selected immunosuppression (as reflected by the degree of neutrope from the group consisting of VRE, pneumococcal species, nia or administration of immunosuppressive medications), methicillin-resistant Staphylococcus species, MDRSA, and concurrent antibiotic therapy, if any. Side effects of phage 25 multi-drug resistant Pseudomonas species, Neisseria sp., administration, and changes in blood counts and renal and Hemophilus sp., Proteus sp., Klebsiella sp. Salmonella, liver function are noted. Campylobacter, Listeria, and Esherichia coli. Sufficient patients should be enrolled in each arm of the 3. The method of claim 2, wherein the bacteria are Salmo study to enable detection of a significant difference between nella. groups (95% confidence, 80% power) if 20% of the group 30 4. The method of claim 2, wherein the bacteria are Esheri receiving phages are VRE positive 3 days after phage admin chia coli. istration, vs. 50% of the group receiving a placebo. For these 5. The method of claim 2, wherein the bacteria are Esheri early Phase II efficacy studies. VRE cases are selected which Chia coli O157:H7. are susceptible to the phage preparation in vitro; a “broad 6. The method of claim 2, wherein the bacteria are Campy spectrum. VRE phage preparation may be tested during Sub 35 lobacter: sequent, more randomized clinical trials (i.e., phase III clini 7. The method of claim 1, wherein the composition con cal trials). VRE counts will be compared before and after tains a plurality of bacteriophage strains. phage/placebo administration, in order to determine whether 8. The method of claim 7, wherein the composition con phage administration, even if not eradicating carriage, results tains bacteriophage strains which produce lytic infections in in a significant (>1 log) decrease in VRE levels in stools. All 40 bacteria of a plurality of bacterial strains. VRE isolates will be screened for susceptibility to the phage 9. The method of claim 7, wherein the composition con preparation. Most patients are expected to be colonized with tains bacteriophage strains which produce lytic infections in only a single strain of VRE, some may have multiple strains; bacteria of a plurality of bacterial species. therefore, for a minimum of patients, 10-20 VRE colonies 10. The method of claim 1, wherein said animal is a farm should be picked from the primary isolation plate, in order to 45 animal. assess clonality (by PFGE) and for screening for phage sus 11. The method of claim 10, wherein said farm animal is ceptibility. poultry or livestock. A successful outcome for the studies consists of the dem 12. The method of claim 11, wherein said poultry is a onstration that (i) significantly more patients receiving chicken. phages became VRE culture-negative than did patients 50 13. The method of claim 10, wherein said farm animal is a receiving the placebo, or (ii) there was a significantly greater COW. decrease (>1 log) in VRE levels in the stools of persons 14. The method of claim 10, where said farm animal is veal receiving phages as compared with persons receiving the calves. placebo. From a clinical standpoint, there would be great 15. The method of claim 1, wherein the composition is value in reducing the levels of intestinal colonization during 55 selected from the group consisting of an oral tablet, capsule or periods of severe neutropenia/immunosuppression, when the liquid, a nasal aerosol, a throat wash, a mouth wash or gargle, risk of bacteremia is greatest. a toothpaste, rinses, creams, aerosols and a topical ointment. For purposes of clarity of understanding, the foregoing 16. The method of claim 1, wherein said animal is a invention has been described in some detail by way of illus chicken and said bacteria are Salmonella. tration and example in conjunction with specific embodi 60 17. The method of claim 1, wherein said animal is a ments, although other aspects, advantages and modifications chicken and said bacteria are E. coli. will be apparent to those skilled in the art to which the inven 18. A method for reducing the level of colonization by tion pertains. The foregoing description and examples are particular bacteria in a population of farm animals compris intended to illustrate, but not limit the scope of the invention. ing administering to farm animals a preparation of bacte Modifications of the above-described modes for carrying out 65 riophage lytic for said bacteria, said farm animals having the invention that are apparent to persons of skill in medicine, been colonized by said bacteria for at least 24 hours, but not bacteriology, infectious diseases, pharmacology, and/or infected with said bacteria, whereby colonization levels in US 8,003,323 B2 23 24 said farm animals are reduced by at least one log, and wherein 26. The method of claim 24, wherein the preparation con the preparation is administered intravesicularly, topically, tains bacteriophage strains which produce lytic infections in orally, rectally, ocularly, otically, nasally, or via inhalation. bacteria of a plurality of bacterial species. 19. The method of claim 18, wherein the bacteria are 27. The method of claim 18, wherein said farm animal is selected from the group consisting of VRE, pneumococcal poultry or livestock. 28. The method of claim 27, wherein said poultry is a species, methicillin-resistant Staphylococcus species, chicken. MDRSA, multi-drug resistant Pseudomonas species, Neis 29. The method of claim 27, wherein said farm animal is a seria sp., Hemophilus sp., Proteus sp., Klebsiella sp. Salmo COW. nella, Campylobacter; Listeria, and Esherichia coli. 30. The method of claim 27, where said farm animal is veal 20. The method of claim 19, wherein the bacteria are Sal 10 calves. monella. 31. The method of claim 18, wherein the preparation is 21. The method of claim 19, wherein the bacteria are selected from the group consisting of an oral tablet, capsule or Esherichia coli. liquid, a nasal aerosol, a throat wash, a mouth wash or gargle, a toothpaste, rinses, creams, aerosols and a topical ointment. 22. The method of claim 19, wherein the bacteria are 15 32. The method of claim 18, wherein said animal is a ESherichia coli O157:H7. chicken and said bacteria are Salmonella. 23. The method of claim 19, wherein the bacteria are 33. The method of claim 18, wherein said animal is a Campylobacter: chicken and said bacteria are E. coli. 24. The method of claim 18, wherein the preparation con 34. The method of claim 1, wherein said composition is tains a plurality of bacteriophage strains. administered periodically or continuously. 25. The method of claim 24, wherein the preparation con 35. The method of claim 18, wherein said preparation is tains bacteriophage strains which produce lytic infections in administered periodically or continuously. bacteria of a plurality of bacterial strains. k k k k k