Killing MRSA in Wounds

Award Number: W81XWH-10-2-0015 TITLE: Killing MRSA in Wounds PRINCIPAL INVESTIGATOR: Vincent$ Fischetti3K' CONTRACTING ORGANIZATION: Rockefeller University New York, NY 10021 REPORT DATE: July 201 TYPE OF REPORT: )LQDO PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for public release; distribution unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. Form Approved OMB No. 0704-0188 REPORT DOCUMENTATION PAGE Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 222024302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) July 2013 4. TITLE AND SUBTITLE 2. REPORT TYPE 3. DATES COVERED (From - To) 1 July 2010 - 30 June 2013 Final 5a. CONTRACT NUMBER Killing MRSA in Wounds 5b. GRANT NUMBER W81XWH-10-2-0015 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Vincent A. Fischetti, Ph.D. 5e. TASK NUMBER E-Mail: [email protected] 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER Rockefeller University New York, NY 10021 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Military personnel have an increased risk of injuries that would be susceptible to infection by Staphylococci (MRSA). The approach uses the rapid killing action of a phage lysin that kills MRSA and all other staphylococci to treat MRSA-infected wounds in a rat model to prevent infection. It is anticipated that the lysin may be used in the field to eliminate MRSA during transport to the field hospital and after. We asked if combination therapy with lysin and vancomycin will be more effective in clearing MRSA from freshly contaminated wounds than the standard of care using vancomycin alone. Results show that wounds of rats treated with buffer alone exhibited 106 CFU/gram of tissue of MRSA while animals treated with both Vancomycin / lysin had an average of 102 CFU/gram of tissue, a reduction of ~4-logs of MRSA. Treatment with vancomycin/buffer or buffer/lysin resulted in a total of 105. Experiments in which a combination of vancomycin and lysin was used on established MRSA wound infections, i.e., 5-day abscesses, show that rats treated with buffer alone exhibited an average of 105 CFU/gram of tissue of MRSA while animals treated with vancomycin and lysin had an average of <102 CFU/gram of tissue, a reduction of >3-logs of MRSA. Treatment with vancomycin/buffer or buffer/lysin resulted in a total of ~103 CFU/gm, reductions of <3 logs. 15. SUBJECT TERMS Phage lysin, MRSA, staphylococci, wound infections, field infections, hospital infections 16. SECURITY CLASSIFICATION OF: a. REPORT U b. ABSTRACT U 17. LIMITATION OF ABSTRACT c. THIS PAGE U UU 18. NUMBER OF PAGES 20 19a. NAME OF RESPONSIBLE PERSON USAMRMC 19b. TELEPHONE NUMBER (include area code) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Table of Contents Page Introduction…………………………………………………………….………..….. 4 Body………………………………………………………………………………….. 6 Key Research Accomplishments………………………………………….…….. 14 Reportable Outcomes……………………………………………………………… 14 Conclusion…………………………………………………………………………… 14 References……………………………………………………………………………. 15 Appendices…………………………………………………………………………… 17 INTRODUCTION: S. aureus is an opportunistic pathogen found on human skin and mucous membranes. It is the causative agent of a variety of skin and soft tissue infections in humans and serious infections such as pneumonia, meningitis, endocarditis, and osteomyelitis. Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a cause of infections in persons within the general community (community-associated methicillin resistant Staphylococcus aureus (CA-MRSA)) [1]. Diseases caused by CA-MRSA range from cutaneous infection to life-threatening systemic illness [2;3]; however, the majority of disease manifests as suppurative skin and soft-tissue infections. CA-MRSA is of particular importance to the military, as soldiers are counted among the epidemiological groups who appear to be particularly at risk [4]. Military personnel have an increased risk of injuries (from skin abrasions to severe wounds) that would be susceptible to infection by these virulent bacteria, thus methods must be devised to treat them quickly and effectively. Furthermore, in the cases of severe trauma under battlefield conditions, wounds may become infected deep within relatively avascular areas. Additionally, in the process of debridement, wounds may become opportunistically infected or colonized deep within the wound that has been closed. Because many staphylococci are resistant to conventional antibiotics, treating such infections is becoming increasingly difficult [4]. The global appearance of methicillin- and vancomycin-resistant clinical isolates of S. aureus has become a serious concern. Currently, 40-60% of nosocomial infections of S. aureus are resistant to oxacillin and greater than 60% of the isolates are resistant to methicillin [5]. Treating infections caused by the drugresistant S. aureus has become increasingly difficult and is therefore a major concern among healthcare professionals. To combat this challenge, development of new and effective antibiotics belonging to different classes are being aggressively pursued. A number of new antimicrobial agents such as linezolid, daptomycin, tigecyline, and ceftobiprole have been introduced or are under clinical development [6]. However, it has been reported that clinical isolates of MRSA have already become resistant to these new classes of antibiotics [7-9]. Consequently, there is an urgent need to develop novel therapeutic agents or antibiotic alternatives against MRSA. Since the beginning of U.S. military operations in Iraq and Afghanistan, there have been more than 40,000 injuries to U.S. service members [10]. Early, aggressive, debridement is the primary tool used to fight contamination and softtissue injuries. Antibiotics are generally not used in early treatment, because antibiotic therapy is initiated when soldiers are admitted to U.S. military hospitals after culture and sensitivity are performed. A major source of concern is that the use of broad-spectrum antibiotics for empirical treating of combat wounds results in selection of more resistant pathogens. Also, the use of broader-spectrum agents to treat multidrug resistant infections of non-U.S. personnel in Iraq may create increasing resistance in this reservoir of patients for potential nosocomial transmission. A survey of infections from hospitals in Iraq treating combat troops     4     showed that the most commonly isolated bacteria from infections in U.S. troops besides MRSA, were other staphylococci and streptococci [11]. The phage lysins will address many of these issues in that it is not an antibiotic, may be used early after the injury to control contaminating staphylococci and more importantly, it is effective against all species of staphylococci. Furthermore, the fact that lysins work synergistically with antibiotics will be useful. Bacteriophages infect their host bacteria to produce more virus particles. At the end of the reproductive cycle (which may last up to an hour) they are faced with a problem, how to release the progeny phage trapped within the bacterium. They solve this problem by producing a peptidoglycan hydrolase enzyme called lysin that degrades the cell wall of the infected bacteria to release the progeny phage [12]. The lytic system consists of a holin [12] and at least one lysin capable of degrading the bacterial cell wall. Lysins can be endo-beta-N-acetylglucosaminidases or N-acetylmuramidases (lysozymes), which act on the sugar moiety, endopeptidases, which cleave the peptide cross bridge, or more commonly, an N-acetylmuramoyl-L-alanine amidase (or amidase), which hydrolyzes the amide bond connecting the sugar and peptide moieties. Typically, the holin is expressed in the late stages of phage infection, forming a pore in the cell membrane, allowing the preformed lysin(s) to gain access to the cell wall peptidoglycan, resulting hypotonic lysis of the cell releasing phage progeny. Significantly, exogenously added lysin can lyse the cell wall of uninfected cells, producing a phenomenon known as lysis from without. However, because of the lack of an outer membrane, this event is observed only in gram-positive bacteria. While lysins have been known for many years [13-15], our laboratory was the first to use these enzymes therapeutically and prophylactically in vivo in their purified form to kill colonizing pathogenic bacteria on mucous membrane surfaces, infected tissues and in blood. So long as contact can be made with the bacteria, lysins have the capacity to kill the cell. In general, lysins are specific for the bacterial species from which they were produced, resulting in targeted killing. For example, we have purified lysins to kill S. aureus (MRSA), S. pyogenes [16] S. pneumoniae [17], Group B streptococci [18], Enterococcus faecalis/faecium [19] and B. anthracis [20]. All of these enzymes are highly evolved molecules designed for a specific purpose, to quickly destroy the bacterial cell wall. Nanogram to sub-microgram quantities of purified lysin per milliliter is sufficient to sterilize a 107 bacterial suspension in seconds to minutes. To date, other than chemical agents, there is no biological compound known that can kill bacteria this quickly. Since nearly all bacteria are or can be infected by bacteriophage, such enzymes may be developed for nearly all disease-causing gram-positive bacteria.     5     BODY: I. ClyS Lysins and Vancomycin in a 10-Day Wound Infection Model We began our studies with animal experiments to determine the effects of the combination of vancomycin and lysin on MRSA wound infections. In these experiments we asked if the combination therapy will be more effective in clearing MRSA from the infected wounds than the standard of care using vancomycin alone. Treatment: Day 0 - Rats were surgically opened and infected with MRSA (strain MW2) as described in previous reports. Day 4 - Rats started treatment with 50mg/kg every 12 hours IP with vancomycin. This treatment with vancomycin continued for the 10 days of the experiment. Day 5 - (36 h after vancomycin began) rat wounds / infections were re-opened, debrided and drained by wiping with sterile gauze and scalpel scraping Wounds were then washed with 500ul of 10mg/ml ClyS or Buffer before closing with surgical staples. Day 10 - Animals were Euthanized, wounds reopened, swabbed, tissue samples of infected muscle and/or abscesses were collected, disrupted and plated for CFU/gram. Results. As can be seen in Figure 1, the wounds of rats that were treated with buffer alone exhibited an average of 5.41 x 106 CFU/gram of tissue of MRSA while animals treated with Vancomycin / ClyS lysin had an average of 8.86 x 102 CFU/gram of tissue, a reduction of ~4-logs of MRSA. Treatment with 5 vancomycin/buffer or buffer/ClyS resulted in a total of 1.37 x10 and 9.44 x 105 respectively, intermediate reductions of about 1- 2 logs. These experiments will be repeated with more animals to determine reproducibility and statistical power. However, these results are consistent with the idea that lysins synergistically with antibiotics to effectively kill MRSA. We plan to repeat these experiments and use an ointment formulation of the lysins in wound infections to determine if efficacy can be increased.     6     1.0!1007 Wound infections treated with lysin and vancomycin 1.0!1006 1.0!1005 1.0!1004 1.0!1003 1.0!1002 Bu ffe r/b uf fe r Bu ffe r/C ly S Va n/ Bu ffe r Va n/ C lyS 1.0!1001 Rx Figure 1. MRSA wound infections were started on day 0, then, on day 4 vancomycin treatment or buffer was initiated and continued every 12 h for 10 days. On day 5 wounds were opened and debrided and treated with 500 ul of 10 mg/ml of ClyS lysine or buffer before closing. On day 10 all animals were euthanized and tissues removed and processed to obtain colony counts. Raw data: IP Vancomycin + ClyS wash upon draining and debridement of wound Vancom ycin/Cly S 1 2 3 4 5 Av     CFU/gra m 9.10E+02 1.92E+01 8.49E+02 1.37E+03 1.28E+03 8.86E+02 Vancom ycin/buf fer 1 2 3 4 5 CFU/gram Buffer /Clys CFU/gra m 5.85E+03 9.46E+03 7.81E+04 1.51E+03 5.92E+05 1.37E+05 1 2 3 4 2.67E+04 1.12E+05 1.38E+05 3.50E+06 7   9.44E+05 Buffer /buffe r 1 2 3 4 CFU/gram 5.42E+06 6.76E+06 3.49E+06 5.95E+06 5.41E+06   II. Effects of Lysin and/or Vancomycin Treatments in a 10-Day Established MRSA Wound Infection In this this set of experiments we continued our animal experiments to determine the effects of the combination of vancomycin and lysin on established MRSA wound infections. This differs from our previous results, where lysin was added early after contamination with MRSA (as would occur in the field) where we clearly show >4-log reduction compared to control given the high 107 dose of MRSA. In the current set of experiments we use lysin alone or in combination with Vancomycin to treat an established infection (abscess formation after 5 days) and compare it to the standard of care, Vancomycin alone. Treatment: Day 0 - Rats were surgically opened and infected with MRSA (MRSA strain MW2) as described in previous reports. Day 4 - Rats started treatment with 50mg/kg every 12 hours IP with vancomycin. This treatment with vancomycin continued for the 10 days of the experiment. Day 5 - (36 h after vancomycin began) rat wound infections were re-opened, debrided and drained by wiping with sterile gauze and scalpel scraping. Wounds were then washed with 500ul of 10mg/ml ClyS or buffer before closing with surgical staples. Day 10 - Animals were euthanized, wounds reopened, swabbed, tissue samples of infected muscle and/or abscesses were collected, disrupted and plated for CFU/gram. Results. As seen in Figure 2, the wounds of rats that were treated with buffer alone exhibited an average of 2x105 CFU/gram of tissue of MRSA while animals treated with Vancomycin and PlySS2 lysin had an average of <102 CFU/gram of tissue, a reduction of >3-logs of MRSA. Treatment with vancomycin/buffer or buffer/PlySS2 resulted in a total of ~103 CFU/gm, reductions of <3 logs. It should be noted that 6/16 animals in the PlySS2/Vanco group and 3/16 in the PlySS2 alone group showed no CFUs at 10 days, so 1 CFU was used for the analysis. Importantly, the results show that one dose of PlySS2 alone on day 5 was more effective than 5 days of treatment with Vancomycin alone. This result also emphasizes the usefulness of lysin treatment (wound irrigation) during the debridement process. Furthermore, a dose of 1 x 107 MRSA was used to infect the wounds, far more than would be expected to contaminate a wound in the field.     8     Day 5 after Debridement & Treatment 1.0!1008 1.0!1007 CFU / Gram 1.0!1006 1.0!1005 1.0!1004 1.0!1003 1.0!1002 Bu ffe r Va nc o Pl yS s2 Va nc o+ Pl yS s2 1.0!1001 Treatment Figure 2. MRSA wound infections were started on day 0 and on day 4 vancomycin treatment or buffer was initiated and continued every 12 h for 10 days. On day 5 wounds were opened and debrided and treated with 500 ul of 10 mg/ml of PlySS2 lysin or buffer before closing. On day 10 all animals were euthanized and tissues removed and processed to obtain colony counts. Based on Mann-Whitney analysis p=0.0001 between buffer and the combination of Vanco and PlySS2, as was PlySS2 alone and buffer.     9     III. Effects of Lysin on high and low dose of MRSA-infected wounds and effects of formulated lysin in a gel on clearance of MRSA-infected wounds. In our previous experiments we used an inoculum of MRSA of around 107 CFU to establish abscesses in the rat wounds. Unfortunately, this may not mimic the exposure to MRSA that may be acquired on the battlefield, which is likely much lower. To address this here, we repeated our lysin treatments using MRSA doses of 104 CFU and compared it to wounds infected with 107 MRSA. Also, we have developed a topical formulation gel that we find increases stability, is easier to manipulate and increases the residence time of the lysin in the wounds. This formulated lysin was tested in our regular wound infection model. A. Low vs High Dose of MRSA in wounds Treatment: Day 0 - Rats were surgically opened and infected with low (104) and high (107) dose of MRSA (strain MW2) as described in previous reports. 15 minutes later the wounds were treated with 500ul of 10mg/ml PlySS2 lysin and the wounds stapled shut. Day 5 - Animals were euthanized, wounds reopened, swabbed, tissue samples of infected muscle and/or abscesses were collected, homogenized and plated for CFU/gram. Results. As seen in Figure 3, the wounds of rats that were infected with low dose (104) of MRSA and treated with buffer alone exhibited an average of 5x104 CFU/gram of tissue of MRSA. The variation is large in this untreated group because this low dose can be somewhat handled by the animals. However, we could recover no CFUs in all the animals treated with PlySS2 in this low dose group. While the CFUs in the high dose MRSA group treated with buffer all clustered around 108 CFUs, 8/11 of the lysin-treated group were below our level of detection. Thus, it is expected that wounds contaminated in the field with MRSA would be around the 104 CFUs or less used in this experiment. Our results show that these wounds would easily be sterilized of these pathogens with one treatment of lysin. Even at doses up to 3-logs higher of MRSA, the single dose of lysin was able to remove all the MRSA in 72% of the infected wounds. B. Using slow release formulated lysin in MRSA wound infections Day 0 - Rats were surgically opened and infected with (107) dose of MRSA (strain MW2) as described in previous reports. The wounds were then treated with 1 ml of 5 mg/ml of PlySS2 lysin formulated in 2% methyl cellulose and 10% glycerol gel and the wounds were stapled shut.     10     Day 5 - Animals were euthanized, wounds reopened, swabbed, tissue samples of infected muscle and/or abscesses were collected, homogenized and plated for CFU/gram. Result: As can be seen in Figure 4, CFU counts clustered around 5x107 in the animals treated with formulated buffer, while 7/11 animals treated with formulated lysin had counts below our detectible limit. This result is comparable to the use of liquid lysin and shows that this type of formulation does not have an adverse effect on the lysin and as such we will be able to better handle and manipulate the lysin in this formulation rather than in a liquid. 5 day Post Wound Infection & Irrigation 1.0!1009 1.0!1008 1.0!1007 CFU/Gram 1.0!1006 1.0!1005 1.0!1004 1.0!1003 1.0!1002 1.0!1001 Bu ffe r C O N C Pl yS s2 C O N C 2x 2x 10 10 7 M R SA 7 M R SA Bu ffe r C O N C M R SA 4 10 2x 2x 10 4 M R SA C O N C Pl yS s2 1.0!1000 Rx 11/11  Below   1/11  Below   8/11  Below   0/11  Below   Figure 3. Comparison of high and low dose of MRSA after treatment with PlySS2 lysin. Numbers “Below” in yellow = animals with counts below detectible limits)     11     PlySs2 -Slow Release Flush Day 5 1.0!1009 1.0!1008 CFU/Gram 1.0!1007 1.0!1006 1.0!1005 1.0!1004 1.0!1003 1.0!1002 1.0!1001 SR -P ly Ss 2 SR -B uf fe r 1.0!1000 Rx 7/11  Below   0/11  Below   Figure 4. The ability of formulated lysin in a 2% methyl cellulose and 10% glycerol gel on the clearance of MRSA from infected wounds. IV. Treatment of mixed bacterial infections with lysins Killing both MRSA and S. pyogenes using a single lysin. Since the PlySS2 lysin we have been using in all of our wound studies not only kills MRSA effectively, but also has a similar effect on S. pyogenes, an organism that also contributes to the wound infections occurring on the battlefield and in military training camps. We examined whether a single lysin having both activities, will be able to control an infection by both pathogens. In order to show this more effectively we developed a model of lethal bacteremia in mice infected by both MRSA and S. pyogenes, and attempted to control both these infections with a single lysin. First we needed to establish the dose of each pathogen that would cause disease on its own and then used the combination of both bacterial infectious doses in the final experiment. As treatment and controls we used the following lysins that are either specific for each organism or the broad acting PlySS2: PlyC – is specific for S. pyogenes and will not kill MRSA ClyS – is specific for MRSA and will not kill S. pyogenes     12     PlySS2 – has broad activity and will kill both MRSA and S. pyogenes Experiment: Mice received 106 MRSA and / or 107 S. pyogenes intraperitoneally (IP) and after 3 hours all animals were bacteremic (based on preliminary experiments). At this time animals were treated IP with: 1. PlySS2 alone 2. ClyS + PlyC 3. ClyS alone 4. PlyC alone 5. Buffer All animals were followed for 7 days for survival. Results: As can be seen in Fig 5, animals with a mixed infection and treated with either PlySS2 or the combination of the PlyC and ClyS lysins were protected from death (left panel). Treatment with only one of the lysins caused death by the second organism. Animals infected with only one of the two pathogens (center and right panels) could be protected with PlySS2 lysin or the single lysin specific for the infecting organism. These results strongly indicate that not only can a lysin with broad activity be used in our wound infection model, but that a mixture of 2 lysins with different lethal specificities can be as effective in preventing infection. 3220% .20% ./0% 120% .50% 1.0% ./0% 160% 120% 0%<@*EBE&:% 420% 620% 820% 520% 720% /20% 340% 320% 330% 20% )*+&,-+',% 9:;#</% !BC% 340% 50% =:;# >9:;=% =:;#% 9:;=% ?@A+*% !BC% !BC% !BC% !BC% 60% B% 9:;#</% =:;#% 9:;=% ?@A+*% !"#$% !"#$% !"#$% !"#$% 20% B% 40% 9:;#</% =:;#% 9:;=% ?@A+*% D*$#% D*$#% D*$#% D*$#% !"#$%&'(%!"#$%&'()(*# Figure 1. Protection from Death After Dual Infection with MRSA and S. pyogenes. Mice received 106 MRSA and / or 107 S. pyogenes IP, and after 3h they were treated with either PlySS2 (n=24), ClyS + PlyC (n=18), ClyS (n=20), PlyC (n=20), or buffer (n=24).     13     KEY RESEARCH ACCOMPLISHMENTS 1. We have established a reliable rat model of wound infection by MRSA 2. We have determined that the addition of a foreign body allows for a reduction in the number of MRSA to cause an abscess 3. We have found that lysin may be used to kill MRSA in a contaminated wound 4. We have found that the combination of vancomycin (the standard of care drug) works synergistically with lysin to achieve a better outcome in the reduction of CFUs of MRSA 5. We have found that formulating the lysin in a slow release form also works to control abscess formation REPORTABLE OUTCOMES: 1. Manuscript in preparation 2. A reliable animal model was developed 3. The MRSA-specific lysins have been licensed by Contrafect Corporation, Yonkers, NY. The lysin is being developed to treat hospital MRSA sepsis CONCLUSIONS. We have clearly shown that phage lysins may be used in wounds to control MRSA from causing infections and abscesses. We show that lysin works more effectively than vancomycin alone and can be used to irrigate wounds that are infected with MRSA to eliminate the pathogen. We also show that a single lysin that has activity against both MRSA and S. pyogenes is able to control both pathogens. In anticipation of formulation studies, we also show that a gel formulation used in the wounds works well in clearing the MRSA and is better manipulated for that application. Thus, based on our animal studies, we are confident that lysins may be used in a field application to prevent infection by MRSA and perhaps also S. pyogenes. It may also be used as a topical treatment of the surface of sutured wounds to prevent infections in a hospital environment.     14     REFERENCES. 1. 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Schuch R, Nelson D, Fischetti VA: A bacteriolytic agent that detects and kills Bacillus anthracis. Science 2002, 418:884-889.     16     CURRICULUM VITAE Vincent A. Fischetti, Ph.D. Rockefeller University 1230 York Avenue New York, NY 10021 Pho: 212-327-8166 Fax: 212-327-7584 Cell: 516-901-8400 E-mail: [email protected] Web site: www.rockefeller.edu/vaf Personal: Married with 2 children Education: Ph.D. New York University, Microbiology, 1970, (with honors) M.S. Long Island University, Microbiology, 1967 B.S. Wagner College, Bacteriology, 1962 Training and Experience: 1990 -Pres. Professor and Chairman, Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, N.Y. 1978 - 1990 Associate Professor, The Rockefeller University, New York, N.Y. 1973 - 1978 Assistant Professor, The Rockefeller University, New York, N.Y. 1970 - 1979 Adjunct Assistant Professor, Adelphi University, Garden City, N.Y. 1972 - 1973 Guest Investigator, The Rockefeller University, New York, N.Y. 1972 - 1973 Postdoctoral Fellow, Albert Einstein College of Medicine, N.Y. 1970 - 1972 Postdoctoral Fellow, The Rockefeller University, New York, N.Y. Membership: American Society for Microbiology American Academy for Microbiology Honors and Awards (selected):   2012 2012 2011 2011 2011 Marie Curie Guest Lecturer, Copenhagen, Denmark Medical Grand Rounds, Weill Cornell Medical College, NY Distinguished Lecturer in Biomedical Science, Harvard University, Boston MA Keynote address, Phage 2011, Oxford England Edwin H. Beachey Distinguished Visiting Professorship, UT, Memphis 2011 2010 2010 2009 2009 2008 2008 2007 2007 2007 2006 2006 2006 2006 2006 2005 Keynote address, Dutch Infectious Diseases Update Course, Amsterdam Invited Speaker, Institute Pasteur Phage Symposium Honorary Doctor of Science, Wagner College, Staten Island, NY Invited Speaker, Nobel Symposium – Sweden ”Gram-Positive Infections” Distinguished Nelson Lecturer, University of Montana Speaker: Royal Society London,” Keynote Speaker, Edinburgh International Phage Conference Keynote Speaker, Pennsylvania ASM Distinguished lecture, CDC, Atlanta OKU Distinguished Lecture, NYU College of Dentistry Fellow, New York Academy of Sciences Lloyd Harris Lecturer, U. of Oklahoma G.F. Heinrich keynote lecture, Lang Center, NY Hospital Division M Keynote Address, ASM Chair, NY Academy of Sciences, Microbiology Section COBRE Visiting Scholar, U. Hawaii 16   2004 2004 2004 2003 2003 2003 2003 2002 2002 2002 2002 2001 2000 1999 1999 1999 1997 - 2007 1996 - 1997 1996 1995 1994 - Pres 1994 - 1995 1992 1992 1987 - 1997 1987 1987 1986 - 1987 1980 1977 - 1982 1973 - 1977 1971 - 1973 1970 Keynote address, Southern California ASM McLaughlin Lecturer, University of Texas, Galveston ASM Lecturer, American Society of Virology, Montreal Ellison Medical Foundation Lecture (Wind River Conference, CO) State of the Art Lecture, Am. Society of Virology, Davis, CA Chair, Gordon Conference, Chemical and Biological Terrorism Defense Invited speaker, ASM Biodefense Conference, Baltimore, MD Invited speaker, Banbury meeting on Bacteriophage Biology, CSH, NY Invited speaker, Boston University, Boston, MA Cover article - Nature, Aug. 22. “Defense against Anthrax” Keynote address, International Organization for Mycoplasmology Guest speaker, Swiss Society of Intensive Medicine Keynote address, Joint German Conference for Microbiology Keynote address, Japanese Lancefield Society Annual Meeting John H. Hanks Memorial Lecture, Johns Hopkins School of Public Health Pfizer Lectureship (U. Pittsburgh) MERIT Award, National Institutes of Health Foundation Lecturer, American Academy for Microbiology Keynote address, International Lancefield Society, Paris Invited speaker, Institut Pasteur Symposia, "The Year of Louis Pasteur" Fellow, American Academy for Microbiology Chairman, Div. B (Microbial Pathogenesis), Am. Society for Microbiology McLaughlin Lecturer, University of Texas, Galveston Burroughs Wellcome Visiting Professor, (University of Arizona) MERIT Award, National Institutes of Health Invited speaker, Institut Pasteur Centennial Shipley Lecturer, Harvard Medical School President, Lancefield Society Alumni Achievement Award, Wagner College Research Career Development Award, National Institutes of Health Senior Investigator, New York Heart Association Helen Hay Whitney Foundation Fellowship NYU Founders Day Award for outstanding scholarship Professional Activities (selected): 2012 – Pres Chairman, Scientific Advisory Board, Avacyn Corp 2012 - Pres Associate Editor, Microbiology Spectrum, an ASM publication 2010 – Pres. Chairman, Scientific Advisory Board, ContraFect Corp. 2009 – Pres. ASM Press Books Committee 2008 – Pres. Advisory Board: The Center for Structural Genomics of Infectious Diseases 1994 – Pres. Board of Scientific Advisors and Trustee, Trudeau Institute 1992 – Pres. Advisory Editor, Trends in Microbiology 1990 – Pres. Advisory Editor, Journal of Experimental Medicine 2009 - 2010 Advisory Board: DTRA (Defense Threat Reduction Agency) 2004 - 2008 Scientific Advisory Board, Great Lakes Regional Center of Excellence 2002- 2010 Chairman, Scientific Advisory Board, Enzybiotics, LLC 1999 - 2019 Microbiology Advisory Board, New York Academy of Sciences 1986 - 1996 2000 - 2003 1996 - 2002 1999 - 2002 1996 - 2000 1996 - 2001 1989 - 1999 1984 - 1998   Advisory Board, New York Hall of Science Awards Advisory Board, American Society for Microbiology Scientific Advisory Board, SIGA Technologies Chairman, Institutional Review Board (IRB), Rockefeller University Advisory Board, Defense Advanced Research Projects Agency (DARPA) Chief Scientific Advisor, SIGA Pharmaceuticals Editor-in-Chief, Infection and Immunity Co-Director, Biotechnology Facility at the Rockefeller University 17   1995 - 1997 1993 - 1995 1988 - 1989 1980 - 1985 1978 - 1983 1978 - 1989 1978 - 1980 Member, Scientific Advisory Board, Spectral Diagnostics Chairman, Scientific Advisory Board, M6 Pharmaceuticals Assistant Editor, Journal of Experimental Medicine Section Editor, Journal of Immunology NIAID Bacteriology and Mycology Study Section (BM2) Editorial Board, Infection and Immunity Editorial Board, Journal of Immunology Patents: US Patent No. 1. Production of streptococcal M protein immunogens # 4,784,948 2. Streptococcal immunoglobulin A binding protein # 5,352,588 3. Immunoglobulin binding protein ML2.2 # 5,556,944 4. Regulation of exoproteins in Staphylococcus aureus # 5,587,288 5. Method for exposing group A streptococcal antigens and an improved diagnostic test for the identification of group A streptococci # 5,604,109 6. Polypeptide of a hybrid surface protein by bacteria # 5,616,686 7. Process, apparatus and reagents for isolating cellular components # 5,634,767 (Commercialized as “Fast-prep” RNA/DNA isolation system by Q-biogene) 8. Gene for serum opacity factor # 5,707,822 9. Delivery and expression of a hybrid surface protein by bacteria # 5,786,205 10. Use of Gram-positive bacteria to express recombinant proteins # 5,821,088 11. Production of streptococcal M protein # 5,840,314 12. Fibronectin/fibrinogen binding protein of group A streptococci # 5,910,441 13. Method for screening inhibitors of the enzyme which cleaves the anchor of surface proteins from Gram-positive bacteria # 5,968,763 14. Regulation of exoprotein in Staphylococcus aureus II # 5,976,792 15. Prophylactic and therapeutic treatment of group A streptococcal infections # 5,985,271 16. Recombinant poxvirus and streptococcal M protein vaccine # 5,985,654 17. Therapeutic treatment of group A streptococcal infections # 5,997,862 18. Therapeutic treatment of group A streptococcal infections # 6,017,528 19. Topical treatment of streptococcal infections # 6,056,955 20. Use of bacterial phage associated lysing enzymes for the prophylactic and therapeutic treatment of various illnesses # 6,056,954 21. Plasmin binding protein and therapeutic use thereof # 6,190,659 22. Use of bacterial phage associated lysing enzymes for treating various illnesses # 6,238,661 23. Bacterial phage associated lysing enzymes for treating dermatological infections # 6,248,324 24. Use of phage associated lytic enzymes for treating bacterial infections of the digestive tract # 6,254,866 25. Parenteral use of bacterial phage associated lysing enzymes for the therapeutic treatment of bacterial infections # 6,264,945 26. Composition incorporating bacterial phage associated lysing enzymes for treating dermatological infections # 6,277,399 27. Use of bacterial phage associated lysing enzymes for treating streptococcal infections of the upper respiratory tract # 6,326,002 28. Receptor for Mycobacterium leprae and methods of use thereof # 6,331,405 29. Use of bacterial phage associated lysing enzymes for treating bacterial infections of the mouth and teeth # 6,335,012 30. Receptor for Mycobacterium leprae and methods of use thereof # 6,331,405 31. Fibronectin/fibrinogen binding protein of group A streptococci # 6,355,477 32. Composition for treatment of a bacterial infection of the digestive tract # 6,399,097 33. Composition for treating dental caries caused by S. mutans # 6,399,098 34. Composition for treatment of ocular bacterial infection # 6,406,692 35. Composition for treatment of a bacterial infection of upper respiratory tract # 6,423,299 36. Vaginal suppository for treating group B streptococcal infections # 6,428,784 37. Use of bacterial phage associated lysing enzymes for treating   18   dermatological infections 38. Synthetic peptides from streptococcal M protein and vaccines prepared therefrom 39. C1 bacteriophage lytic system 40. Chewing gum containing phage associated lytic enzymes for treating streptococcal A infections 41. Method for the treatment of bacterial eye infections 42. Tampon for the treatment of streptococcal group B infections 43. Therapeutic treatment of upper respiratory infections using a nasal spray 44. Method of treatment of vaginal infections 45. Nasal spray for treating streptococcal infections 46. Syrup composition containing phage associated lytic enzymes 47. Use of bacterial phage associated lysing enzymes for treating upper respiratory illness 48. Phage-associated lytic enzymes for the treatment of Bacillus anthracis and related conditions 49. Phage associated lytic enzymes for treatment of pneumonia 50. Nucleic acid and polypeptides of C1 bacteriophage and uses thereof 51. Bacteriophage lysins for Enterococcus faecalis, Enterococcus faecium and other bacteria 52. Glycosylated LPXTGases and uses thereof 53. PlyGBS mutant lysin Societies: American Society for Microbiology, Kunkel Society, New York Academy of Sciences   19   # 6,432,444 # 6,602,907 # 6,608,187 # 6,685,937 # 6,875,431 # 6,881,403 # 6,893,635 # 6,899,874 # 7,014,850 # 7,063,837 #7,141,241 #7,402,309 #7,569,223 #7,582,729 #7,582,281 #7,604,975 #8,105,585