Introduction

The development of antibiotics is one of the major milestones of medical science. But the efficacy of these miracle drugs has been threatened by microbial resistance, a natural response to widespread, and often indiscriminate use of antibiotics. PHRI scientists are seeking new ways to preserve antimicrobial agents from resistance and to develop new antimicrobials that overcome resistance through an integrated research and training program. Five areas are being studied:

    - New dosing strategies to severely limit the acquisition of bacterial resistance.

    - Identification of new intracellular targets for small-molecule enhancers of antimicrobials.

    - Mechanisms of drug resistance in bacteria and fungi.

    - Development of new, DNA-based methods for identifying drug-resistant microorganisms.

    - Development of new antimicobials that will overcome existing resistance.



This work focuses on a variety of pathogens that include Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, and a variety of pathogenic fungi. These organisms, as well as laboratory strains of Escherichia coli, serve to study the mechanism of action of fluoroquinolones and echinofungins, a new class of antifungal agent. The PHRI group has one of the largest collections of multidrug-resistant M. tuberculosis, and it originated the mutant prevention window hypothesis, a new strategy for blocking the acquisition of resistance. It has also pioneered the diagnostic use of molecular beacons, a PHRI discovery. Specific research interests of five PHRI laboratories are described below.



Participating Investigators

Karl Drlica, Ph.D. (Univ. of California, Berkeley)

Dr. Drlica’s research has focused on fluoroquinolones and their intracellular targets, the type II bacterial DNA topoisomerases (eg. DNA gyrase). Early work revealed that gyrase is responsible for maintaining negative supercoils in bacterial DNA and that the level of supercoiling is affected by a variety of perturbations including transcription and cellular energetics. Current work on the lethal mechanism of fluoroquinolones has revealed two pathways that lead to fragmentation of the bacterial chromosome. Work on quinolone resistance has focuses Mycobacterium tuberculosis, since the fluoroquinolones are agents of last resort with this pathogen. In collaborative work, Drs. Drlica and Zhao formulated the mutant selection window hypothesis, which provides a general understanding of how antimicrobial dosing relates to the acquisition of resistance.

Funding:

1R01 AI073491 (Drlica, PI) Lethal action of fluoroquinolones with non-growing Mycobacterium tuberculosis

Selected publications:

Zhou, J., Dong, Y., Zhao, X., Lee, S., Amin, A., Ramaswamy, S., J. Domagala, J. Musser, and Drlica, K. 2000. Selection of antibiotic-resistant bacterial mutants: allelic diversity among fluoroquinolone-resistant mutants. J. Infect. Dis. 182: 517-525.

Malik, M., Zhao, X., and Drlica, K. 2006. Lethal fragmentation of bacterial chromosomes mediated by DNA gyrase and quinolones. 2006. Mol. Microbiol. 61: 810-825.

Drlica, K. and Zhao, X. 2007. Mutant selection window hypothesis updated. Clin. Inf. Dis 44: 681-688.

Drlica, K., Malik, M., Kerns, R., and Zhao, X. 2008. Quinolone-mediated cell death. Antimicrobial Agents Chemother. 52: 385-392.

German, N., Malik, M., Drlica, K., and Kerns, R. 2008. Use of gyrase resistance mutants to guide synthesis of 8-methoxy-quinazoline-2,4-diones. Antimicrob. Agents Chemother. 52: 3915-3921.

Hussain, S., Malik, M., Shi, L., Gennaro, M., and Drlica, K. 2009. An in vitro model of mycobacterial growth arrest using nitric oxide with limited air. Antimicrob. Agents Chemother. 53: 157-161.



Barry Kreiswirth, Ph.D. (New York University)

In response to tuberculosis outbreaks in New York City, the PHRI TB Center was established in 1992 under Dr. Kreiswirth’s direction as a genotyping laboratory to study the molecular epidemiology of tuberculosis. The Center characterized the highly multidrug resistant strain W and created the nation’s largest M. tuberculosis strain and DNA fingerprint library (26,000 clinical isolates). Since its inception, the PHRI TB Center has worked closely with the Centers for Disease Control and Prevention and the New York City Department of Health to integrate the tools of molecular biology with tuberculosis control efforts. Local collaborations include the New Jersey Department of Health and Senior Services and the Wadsworth Center in Albany, NY; global interactions involve Russia, South Africa, Shanghai, Tanzania, and India. The molecular epidemiology of tuberculosis is being used as a platform to study both the evolution and the pathogenesis of M. tuberculosis.

The PHRI TB Center M. tuberculosis database includes over 26,000 strains, including over 5,000 multidrug resistant strains. The Center has been involved in studies of multidrug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB), and the molecular basis of resistance to streptomycin, rifampin, isoniazid, ethambutol, pyrazinamide, fluroquinolones and kanamycin.

Dr. Kreiswirth has also been involved in molecular characterization of nosocomial bacterial pathogens. He co-directed in the mid-1990s the Bacterial Antibiotic Group (BARG), a large consortium of New York City hospitals evaluating drug resistance in laboratory isolates. In 2003, he established the Molecular Outbreak Center to support hospital infection control activities with more than 45 participating hospitals in New Jersey. The genotyping of methicillin resistant S. aureus (MRSA) in outbreak investigations, both in the hospital and in the community setting, has become a major focus. His group developed spa typing, which has become the standard methodology to differentiate MRSA isolates. There are currently two active surveillance studies underway in Northern NJ and in New York City to understand the strain genotypes and patient risk factors associated with the spread of community acquired MRSA.

Funding:

U01 AI066561-01 (Perlin, PI) A Rapid and Expendable Nucleic Acid Platform to Detect Bloodstream Infections; 07/01/05 – 06/30/10

AI 066046-10A1 (Kaplan, PI) Host-Pathogen Interactions and Mtb Drug Resistance
The aim of this work is study MDR Mtb strains from S. Africa and evaluate potential virulence determinants; 8/15/06 – 7/31/10

Cepheid (Kreiswirth, PI) characterize a collection of MRSA isolates that have given false positive or false positive results in rapid MRSA diagnostics; 01/01/09 -12/31/09

New York City Depart of Health (Kreiswirth, PI) NYC Molecular Epidemiology Program; This public health program genetically characterizes all culture-positive M. tuberculosis clinical isolates from New York City patients in a Southern blot DNA fingerprint assay; 11/01/08 – 10/30/11

New York Community Trust (Kreiswirth, PI) Genotyping MRSA isolates among the Continuum Health Care hospital network MRSA from both surveillance cultures and infecting isolates will be genetically determined; 11/01/08 – 10/31/09

Heiser Foundation (Kreiswirth, PI) Characterization of M. tuberculosis strains among gold miners in Johannesburg; 01/01/09 – 12/31/11

NIH -2R44-AI078694-02 (Ecker, PI) Rapid High-Throughput Mycobacterium tuberculosis Genotyping drug resistant genes by mass spectrometry; 05/01/08 – 04/03/10

1R01 AI075463-01 (Xu, PI) Social and biomedical risk factors for multi-drug resistant TB in rural China Genotyping MDR M. tuberculosis strains from three different sites in rural China; 9/1/07 – 8/31/10

Selected publications:

Sinsimer, D., Leekha, S., Park, S., Marras, S.A., Koreen, L., Willey, B., Naidich, S., Musser, K.A., Kreiswirth, B.N. Use of a multiplex molecular beacon platform for rapid detection of methicillin and vancomycin resistance in Staphylococcus aureus. J Clin Microbiol 2005;43:458.

Mathema, B., Kurepina, N.E., Bifani, P.J., Kreiswirth, B.N. Molecular epidemiology of tuberculosis: current insights. Clin. Microbiol. Rev. 2006; 19: 658-685.

Fowler, V.G. Jr, Nelson. C.L., McIntyre, L.M., Kreiswirth, B.N., Monk, A., Archer, G.L., Federspiel, J., Naidich, S., Remortel, B., Rude, T., Brown, P., Reller, L.B., Corey, G.R., Gill, S.R.. Potential associations between hematogenous complications and bacterial genotype in Staphylococcus aureus infection. J Infect Dis. 2007;196:738-47.

Kennedy, A.D., Otto, M., Braughton, K.R., Whitney, A.R., Chen, L., Mathema, B., Mediavilla, J.R., Byrne, K.A., Parkins, L.D., Tenover, F.C., Kreiswirth, B.N., Musser, J.M., DeLeo, F.R..Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proc Natl Acad Sci U S A. 2008;105:1327-32.

Shet, A., Mathema, B., Mediavilla, J.R., Kishii, K., Mehandru, S., Jeane-Pierre, P., Laroche, M., Willey, B.M., Kreiswirth, N., Markowitz, M., Kreiswirth, B.N. Colonization and subsequent skin and soft tissue infection due to methicillin-resistant Staphylococcus aureus in a cohort of otherwise healthy adults infected with HIV Type 1. Infect Dis. 2009;200:88-93.



Arkady Mustaev, Ph.D (Novosibirsk State University, Russia)

This program focuses on molecular interactions between drugs and their protein targets. With RNA polymerase (RNAP) the goal has been to understand the functioning of the enzyme as a dynamic molecular machine at the atomic level of resolution in terms of (a) structural-functional studies of RNAP active center, (b) conformational transitions associated with RNAP catalytic cycle, (c) structural aspects of initiation, (d) aptamers to RNAP. Action of the antibiotic rifampicin is integrated into this work. Studies of DNA gyrase involve modeling of the fluoroquinolone-gyrase-DNA complex and use of new crosslinking as well as chemically modified drug derivatives to test the models. Dr. Mustaev is also developing fluorescence-based assays to study drug-protein interactions.

Funding:

R01-GM30717-21 (PI Mustaev) Structure and function of RNA polymerase in E. coli. (1983-2012)

Selected publications:

Mustaev A, Zaychikov E, Grachev M, Kozlov M, Severinov K, Epshtein V Korzheva N, Bereshchenko O, Markovtsov V, Lukhtanov E, Tsarev I, Maximova T, Kashlev M, Bass I, Nikiforov V, Goldfarb A. (2003) Strategies and methods of cross-linking of RNA polymerase active center. Methods Enzymol. 371, 191-206

Mustaev A, Goldfarb A. (2004) RNA polymerase reaction in bacteria. Encyclopedia of Biological Chemistry. Elsevier Inc. 775-780.

Kozlov M, Bergendahl V, Burgess R, Goldfarb A, Mustaev A. (2005 ) Homogeneous fluorescent assay for RNA polymerase. Anal Biochem.

Sosunov V, Morozov S, Sosunova E, Bass I, Nikolaev A, Goldfarb A, Nikiforov V, Severinov K, Mustaev A. (2005) Involvement of the aspartate triad of the active center in all RNA polymerase catalytic activities. Nucleic Acids Res. 33, 4202-11.

Kulbachinskiy A, Mustaev A.( 2006 ) Region 3.2 of the sigma subunit contributes to the binding of the 3'-initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation. J Biol Chem. 281, 18273-6

Feklistov A, Mekler V, Jiang Q, Westblade L, Irschik H, Jansen R, Mustaev A,.Darst S and Ebright R. (2008) Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center. PNAS 105:14820-12825.



David Perlin, Ph. D. (Cornell University)

Fungal infections are a significant cause of morbidity and mortality in severely ill patients, and their impact is exacerbated by a failure to rapidly diagnose and effectively treat these infections. The widespread use of antifungal agents has resulted in selection of naturally resistant fungal species, as well as the emergence of resistance in susceptible species. Treatment of fungal disease is hampered by the availability of few classes of antifungal drugs. Recently, a new class of echinocandin drugs was introduced clinically that target the fungal cell wall by blocking B-(1,3)-D-glucan synthase. The echinocandins are the first new major antifungal drug class to enter the market in decades, and it is vital to understand the nature of resistance mechanisms. Echinocandin resistance resulting in patient failure due to high MIC infecting strains is uncommon among susceptible species, such as the Candida spp. Yet, increasingly, there are reports of resistance in C. albicans, C. glabrata and C. krusei due to mutations in Fks1p, the major subunit of glucan synthase. In most cases, the patients experienced repeated and prolonged exposure to the drug prior to the emergence of resistant break-through strains. We have firmly established that resistance to echinocandin drugs in a wide range of fungi is associated with amino acid substitutions in two “hot-spot” regions of Fks1p, the catalytic subunit of glucan synthase. The mutations result in elevated MIC values and confer cross-resistance among the class of echinocandin drugs. Most significantly, Fks1 mutations diminish the biochemical drug sensitivity of glucan synthase by more than a thousand-fold, which is also observed as a comparable reduction in efficacy in animal models. The Fks1 resistance mechanism accounts for intrinsic reduced susceptibility of the C. parapsilosis group and it confers resistance in A. fumigatus. Often, Fks1 mutations resulting in resistance decrease the kinetic capacity of glucan synthase. As this enzyme is critical to cell physiology, the mutant strains show attenuated virulence in a range of animal models. This attenuated growth phenotype may help explain why echinocandin resistance is relatively rare in the clinic.

In addition, the Perlin group has adapted molecular beacons for both PCR and NASBA amplification platforms to establish novel molecular tools to rapidly diagnose drug-resistant infections. This technology is capable of detecting less than one colony-forming unit and can identify a drug-resistant strain in under 2 hours.

Funding:

1R01AI069397-01 (PI-Perlin) 12/01/06 - 11/30/11 Mechanism of clinical resistance to echinocandin antifungal drugs

U01 AI066561-01 (Perlin, PI) A Rapid and Expendable Nucleic Acid Platform to Detect Bloodstream Infections; 07/01/2005 – 06/30/2010

Pfizer (PI-Perlin) 05/01/2006-4/40/08 Echinocandin Reference Lab

Selected publications:

Arendrup, MC, Perkhofer, S, Howard, S.J., Garcia-Effron, G., Vishukumar, A., Perlin D., and Lass-Flörl, C. 2008. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins. Antimicrob Agents Chemother. 52(10):3504-11. PMC2565905

Cramer, R.A., Perfect, B.Z., Pinchai, N., Park, S., Perlin, D.S., Heitman, J., Perfect, J.R. and Steinbach, W.J. 2008. The Aspergillus fumigatus crzA gene is involved in conidia germination, hyphal growth, and required for fungal pathogenesis in an experimental murine model of invasive pulmonary aspergillosis. Eukaryot Cell. 7:1085-97. PMC2446674

Garcia-Effron, G., Park, S. and Perlin, D.S. 2009. Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints. Antimicrob Agents Chemother. Jan;53(1):112-22. PMC2612148

Arendrup,M.C., Garcia-Effron , G., Buzina, W., Mortensen, K.L., Reite, N., Lundin, C., Jensen, H.E., Lass-Flörl, C., Perlin, D.S. and Bruun, B. 2008. Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implication for susceptibility testing. In press. PMC Journal-In Process

Howard, S.J., Andersen, M.J., Albarrag, A., Fisher, M.C., Pasqualotto, A.C., Laverdiere, M., Perlin, D.S., Dennining, D.W. 2009. Frequency and Evolution of Azole Resistance in Aspergillus fumigatus Associated with Treatment Failure. Emerg. Infect. Dis. In press.

Garcia-Effron G, Lee S, Park S, Cleary JD, Perlin DS. 2009.Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-{beta}-D-glucan synthase: implication for the existing susceptibility breakpoint. Antimicrob Agents Chemother. 2009 Jun 22. [Epub ahead of print]



Xilin Zhao, Ph.D. (Univ. of East Anglia, UK)

Dr. Zhao is studying the bacterial stress response. His immediate goal is to identify protective gene products that can be inactivated with small-molecule inhibitors that will simultaneously increase the lethality of multiple antimicrobials. Current work focuses on making connections among bacterial toxin-antitoxin modules, reactive oxygen species, and a novel protein kinase that when deficient causes many antibacterial agents and environmental stressors to be more lethal.

In separate collaborative work, Drs. Zhao and Drlica developed the mutant selection window hypothesis, an idea that explains the acquisition of resistance. The hypothesis provides a way to severely restrict the development of resistance through adjustment of antimicrobial dosing. To validate the hypothesis Dr. Zhao has directed both animal and clinical tests.

In a third line of work, Dr. Zhao has been exploring anaerobic shock as a novel way to rapidly cure tuberculosis regardless of drug susceptibility and physiological state (growing or dormant) of the causative organism.

Funding:

NIH R21 AI068014-01 (PI-Zhao) 7/1/07-6/30/09 New genes involved in cellular responses to quinolone treatment.

Gates Foundation GCE 51963 (PI-Zhao) 10/1/08-9/30/09 Anaerobic shock as a novel treatment for tuberculosis.

Selected publications:

Zhao, X., and Drlica, K. 2001. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clin. Inf. Dis. 33(Suppl. 3): S146-S157.

Zhao, X. and Drlica, K. 2002. Restricting the selection of antimicrobial-resistant mutants: measurement and potential use of the mutant selection window. J. Inf. Dis. 185: 561-565.

Liu, Y., Cui, J., Wang, R., Wang, X., Drlica, K. and Zhao, X. 2005. Selection of rifampicin-resistant Staphylococcus aureus during tuberculosis therapy: concurrent bacterial eradication and acquisition of resistance. J. Antimicrobial Chemother. 56:1172-1175.

Cui, J., Liu, Y., Wang, R., Tong, W., Drlica, K., and Zhao, X. 2006. The mutant selection window in rabbits infected with Staphylococcus aureus. J. Inf. Dis. 194: 1601-1608.

Drlica, K. and Zhao, X. (2007). Mutant selection window hypothesis updated. Clin. Inf. Dis 44: 681-688.

Wang, X. and Zhao, X. 2009. Contribution of oxidative damage to antibiotic lethality. Antimicrob. Agents Chemother. 53:1395-1402.