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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 guard 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 selection 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 four 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 focuses on 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 AI35257 (Drlica, PI) DNA gyrase and fluoroquinolone resistance in tuberculosis
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.
Malik, M., and Drlica, K. 2006. Moxifloxacin lethality with Mycobacterium tuberculosis in the presence and absence of chloramphenicol. Antimicrobial Agents Chemother 50: 2842-2844.
Malik, M., Hussain, S., and Drlica, K. 2007. Effect of anaerobic growth on quinolone lethality with Escherichia coli. Antimicrobial Agents Chemother 51: 28-34.
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. Quinolone-mediated cell death. Antimicrobial Agents Chemother. (in press).
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 (23,000 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, China, 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 23,000 strains, including 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), 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 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; 07/01/2005 - 06/30/2010
Cepheid (Kreiswirth, PI) Genotyping the SCCmec of MRSA; 03/01/2007 - 02/28/2008
New York City Depart of Health (Kreiswirth, PI) NYC Molecular Epidemiology Program; 07/01/2005 - 10/30/2007
Gates Foundation (Sub-Contract) Natural Products Inhibit Intracellular Microorganisms Via Cellular Mechanisms 08/01/2005 - 07/31/2007
NIH (Sub-contract) Genotyping of M. tuberculosis using SSRs; 03/01/2005 - 2/28/2008
Eurofin (subcontract) Genotype the NARSA collection; 02/01/2007 - 01/31/2008
Johnson & Johnson (Kreiswirth, PI) The molecular epidemiology and pathogenesis of community-acquired MRSA; 12/01/2005 - 11/30/2007
Healthcare Foundation of New Jersey (Kreiswirth, PI) Genotyping M. tuberculosis from NJ patients; 03/01/2007 - 02/28/2008
Selected publications:
Bifani, P., Mathema, B., Kurepina, N., Kreiswirth, B.N. The Global dissemination of the W family of M. tuberculosis strains. Trends in Microbiology. 2002;10:45-52.
Munsiff SS, Nivin B, Sacajiu G, Mathema B, Bifani P, Kreiswirth BN. Persistence of a highly resistant strain of tuberculosis in New York City during 1990-1999. J Infect Dis. 2003;188:356-63.
Musser, J.M., Kapur, V., Williams, D.L., Kreiswirth, B.N., van Soolingen, D., van Embden, J.D.A. Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J. Infect. Dis. 1996;173:196-202.
Koreen, L., Ramaswamy, S.V., Graviss, E.A., Naidich, S., Musser, J.M., Kreiswirth, B.N. spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macroevolution. J Clin Microbiol 2004;42:792-799.
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 NE, Bifani PJ, Kreiswirth BN. Molecular epidemiology of tuberculosis: current insights. Clin. Microbiol. Rev. 2006; 19: 658-685.
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. The Perlin laboratory has been active in characterizing mechanisms of triazole resistance in Candida spp. and Aspergillus spp. including identification of target-site mutations and expression of multidrug efflux transporters. Recently, a new class of echinocandin drugs was introduced clinically that target the fungal cell wall by blocking β-(1,3)-D-glucan synthase. As patient exposure to echinocandin class drugs expands, it is anticipated that the number of clinical isolates with drug resistance will rise. 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. Recently, the Perlin laboratory reported that amino acid substitutions in two regions of Fks1p, a primary component of the glucan synthase complex, account for resistance in laboratory and clinical isolates of Candida spp. and Aspergillus fumigatus. They are exploring in detail by genetic and biochemical approaches the role of Fks1p as an important new mechanism for clinical resistance to echinocandin drugs.
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 (Perlin, PI) 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 (Perlin, PI) 05/01/2006 - 4/40/08 Echinocandin Reference Lab
Selected publications:
Park, S. and Perlin, D.S. 2005. Establishing surrogate markers for triazole resistance in Candida albicans. Microbiol. Drug. Res. 11(3):232-8
Park, S., Kelly, R., Nielsen-Kahn, J., Robles,J., Hsu,M-J., Register, E., Li, W., Vyas, V., Fan, H., Abruzzo, G., Flattery, A., Gill, C., Chrebet, G., Parent, S., Kurtz, M., Teppler, H., Douglas, C.M. and Perlin, D.S. 2005. Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida isolates. Antimicrob. Agents Chemother. 49(8):3264-73.
Balashov, S.V., Park, S. and Perlin, David S. 2006 Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by profiling mutations in FKS1. Antimicrob Agents Chemother. 50:2058-63.
Nielsen-Kahn, J., Garcia-Effron,G., Park,S., Marr, K.A. and Perlin, D.S. 2007 Acquired echinocandin resistance in a Candida krusei isolate due to modification of glucan synthase. Antimicrob Agents Chemother. 51(5):1876-8
Perlin, D.S. 2007. Resistance to echinocandin-class antifungal drugs. Drug Resis.Updates. 10(3):121-130.
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 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.
Funding:
NIH R21 AI068014-01 (Zhao, PI) 7/1/2007-6/30/2009 New genes involved in cellular responses to quinolone treatment
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.
Zhao, X., Malik, M., Chan, N., Drlica-Wagner, A., Wang, J.-Y., Li, X., and Drlica, K. 2006. Lethal action of quinolones with a temperature-sensitive dnaB replication mutant of Escherichia coli. Antimicrobial Agents and Chemotherapy 50: 362-364.
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