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Program in Antimicrobial Resistance
 





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 to better understand the mechanisms responsible for drug resistance, find new ways to protect antimicrobial agents from resistance, and ultimately develop new antimicrobials that overcome resistance. An integrated basic science, translational research, training program is being pursued in five key areas:

    - 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 pathogenic fungi such as Candida spp. and Aspergillus spp. These organisms, as well as laboratory strains of Escherichia coli, are used to study the mechanism of action of fluoroquinolones and echinocandins, a new class of antifungal agent. The PHRI group has one of the largest global collections of multidrug-resistant M. tuberculosis, and it originated the mutant selection window hypothesis, a new dosing strategy for blocking the acquisition of resistance. PHRI has also pioneered the diagnostic use of molecular beacons, a PHRI discovery.

PHRI scientists are actively involved in communicating new results to the lay and scientific communities. Among the recent publications is Antibiotic Resistance: Understanding and Responding to an Emerging Crisis by Drlica and Perlin (FT Press, scheduled for publication in 2011).

Specific research interests of five PHRI laboratories are described below.



Participating Investigators

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

Dr. Drlica’s research focuses on fluoroquinolones and their intracellular targets, the type II bacterial DNA topoisomerases (e.g. 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. One pathway, which is common to all quinolones and requires ongoing protein synthesis, involves the production of toxic reactive oxygen species. The other does not. Understanding the mechanism of the second pathway is a major priority for the Drlica laboratory, since that may lead to new derivatives that will actively kill non-growing bacterial cells.

Work on quinolone resistance has focused 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 from studies of mycobacteria and fluoroquinolones. This hypothesis provides a general framework for how antimicrobial dosing relates to the acquisition of resistance for many antibiotics and many pathogens.



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 (29,000 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 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) as well as 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 hospital and community settings, 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.



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, and (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.



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. Moreover, 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. The Perlin group focuses on the echinocandins, the first new, major antifungal drug class to enter the market in decades.

The echinocandins target the fungal cell wall by blocking β-(1,3)-D-glucan synthase. This knowledge forms the basis for understanding resistance mechanisms. So far, echinocandin resistance resulting in patient failure due to infecting strains having high MIC 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. The Perlin laboratory firmly established that resistance to echinocandin drugs for a wide range of fungi is associated with amino acid substitutions in two “hot-spot” regions of Fks1p or Fks2p, the catalytic subunits of glucan synthase. The mutations result in elevated MIC 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. This behavior of enzyme kinetics, along with clinical and pharmacokinetic/pharmacodynamic studies, has helped establish new CLSI breakpoints for susceptibility and resistance.

The Fks1 resistance mechanism accounts for intrinsic reduced susceptibility found in 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.

The Perlin group also studies triazole resistance in Aspergillus fumigates, a serious and emerging problem for patients suffering invasive fungal disease. Their goal is to develop a mechanistic view of emerging drug resistance to highly active triazole antifungal drugs used in the care of patients with acute and chronic Aspergillus infections. The hypothesis being explored is that both common (known) and new mechanisms contribute to clinical resistance. The program provides a comprehensive evaluation of existing resistance mechanisms accounting for a majority of clinical resistance, and novel mechanisms that are clinically significant and emerging. This work takes advantage of unique access to novel clinical resources present at the National Aspergillosis Center in South Manchester, United Kingdom, where Dr. Perlin serves as a visiting Professor. This program will establish a detailed understanding of target site modifications in Cyp51Ap that result in triazole resistance through a comprehensive genetic and biochemical evaluation of mutations obtained from clinical and laboratory isolates. They are also exploring the role of up-regulation of the target site and drug pumps in response to antifungal challenge in susceptible and resistant strains. New mechanisms of resistance in clinical isolates are being examined using a novel transposon insertion system. Overall, this program provides a comprehensive evaluation of clinically relevant triazole antifungal resistance in A. fumigatus.

Finally, the Perlin group has developed both PCR- and NASBA-based diagnostic detection platforms to rapidly diagnose drug-resistant infections in fungi and bacteria. This technology is capable of detecting less than one colony-forming unit and can identify a drug-resistant strain with its associated resistance mechanism in less than 2 hours.



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.

Dr. Zhao is also developing a gas-based treatment of tuberculosis that causes rapid and extensive cell death even when M. tuberculosis is resistant to multiple antimicrobials. One line of work seeks to validate treatment of infected lungs using animal models, while another focuses on understanding the biochemical events underlying rapid bacillary death. Gas-based cell death is expected to radically reduce bacterial load and increase the efficacy of all anti-tuberculosis agents.

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.



 
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