Program in Antimicrobial Resistance
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.
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.
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.
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.