Because of B. pseudomallefs (Bp's) intrinsic resistance to many antibiotics, melioidosis therapy is difficult and must be continued for extended periods of time. The limited spectrum of antibiotics available for melioidosis treatment, and the emergence of resistant strains during antibiotic therapy call for a better understanding of underlying resistance mechanisms to enable proper therapeutic interventions. Furthermore, successful and proper treatment of infections caused by bioterrorism events involving strains having acquired resistance determinants, whether by natural means or by malicious genetic engineering, may be impossible if the underlying resistance mechanism(s) cannot be readily identified. The hypothesis is that a definition of the mechanisms governing resistance to frontline clinical drugs will allow design of strategies for rapid detection of resistance mechanisms in clinical isolates or in maliciously engineered strains. In turn, then, resistance can be detected early (bioterrorism events) and appropriate treatment initiated or redirected (in clinical settings during melioidosis therapy).
Three aims will be pursued to test the hypothesis:
Aim 1 - Definition of resistance mechanisms in clinical and environmental isolates. RT-PCR, expression of cloned gene products and biochemical approaches will be employed to characterize resistance determinants from clinical and environmental isolates resistant to p-lactams, doxycyline, trimethoprim and sulfamethoxazole.
Aim 2 - Definition of resistance mechanisms in genetically engineered strains. This will be achieved by characterizing transposon-induced resistant mutants, as well as by selecting and characterizing spontaneously resistant mutants to the antibiotics tested in aim 1.
Aim 3 - Design of a directed DNA microarrav as a tool for rapid identification of resistance determinants. Utilizing the information gleaned from aims 1 and 2, a directed DNA microarray will be generated which, in concert with a set of specific PCR primers, will provide a set of diagnostic tools for rapid determination of resistance mechanism in Bp isolates from diverse sources. Bp is an integral part of the RMRCE Bacterial Therapeutics Integrated Research Focus , whose main efforts are geared towards identification of novel therapeutics for this pathogen and a few other Select Agents. The tools, mutants and knowledged gained in this study will directly benefit these efforts.
Overcoming Gram-negative antibiotic resistance will be the major challenge for drug discovery efforts over the next decade;but how can we control antibiotic resistance when we don't understand it? What we learn about antimicrobial resistance with Bp will be directly applicable to similar problems faced with other nonenteric Gram negative pathogens.
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