Using Genomics to Identify Antibiotic Sensitivity Genes

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DESCRIPTION (provided by applicant): Antibiotic resistance is a growing problem that affects the treatment of almost all infectious diseases. The cost of treating antibiotic resistant infections is close to $30 billion/year in the United States alone. The identification of genes and mechanisms underlying resistant phenotypes is a focus of current drug discovery efforts wherein the goal is to develop combination therapies that target a process essential for bacterial survival and simultaneously render resistance mechanisms ineffective. On the way to this goal we believe it is necessary to understand the mechanisms by which resistant pathogens can once again be made sensitive to antibiotics and then exploit these mechanisms in the development of combination therapy. The objective of this study is to develop a new genomics based approach for the efficient identification of genes whose overexpression restores sensitivity to antibiotics in resistant pathogens and apply that approach. In the case of aminoglycoside resistant Pseudomonas aeruginosa, a significant cause of morbidity and mortality, particularly in cystic fibrosis patients, in addition to studying naturally occurring resistant clinical isolates of P. aeruginosa, two clinically relevant mechanisms of resistance with be engineered into PAO1 a sensitive strain of P. aeruginosa. This will be done with a plasmid either expressing the enzyme 6"-N-aminoglycoside acetyltransferase7, which modifies aminoglycosides or expressing the phoP gene, which reduces bacterial permeability to aminoglycosideds. Transforming these resistant strains with a genomic library will then allow for a DNA microarray based method to identify clones expressing genomic inserts which are absent in a bacterial population grown in the presence of aminoglycosides. It is these inserts which presumably restore a sensitive phenotype to these resistant strains, causing bacterial death and an absence of these inserts in the population. Confirmatory and then mechanistic studies will be carried out to further elucidate the mechanisms behind the sensitivity phenotypes discovered.