Significance of efflux pumps in multidrug resistance and pathogenesis of Acinetobacter spp.

Hospital-acquired infections caused by Acinetobacter spp. are a serious problem especially in the burns and intensive care units because of the rapid increase in Acinetobacter spp. that have become resistant to multiple antibiotics. In this project, we will perform experiments that will allow us to understand how multi-resistance to antibiotics is caused. We will focus on broad substrate-specificity efflux pumps that actively export the drugs, thus rendering them less effective. We want to know the contribution of increased efflux pump activities in the development of multi-drug resistance in Acinetobacter spp. in the clinical environment. We anticipate that we will discover physiological substrates of these pumps and the biochemical pathways that produce them. This could lead to the discovery of novel antimicrobials and inhibitors of the broad-spectrum efflux pumps.

Nosocomial infections by Acinetobacter spp. have become increasingly difficult to treat because of the rapid development of multiple drug resistance (MDR) bacteria. The three members of the Acinetobacter calcoaceticus, Acinetobacter baumannii complex comprising A. genomospecies 2 (A. baumannii), A. genomospecies 3, and 13TU, are very closely related and not easily distinguishable in the clinical microbiology laboratory. In Singapore, 79% of Acinetobacter spp. isolated in hospitals are A. baumannii, 13% are A. genomosp. 13TU and 8% are A. genomosp. 3 but some non-baumannii isolates are also MDR and cause serious infections. Sequenced genomes of A. baumanii, A. genomosp. 3 and A. genomosp. 13TU have revealed many efflux pumps, but the substrates of only a few are known, and none of these efflux carbapenems (a marker for resistance). Homologues of these pumps in other bacteria are known to contribute to innate MDR and some pumps, such as AdeXY in A. genomospecies 3, appear physiologically important but this needs to be proven. Mechanisms regulating pump expression and the nature of the signals and environmental stimuli of the two-component systems (e.g. AdeSR) have not been determined. The physiological and pathological role of Acinetobacter efflux pumps is also not known. This project will identify the pumps that contribute to MDR by comparing expression of putative efflux pumps in drug-resistant versus drug-susceptible clinical Acinetobacter isolates. The hypotheses to be tested in this project are (1) efflux pumps that belong to the resistance-nodulation-division (RND) family have broad substrate-specificity and their over-expression will contribute significantly to increased drug resistance in Acinetobacter; (2) knowing the molecular mechanisms that activate the expression of these pumps will help manage the emergence of drug resistance; (3) expression profiling to map genes that are perturbed in the pump mutants will reveal biochemical pathway(s) that are pertinent to the efflux activities (e.g. the production of a toxic metabolite) and discover targets for development of efflux pump inhibitors or antimicrobials; and (4) identification of synergistic drug-antibiotic interactions will improve therapeutic options using existing antimicrobials. Our approach involves molecular genetics, microbiology, biochemistry, genome-wide expression profiling and cell-based virulence assays.