Systematic analysis of multidrug efflux pumps in multidrug resistant bacteria and development of pump inhibitor

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Infections by multidrug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Pseudomonas aeruginosa, Klebsiella pneumoniae are serious problems. Multidrug efflux pumps are most importantly involved in the multidrug resistance in multidrug resistant bacteria. We systematically investigated the multidrug efflux pumps of such bacteria. Based on genome sequence of MRSA N315, we cloned all of the 37 genes for tentative multidrug efflux pumps, and characterized the pumps. Also we investigated expression of the 37 genes in MRSA N315, and made clear which ones are really expressed in N315 cells and really contributing to the multidrug resistance. Thus, total picture of multidrug efflux pumps was made clear in MRSA N315. RND-type multidrug efflux pumps play major role in multidrug resistance in P aeruginosa. We cloned all of the 11 possible genes for RND-type multidrug efflux pumps in P aeruginosa PAO1 with which genome sequence is available. We characterized all of the hitherto uncharacterized pumps. Thus, total picture of RND-type multidrug efflux pumps was made clear. We isolated mutants of P.aeruginosa with elevated resistance to multiple antimicrobial agents. We cloned genes responsible for the multidrug resistance from chromosome of the mutants, expressed the genes and characterized the pumps. We identified the mutated portion of each gene for multidrug efflux pump. Genes for multidrug efflux pumps were also cloned from chromosome of K.pneumoniae, VRE, Vibrio parahaemolyticus and V.cholerae non-O1. We characterized many multidrug efflux pumps. We constructed mutants lacking genes for the multidrug efflux pumps, and investigated physiological roles of the pumps.We found inhibitors for the multidrug efflux pumps, skyrin and baicalein.