Nature Acta Medica Okayama 2045-2322 10 1 2020 The role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae 10876 EN Rui Ting Ni Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Motoyasu Onishi Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Minako Mizusawa Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ryoko Kitagawa Department of Microbiology, Faculty of Pharmaceutical Sciences,Okayama University Takanori Kishino Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Futoshi Matsubara Department of Microbiology and Biochemistry, Daiichi University of Pharmacy Tomofusa Tsuchiya Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teruo Kuroda Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Wakano Ogawa Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10(-7)-10(-9). Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 9 2019 Comprehensive analysis of resistance-nodulation-cell division superfamily (RND) efflux pumps from Serratia marcescens, Db10 4854 EN Shinsuke Toba Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yusuke Minato Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuma Kondo Department of Microbiology, Institute of Biomedical & Health Sciences, Hiroshima University Kanami Hoshikawa Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shu Minagawa Shiho Komaki Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Kumagai Department of Microbiology, Institute of Biomedical & Health Sciences, Hiroshima University Yasuyuki Matoba Department of Microbiology, Institute of Biomedical & Health Sciences, Hiroshima University Daichi Morita Department of Microbiology, Institute of Biomedical & Health Sciences, Hiroshima University Wakano Ogawa Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naomasa Gotoh Department of Microbiology and Infection Control Science, Kyoto Pharmaceutical University Tomofusa Tsuchiya Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Teruo Kuroda Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coil. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6G efflux assay. When expressed in the toiC-deficient strain of E. coil, KAM43, none of the genes conferred resistance on E. coil. When hasF, encoding the S. marcescens ToIC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coil, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2003 細菌細胞におけるイオン共役型輸送系の解析 EN Wakano Ogawa No potential conflict of interest relevant to this article was reported.