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, hypermultidrug-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. Klebsiella pneumoniae is an important pathogen that causes urinary tract infection, opportunistic infection, and nosocomial infection. This bacterium can be isolated from not only the mammalian intestine but also environment 1,2. It belongs to the same Enterobacteriaceae as Citrobacter and Enterobacter. Recently, antibiotic resistance levels of these bacteria are increasing, and emerging multidrug-resistant bacteria are a serious problem at clinical sites. Multidrug efflux pumps are one of the mechanisms causing elevated resistance against many kinds of antimicrobial chemicals, such as antibiotics, dyes, antiseptics, and detergents in bacteria. So far, AcrAB 3,4 OqxAB 5 , EefAB 6 , KexD 7 , KmrA 8 , KdeA 9 , and CepA 3 have been reported as multidrug efflux pumps in K. pneumoniae. Of them, AcrAB, OqxAB, EefAB, and KexD are classified into the RND family, which is often the most important group of multidrug efflux pumps to protect against harmful chemicals in Gram-negatives. It was revealed that RND-type multidrug efflux pumps function with three components: in...

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