Plasmid-mediated Qnr and AAC(6)-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrBTolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.Fluoroquinolones (FQs) are synthetic chemical agents and among the most commonly prescribed antimicrobials because of their broad-spectrum antimicrobial activity. Extensive clinical and agricultural use of FQs has led to high rates of resistance to these agents among pathogenic microbes (6, 9). The most common mechanism for resistance to FQs among pathogenic microbes is the mutation of chromosomal genes encoding DNA gyrase and/or topoisomerase IV (12, 13). Changes in the expression of efflux pumps and porin proteins are also a common FQ resistance mechanism in bacteria (1), but no plasmid-mediated FQ efflux pump has been documented to date. Four chromosome-dependent efflux systems responsible for FQ resistance have so far been reported, e.g., the resistance nodulation division family, AcrAB-TolC in Escherichia coli (7,28) and MexAB-OprM in Pseudomonas aeruginosa (24); the major facilitator superfamily (MFS), NorA of Staphylococcus aureus (34); the multidrug and toxic compound extrusion family, NorM of Vibrio parahaemolyticus (19); and the ATP-binding cassette family, VcaM of non-O1 Vibrio cholerae (14).Two molecular groups have been identified as plasmid-mediated FQ resistance mechanisms to date. Qnr peptides, QnrA (30), QnrB (15), and QnrS (11), were identified from Klebsiella pneumoniae, Klebsiell...

The emergence of carbapenem resistance in Enterobacteriaceae clinical isolates is becoming a substantial clinical concern, because carbapenem antibiotics remain important agents for the treatment of infectious diseases caused by pathogenic Enterobacteriaceae in clinical settings (2, 7). The carbapenem resistance of these bacterial strains is due mostly to the production of horizontally acquired ␤-lactamases that are capable of hydrolyzing carbapenems, like IMP-1 metallo-␤-lactamase (MBL), which was first characterized in a Serratia marcescens clinical isolate in Japan (18), and a KPC ␤-lactamase was first identified from a Klebsiella pneumoniae clinical isolate in the United States (31). As for MBLs, the identification of IMPand VIM-type MBL genes, mediated by specific genetic elements like integrons, have increasingly been reported worldwide. In addition, SPM-1, SIM-1, GIM-1, KHM-1, and DIM-1 MBL genes have been found sporadically in members of the family Enterobacteriaceae, Pseudomonas spp., and Acinetobacter baumannii (4,16,20,22,28).Recently, a novel MBL, NDM-1, was identified from a K.pneumoniae strain recovered from a Swedish patient who had come back from India (32). After that, several reports indicate the further worldwide dissemination of NDM-1 producers (5,15,19,26), and it is becoming a great threat to human health together with the fact that NDM-1 producers also often possess a multidrug-resistant nature (14,19). In consideration of the rapid worldwide spread of NDM-1 producers, a nationwide survey in Japan was performed to determine whether or not the NDM-1-producing bacterial strain resided in imipenemnonsusceptible or ceftazidime-resistant Enterobacteriaceae strains collected from clinical facilities in Japan between September and December in 2010. In this survey, the presence of bla NDM-1 , bla KPC , bla IMP-1 , bla IMP-2 , and bla VIM-2 was detected by PCR in the collected strains. As a result, a small number of bla NDM-1 -positive strains were identified, although most of the MBL genes found in the collected strains were IMP-1-type MBL genes (unpublished data). Several strains, including S. marcescens strain 10mdr148, were found to be negative for the five carbapenemase genes described above, despite showing resistance to carbapenems. These results indicate the possibility that the carbapenem resistance of these isolates, including S. marcescens strain 10mdr148, depends on an unknown molecular mechanism, such as the production of a novel MBL. This study aimed to characterize the molecular mechanism underlying the carba-* Corresponding author. Mailing address:

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The complete 41 268 bp nucleotide sequence of the IncP-1b plasmid pBP136 from the human pathogen Bordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1b backbone, and 2 ORFs similar to the XF1596 and XF1597 genes with unknown function of the plant pathogen Xylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of the kle genes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid in Pseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of the kle genes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1b 'R751 group'. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1b plasmids, especially those of the R751 group, and (ii) the kle genes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells. INTRODUCTIONThe bacterial incompatibility group P-1 (IncP-1) plasmids can replicate and be stably maintained in almost all Gramnegative bacteria, and these plasmids are very promiscuous as either antibiotic-resistance or xenobiotic-degradative plasmids (Adamczyk & Jagura-Burdzy, 2003;Dennis, 2005;Thomas, 2000;Thomas & Smith, 1987). From the 1970s, many new members of the IncP-1 group have been discovered in bacteria from aquatic and soil environments (pB3, Heuer et al., 2004; pADP-1, Martinez et al., 2001; pB10, Schlüter et al., 2003; pB8, Schlüter et al., 2005; pUO1, Sota et al., 2003; pB4, Tauch et al., 2003; pTB11, Tennstedt et al., 2005; pJP4, Trefault et al., 2004; pEST4011, Vedler et al., 2004). The complete genome sequences of these newly identified IncP-1 plasmids have been determined, and the sequence data revealed that these plasmids have IncP-1-specific backbone modules for their replication, stable inheritance and conjugative transfer which show high similarity to the corresponding modules of two wellcharacterized IncP-1 plasmids, the IncP-1a subgroup plasmid RP4 (Pansegrau et al., 1994) and the IncP-1b subgroup plasmid R751 (Thorsted et al., 1998). Their genomes have various mobile genetic elements (transposons and their remnants) that carry genes encoding antibiotic resistance or degradation of xenobiotic compounds. Most of these accessory genes are inserted in one or both of two specific regions of the backbone, e.g. the IncP-1b plasmid pUO1 contains two haloacetate-catabolic transposons in the oriV-trfA region and two mercury-resistance transposons in the trb-tra region (Sota et al., 2003). In general, IncP-...

To investigate the exact isolation frequency of 16S rRNA methylase-producing, gram-negative pathogenic bacteria, we tested 87,626 clinical isolates from 169 hospitals. Twenty-six strains from 16 hospitals harbored 16S rRNA methylase genes, which suggests sparse but diffuse spread of pan-aminoglycoside-resistant microbes in Japan.