No abstract

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla CMY-2-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla NDM-1 gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla NDM-1 gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla NDM-1-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla NDM-1 gene. The complete sequence of pNDM-1_Dok01 suggests that the bla NDM-1 gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.

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:

cHelicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method involving multilocus sequence typing (MLST) was developed and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulsed-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from strain CCUG18818, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5 to 100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals belonged to the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar, but not identical, patterns between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, had the same pattern. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi. These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.