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Aim: This study aims to evaluate the genetic and population structure of Vibrio parahaemolyticus in the major coastal regions of China., Materials & Methods: Multilocus sequence typing was performed., Results: Insertion of large sequence into recA happened in nearly 30 strains, which were untypeable by multilocus sequence typing. A collection of 307 V. parahaemolyticus isolates were typed into 160 sequence types, including 117 novel ones. eBURST analysis revealed five clonal complexes, 11 doublets, and 108 singletons. The 160 sequence types formed two main lineages in the phylogenetic analysis., Conclusion: V. parahaemolyticus along the Chinese coastal regions exhibits high levels of genetic diversity and has undergone significant purifying selection and frequent recombination. A deeper understanding of V. parahaemolyticus genetic diversity could be obtained at the level of genome sequences.

Here, we constructed an Escherichia coli recA::luxCDABE bioreporter for genotoxicity assessments. The recA promoter was cloned from the marine bacterium Vibrio natriegens. This bioreporter showed a dose-response relationship following induction by mitomycin C, and other pollutants or environmental samples could be calculated as mitomycin C equivalents, which provided a way to quantitatively compare the genotoxicities of different environmental samples. This bioreporter was used to evaluate the genotoxicity under a wide range of external environmental conditions, like temperatures ranging from 15 °C to 42 °C, pH between 4.0 and 9.0, and salinity ranging from 0% to 3%. This successfully extended its application from the laboratory to the field, and allowed the bioreporter to assess the genotoxicity and bioavailability of genotoxins in various environmental media, including surface water, groundwater, seawater, and soil matrix. Expression of V. natriegens recA in E. coli indicated a LexA-like regulator in V. natriegens, and the putative SOS box of V. natriegens recA was similar to that of E. coli. The genotoxicities of groundwater samples from a petrochemical-contaminated site in northern China were evaluated by this bioreporter assay, and the genotoxic levels were in accordance with contamination levels obtained by chemical analyses.

A Gram-negative, oxidase-positive, facultatively anaerobic bacterium, designated strain E20121, was isolated from the digestive tract of a Japanese prawn (Marsupenaeus japonicus) collected from the coastal sea water area of Zhuhai, Guangdong province, China. The new isolate was determined to be closely related to Vibrio ponticus DSM 16217(T), having 97.6 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on recA, pyrH and rpoA also showed low levels of sequence similarities (72.6-96.6 %) with all species of the genus Vibrio. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) clearly showed that the new isolate is different from the currently known Vibrio species. DNA-DNA hybridization experiments revealed similarity values below 70 % with the closest related species V. ponticus DSM 16217(T). Several phenotypic traits enabled the differentiation of strain E20121 from the closest phylogenetic neighbours. The DNA G+C content of strain E20121 was determined to be 47.6 mol % and the major fatty acid components identified were C16:1ω7c and/or C16:1ω6c (39.8 %), C18:1ω7c (13.6 %) and C16:0 (9.6 %). Based on genotypic, phenotypic, chemotaxonomic, phylogenetic and DNA-DNA hybridization analyses, strain E20121 is proposed to represent a novel species of the genus Vibrio for which the name Vibrio zhuhaiensis sp. nov. is proposed. The type strain is E20121(T)(=DSM 25602(T) = CCTCC AB 2011174(T)).

Objectives: This study screened hospital sewage for the presence of blaNDM., Methods: Colonies grown from plates containing meropenem streaked with hospital sewage were screened for blaNDM by PCR. Species identification was performed by sequencing 16S rRNA genes. Clonal relatedness of isolates was determined by Enterobacterial repetitive intergenic consensus sequence-PCR, sequencing recA and PFGE. Mating was attempted to obtain self-transmissible plasmids carrying blaNDM. The genetic context of blaNDM was investigated by PCR mapping using pNDM-BJ01, a blaNDM-1-carrying plasmid from Acinetobacter lwoffii as the reference., Results: Two Acinetobacter johnsonii isolates, designated XBB1 and XBC1, from sewage were found carrying blaNDM-1, but were of different clonal origins. In both cases, blaNDM-1 was carried by a self-transmissible plasmid. PCR mapping and sequencing revealed that the blaNDM-1-carrying plasmid of XBB1, pXBB1, was the same as pNDM-BJ01, whereas that of XBC1, pXBC1, was a variant of pNDM-BJ01. A large region downstream of blaNDM-1, including groES/groEL, ISCR27 and ISAba125, was absent from pXBC1. On pXBB1, blaNDM-1 was carried by Tn125, an ISAba125-formed composite transposon, which was inserted into a sequence downstream of aphA6., Conclusions: Sewage of a hospital in China was found to contain blaNDM-1, suggesting that hospital sewage is an important but often overlooked reservoir of antimicrobial resistance determinants. A variety of Acinetobacter species in different locations have been found to harbour blaNDM-1, so the introduction of blaNDM-1 into Acinetobacter is unlikely to be a single event. The identification of blaNDM-1-carrying plasmid pNDM-BJ01/pXBB1 and its variants suggests that a common plasmid has been transferred among different host species at different locations.

During a study of the diversity and phylogeny of rhizobia isolated from root nodules of Oxytropis ochrocephala grown in the northwest of China, four strains were classified in the genus Rhizobium on the basis of their 16S rRNA gene sequences. These strains have identical 16S rRNA gene sequences, which showed a mean similarity of 94.4 % with the most closely related species, Rhizobium oryzae. Analysis of recA and glnA sequences showed that these strains have less than 88.1 and 88.7 % similarity with the defined species of Rhizobium, respectively. The genetic diversity revealed by ERIC-PCR fingerprinting indicated that the isolates correspond to different strains. Strain CCNWQLS01(T) contains Q-10 as the predominant ubiquinone. The major fatty acids were identified as feature 8 (C18: 1ω7c and/or C18: 1ω6c; 67.2 %). Therefore, a novel species Rhizobium qilianshanense sp. nov. is proposed, and CCNWQLS01(T) (= ACCC 05747(T) = JCM 18337(T)) is designated as the type strain.

During a study of the diversity and phylogeny of rhizobia in the root nodules of Kummerowia striata grown in north-western China, four strains were classified in the genus Rhizobium on the basis of their 16S rRNA gene sequences. The 16S rRNA gene sequences of three of these strains were identical and that of the other strain, which was the only one isolated in Yangling, differed from the others by just 1 bp. The16S rRNA gene sequences of the four strains showed a mean similarity of 99.3 % with the most closely related, recognized species, Rhizobium vitis. The corresponding recA and glnA gene sequences showed similarities with established species of Rhizobium of less than 86.5 % and less than 89.6 %, respectively. These low similarities indicated that the four strains represented a novel species of the genus Rhizobium. The strains were also found to be distinguishable from the closest related, established species (R. vitis) by rep-PCR DNA fingerprinting, analysis of cellular fatty acid profiles and from the results of a series of phenotypic tests. The level of DNA-DNA relatedness between the representative strain CCNWSX 0483(T) and Rhizobium vitis IAM 14140(T) was only 40.13 %. Therefore, a novel species, Rhizobium taibaishanense sp. nov., is proposed, with strain CCNWSX 0483(T) ( = ACCC 14971(T) = HAMBI 3214(T)) as the type strain. In nodulation and pathogenicity tests, none of the four strains of Rhizobium taibaishanense sp. nov. was able to induce any nodule or tumour formation on plants. As no amplicons were detected when DNA from the strains was run in PCR with primers for the detection of nodA, nifH and virC gene sequences, the strains probably do not carry sym or vir genes.

The purpose of this study was to determine the existence of Burkholderia cepacia complex (Bcc) at species level and the predominant species in the environment of moso bamboo plantations in Hangzhou, China. A total of 423 isolates were recovered from moso bamboo rhizhosphere soil samples of three sites on the selective medium during 2007-2008. Isolates were identified by Bcc-specific PCR assays, followed by recA-restriction fragment length polymorphism assays, species-specific PCR analysis, recA gene sequencing, multilocus sequence typing (MLST) scheme, and BOX-PCR fingerprinting for genomic diversity. Out of 423 isolates, 278 isolates were assigned to the following Bcc species, eight B. stabilis, 26 B. anthina, 193 B. pyrrocinia, and 51 B. arboris, which indicated B. pyrrocinia as the most dominant species followed by B. arboris. Moreover, false positives were observed in certain isolates of B. arboris while performing species-specific PCR test. Furthermore, the results of recA gene sequence similarity and MLST data demonstrated that nine isolates formed a single discrete cluster but were PCR negative to species-specific primers representing novel species may exist within the Bcc. In addition, BOX-PCR fingerprinting for all the Bcc isolates also showed the strain diversity. It is the first report of the existence of B. arboris and predominance of B. pyrrocinia in the moso bamboo environment.

A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Thirteen novel strains were isolated from root nodules of three different leguminous plants of the genera Albizia, Kummerowia and Dalbergia grown in China. Cells of these strains were Gram-negative, strictly aerobic, non-spore-forming, motile rods. Phylogenetic analysis of 16S rRNA gene sequences revealed that they belong to the genus Rhizobium. A representative strain, CCBAU 25010T, showed 98.8% 16S rRNA gene sequence similarity to its closest phylogenetic relative, Rhizobium sullae IS123T. The 16S-23S intergenic spacer (ITS) sequence of CCBAU 25010T shared 91.5 and 87.2% sequence similarity, respectively, with those of Rhizobium etli CFN 42T and Rhizobium leguminosarum LMG 14904T. Analysis of the sequences of the housekeeping genes atpD and recA was in agreement with the results of ITS sequence analysis. The nodC gene sequence of CCBAU 25010T was identical to that of Rhizobium tropici CFN 299. DNA-DNA hybridization values for strain CCBAU 25010T ranged from 20.7% (with Rhizobium mongolense USDA 1844T) to 34.4% (with R. leguminosarum USDA 2370T). Cell protein SDS-PAGE, BOX-PCR and several phenotypic characteristics, such as use of sole carbon sources and antibiotic resistance, could differentiate the novel strains from defined Rhizobium species. We therefore propose that the novel strains reported in this study form a novel species, Rhizobium mesosinicum sp. nov., with the type strain CCBAU 25010T (=LMG 24135T =JCM 14777T).

Objective: To analyze sequences of the housekeeping genes including recA, dnaE, and mdh in different serogroups or different biotypes of Vibrio cholerae (VC) strains isolated from China., Methods and Results: recA, dnaE, and mdh genes of Vibrio cholerae were obtained by PCR, sequenced and analyzed. Forty-four variable bases were identified in the 500 bases of recA gene of 18 VC strains, and the mutation of only 3 variable bases could result in changes of 2 amino acids. In the 600 bases of dnaE genes of 18 strains, 32 variable bases were found and only 1 contributed to an amino acid change. In the 367 bases of mdh genes of 18 strains, only 1 variable base was identified whose mutation involved an amino acid convertion. Toxic EI Tor biotype (EVC) strains and toxic O139 serogroup strains were closely related. Non-toxic strains of different serogroups or types were lowly related. Non-toxic and toxic strains of different serogroups or types were lowly related., Conclusion: Toxic EVC and toxic O139 serogroup strains isolated from different areas of China may evolve from a common original strain, and toxic O139 VC strains may come from toxic EVC strains.