Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification

<p>Abstract</p> <p>Background</p> <p><it>Vibrio cholerae </it>is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of <it>V. cholerae</it>. Detection of CT-producing <it>V. cholerae </it>using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of cholera toxin (CT)-producing <it>Vibrio cholerae</it>.</p> <p>Results</p> <p>The assay provided markedly more sensitive and rapid detection of CT-producing <it>V. cholerae </it>strains than conventional biochemical and PCR assays. The assay correctly identified 34 CT-producing <it>V. cholerae </it>strains, but did not detect 13 CT non-producing <it>V. cholerae </it>and 53 non-<it>V. cholerae </it>strains. Sensitivity of the LAMP assay for direct detection of CT-producing <it>V. cholerae </it>in spiked human feces was 7.8 × 10²CFU per g (1.4 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay for detection of CT-producing <it>V. cholerae </it>required less than 35 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 70 min with human feces from the beginning of DNA extraction to final determination.</p> <p>Conclusion</p> <p>The LAMP assay is a sensitive, rapid and simple tool for the detection of CT-producing <it>V. cholerae </it>and will be useful in facilitating the early diagnosis of human <it>V. cholerae </it>infection.</p

Investigation of stx2 eae+ Escherichia coli O157:H7 in beef imported from Malaysia to Thailand

To gain insight into the microbiological safety of food products routinely traded across international borders in Southeast Asian countries, beef imported from Malaysia to southern Thailand was examined for contamination with Escherichia coli O157 and its subsequent spread into the imported areas. We screened 31 samples exported from Malaysia and 36 domestic Thai samples. Isolation methods including an O157 antigen-targeted immunomagnetic separation technique, screening on CHROMagar O157 medium, and serotype confirmation of E. coli isolates by specific agglutination tests were employed. Fourteen strains of E. coli O157:H7 were isolated from eight Malaysian samples (25.8%) and six strains from four Thai samples (11.1%). These strains were of the stx1- stx2+ eae+ genotype except one Malaysian strain which was of the stx1 - stx2-eae+ genotype. All 19 O157:H7 strains possessing the stx2 gene produced little or no Stx2 (reversed passive latex agglutination titer ≤ 4). Of the 19 strains, five Malaysian (38.5%) and two Thai (33.3%) strains exhibited resistance to a set of antibiotics. Finally, the results of two DNA fingerprinting analyses (O157 IS-printing targeted to IS629 and pulsed-field gel electrophoresis, PFGE) of the O157:H7 strains possessing the stx2 gene, indicated that the Malaysian and Thai strains are closely related. Therefore, E. coli O157:H7 might be transferred from Malaysia to southern Thailand through beef trade

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

peer reviewedAmong Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified

Wide Distribution of O157-Antigen Biosynthesis Gene Clusters in Escherichia coli

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci

鹿児島県下の高校生の環境問題に関する認識(第3報) : 知識の情報源,環境問題の授業の評価,環境を守るために必要な事項および今後の課題

In September 1998, questionnaires on the source of environmental issues, the evaluation and the handling of the classes on the issues, and what was necessary for conserve the environment were investigated of 666 senior high school students in Kagoshima Prefecture. The following results were clarified : 1. Sources of knowledge concerning environmental issues were television (75%), newspaper (33%), other classes (29%), and home economics classes (8%). When students made the newspaper a source, they had awareness, knowledge, activity experiences, and responsible concern for the environment more than others significantly. 2. Eighty nine percent of the respondents answered that the classes on environmental issues were "Insufficiency". However, only 45% wanted to learn the issues in the class positively, and few items were wanted to study. 3. Forty eight percent of students gave the knowledge and the desire as a necessity to conserve the environment, and 35% gave oractice. Furthermore, 13% gave consideration and the action of the individual

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored bla CMY-2, a plasmid-mediated AmpC β-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring bla CMY-2