MLST/MVLST Analysis and Antibiotic Resistance of Vibrio cholerae in Shandong Province of China

Background: Vibrio cholerae is an important bacterium causing profuse watery diarrhea. Cholera had swept the whole Shandong province from 1975 to 2013. Methods: From epidemiological data and pulsed-field gel electrophoresis data, we selected 86 V. cholerae isolates appearing in Shandong Province in China from 1975 to 2013 and characterized them by multilocus sequence typing (MLST)/multi-virulence locus sequence typing (MVLST), antibiogram and analysis of genes related to antibiotic resistance. Results: Combined MLST/MVLST data revealed 33 sequence types and a major group. Within the group, 3 subgroups (ST1, ST24 and ST29) were revealed, prevalent in the strains isolated during the 1980s, 1990s and 21st century, respectively. All the O1 isolates after 1990 were found to be El Tor variants harboring the classical ctxB gene. The tcpA gene of O139 strains had a mutation at amino acid position 62 (N→D). Antibiotic resistance of V. cholerae increased over time. Most El Tor variants between 1998 and 1999 were resistant to trimethoprim/sulfamethoxazole. The O139 strain, since its appearance in 1997, had significantly broader spectrum of antibiotic resistance than O1 variants. The presence of the SXT element corresponds to the trend of growing drug resistance. Conclusion: The analysis of genotypic polymorphism and enhanced resistance of V. cholerae indicated continuous variation and evolution of this pathogenic agent in Shandong Province.

Characterization of V. cholerae O1 biotype El Tor serotype Ogawa possessing the ctxB gene of the classical biotype isolated from well water associated with the cholera outbreak in Kerala, South India

We investigated 22 water samples (17 well water and five pipe water – both chlorinated) and six soil samples from the surroundings of wells of the households of suspected patients from Palakkad district, Kerala (India), from where a cholera outbreak was reported during June–July 2016. A total of 25 Vibrio cholerae isolates were collected from three well water samples during a recent cholera outbreak. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. PCR assays confirmed the occurrence of ctxB, ctxA, hlyA, tcpA El Tor,VPI, ace, zot, ompW, rfbO1 and toxR genes in all isolates. The presence of the ctxB gene of the classical biotype in all of the El Tor isolates suggests that it is a new variant of El Tor biotype. Antibiogram profile of all V. cholerae O1 isolates revealed resistance towards five classes of antibiotics island and indicates that they were multidrug resistant. ERIC-PCR and PFGE finger prints showed the clonal relationship among the V. cholerae O1 isolates. The results of this study revealed the emergence of a new variant of El Tor biotype in the water samples from Palakkad district, from where a cholera outbreak was reported.

Detection of toxigenic Vibrio cholerae by PCR

holera toxin (CT) is a major virulence factor of V. cholerae causing water diarrhea. The detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming, laborious, and requiring more than three days perform. In this work a specific primers for ctxB gene were used for detection of V. cholera in water samples. Few colonies of V. cholera were suspended in water and used as a template in PCR reaction for the detection of ctxB gene. The 391-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. Direct use of V. cholerae pure culture for PCR replaces the need for DNA extraction or boiling. Increase the concentration of MgCl2 enhances the efficiency of amplification. The specificity of the assay was determined to be specific for V. cholerae but not for, vibrio related bacteria, E. coli, Non-Agglutinable (NAG) V. cholerae, and Aeromonas sp.

Co-expression and Immunity of Legionella pneumophila mip Gene and Immunoadjuvant ctxB Gene

The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplified by PCR respectively. The amplified cDNA was ligated to the pcDNA3.1(+) vector. The recombinant plasmids pcDNA3.1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR, and further confirmed by sequencing analysis. NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB according to the Lipofection method. Transient and stable products of the co-expression of the mip gene and ctxB gene were detected by immunofluorescence and Western blotting. The results showed that NIH3T3 cells were successfully transfected, and that the transiently and stably co-expressed products can be detected in the transfected cells. To detect the humoral and cellular immune response in immunized mice induced by the coimmunization of the mip and ctxB genes, female BALB/c mice were immunized intramuscularly with pcDNA3.1-mip and pcDNA3.1-ctxB. The results showed that the specific antibody titer and the cytotoxic T-lymphocyte response for pcDNA3.1-mip immunization and co-immunization were increased compared with that of pcDNA3.1(+) immunization. Furthermore, the specific antibody titer and cytotoxic T-lymphocyte response for co-immunization were increased compared with that of pcDNA3.1-mip immunization. Statistical analysis using one-way analysis of variance (ANOVA) showed that there was a significant difference between the groups (P<0.01). The results indicated that the ctxB gene enhanced the humoral and cellular immune response to the mip gene immunization. These findings provide experimental evidence to support the development of the L. pneumophila DNA vaccine.