Triplex reverse transcription-PCR for detecting viable toxigenic Vibrio cholerae in water samples in Thailand.

Abstract. Detection of toxigenic Vibrio cholerae O1/O139 in aquatic environment is difficult to achieve using the culture method. For direct detection of viable toxigenic V. cholerae in aquatic environment, we developed a triplex reverse transcription (RT)-PCR, targeting genes for the outer membrane protein (ompW), cholera toxin A (ctxA) and toxin-coregulated pilli (tcpA) and compared the assay with the culture method. After enrichment of the bacteria-containing filters in alkaline peptone water for 6 hours, the sensitivity of triplex RT-PCR for detecting V. cholerae was 7 cfu/ml. Of the 80 environmental water samples collected from various regions in Northeast Thailand, triplex RT-PCR detected 15 toxigenic and 20 non-toxigenic V. cholerae, whereas the culture method detected only 3 toxigenic V. cholerae--containing water samples. These results show that this triplex RT-PCR method could be used as an alternative tool for rapid and sensitive identification of viable toxigenic V cholerae in environmental water samples.

ROLE OF HLYA-POSITIVE VIBRIO CHOLERAE NON-O1/NON-O139 ON APOPTOSIS AND CYTOTOXICITY IN A CHINESE HAMSTER OVARY CELL LINE.

Vibrio cholerae non-O1/non-O139 is capable of producing sporadic outbreaks of cholera-like diarrhea; however, the pathogenic mechanisms of this bacterium remain unclear. The objectives of this study were to: 1) compare the apoptosis induction and cytotoxicity between hlyA-positive and hlyA-negative strains of V. cholerae non-O1/non-O139; 2) clarify the molecular mechanisms by which these strains induce apoptosis; and 3) compare clinical and environmental V. cholerae non-O1/non-O139 isolates with respect to cytotoxicity and ability to induce apoptosis. Using cytotoxicity and apoptosis assays, it was shown that hlyA-positive strains of V. cholerae non-O1/non-O139 had significantly higher cytotoxic activity (70.6%) and levels of apoptosis induction (59.6%) than hlyA- negative strains (37.0% and 37.5%, respectively). Western blot analyses revealed that hlyA-positive strains had significantly increased expression of Bax; active caspase-3 and -9; and significantly decreased expression of NF-κB and Bcl-2 relative to hlyA-negative strains. Expression of BID did not differ significantly between hlyA-positive and negative strains. The truncated BID was not found, indicating that V. cholerae non-O1/non-O139 induces apoptosis through a mitochondria- dependent apoptosis pathway and not an extrinsic pathway. V. cholerae non-O1/ non-O139 isolated from clinical sources exhibited significantly higher cytotoxic activity (79%) and levels of apoptosis induction (65.2%) than bacteria isolated from environmental sources (63% and 54.6%, respectively), suggesting that the clini- cal isolates may have other virulence-associated genes besides hlyA. Our results indicate that hlyA products play a role in cytotoxicity and apoptosis induction and that a mitochondria-dependent apoptosis pathway is involved.

Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003-2011.

Cholera, caused by Vibrio cholerae, results in significant morbidity and mortality worldwide, including Thailand. Representative V. cholerae strains associated with endemic cholera (n = 32), including strains (n = 3) from surface water sources, in Khon Kaen, Thailand (2003-2011), were subjected to microbiological, molecular and phylogenetic analyses. According to phenotypic and related genetic data, all tested V. cholerae strains belonged to serogroup O1, biotype El Tor (ET), Inaba (IN) or Ogawa (OG). All of the strains were sensitive to gentamicin and ciprofloxacin, while multidrug-resistant (MDR) strains showing resistance to erythromycin, tetracycline, trimethoprim/sulfamethoxazole and ampicillin were predominant in 2007. V. cholerae strains isolated before and after 2007 were non-MDR. All except six diarrhoeal strains possessed ctxA and ctxB genes and were toxigenic altered ET, confirmed by MAMA-PCR and DNA sequencing. Year-wise data revealed that V. cholerae INET strains isolated between 2003 and 2004, plus one strain isolated in 2007, lacked the RS1 sequence (rstC) and toxin-linked cryptic plasmid (TLC)-specific genetic marker, but possessed CTX(CL) prophage genes ctxB(CL) and rstR(CL). A sharp genetic transition was noted, namely the majority of V. cholerae strains in 2007 and all in 2010 and 2011 were not repressor genotype rstR(CL) but instead were rstR(ET), and all ctx(+) strains possessed RS1 and TLC-specific genetic markers. DNA sequencing data revealed that strains isolated since 2007 had a mutation in the tcpA gene at amino acid position 64 (N→S). Four clonal types, mostly of environmental origin, including subtypes, reflected genetic diversity, while distinct signatures were observed for clonally related, altered ET from Thailand, Vietnam and Bangladesh, confirmed by distinct subclustering patterns observed in the PFGE (NotI)-based dendrogram, suggesting that endemic cholera is caused by V. cholerae indigenous to Khon Kaen.

Molecular analysis and antimicrobial resistance of Vibrio cholerae O1 in northeastern Thailand.

A total of 84 clinical Vibrio cholerae O1 isolates were collected from Khon Kaen (KK), Udon Thani (UT), Loei (LI), and Nong Khai (NK), northeastern Thailand during cholera outbreaks in 2007 and 2008. The majority of V. cholerae O1 strains carried nearly all the virulence-associated genes (ctxA, zot, and ace) except for four isolates and one isolate from UT and NK, respectively, which carried only tcpA, ompU, hlyA and toxR. None of the V. cholerae O1 strains carried sto. Pulsed field gel-electrophoresis (PFGE) profiling of 16 randomly chosen isolates showed the same PFGE pattern, except for one NK isolate, which was sensitive to all seven antibiotics used in the antimicrobial susceptibility tests. The tests revealed that multi-drug resistance to tetracycline and co-trimoxazole were present in KK strains (92%), followed by LI (75%) and UT (52%) strains. All strains were sensitive to norfloxacin but intermediate resistance to ciprofloxacin was found in a single strain from KK and LI. Differences in antimicrobial resistance among V. cholerae strains with the same PFGE pattern reflect differences in the antimicrobial agents used in each area of northeastern Thailand.