Characterization and genetic variation of vibrio cholerae isolated from clinical and environmental sources in Thailand

Cholera is still an important public health problem in several countries, including Thailand. In this study, a collection of clinical and environmental V. cholerae serogroup O1, O139, and non-O1/non-O139 strains originating from Thailand (1983 to 2013) was characterized to determine phenotypic and genotypic traits and to investigate the genetic relatedness. Using a combination of conventional methods and whole genome sequencing (WGS), 78 V. cholerae strains were identified. WGS was used to determine the serogroup, biotype, virulence, mobile genetic elements, and antimicrobial resistance genes using online bioinformatics tools. In addition, phenotypic antimicrobial resistance was determined by the minimal inhibitory concentration (MIC) test. The 78 V. cholerae strains belonged to the following serogroups O1: (n = 44), O139 (n = 16) and non-O1/non-O139 (n = 18). Interestingly, we found that the typical El Tor O1 strains were the major cause of clinical cholera during 1983-2000 with two Classical O1 strains detected in 2000. In 2004-2010, the El Tor variant strains revealed genotypes of the Classical biotype possessing either only ctxB or both ctxB and rstR while they harbored tcpA of the El Tor biotype. Thirty O1 and eleven O139 clinical strains carried CTXϕ (Cholera toxin) and tcpA as well four different pathogenic islands (PAIs). Beside non-O1/non-O139, the O1 environmental strains also presented chxA and Type Three Secretion System (TTSS). The in silico MultiLocus Sequence Typing (MLST) discriminated the O1 and O139 clinical strains from other serogroups and environmental strains. ST69 was dominant in the clinical strains belonging to the 7th pandemic clone. Non-O1/non-O139 and environmental strains showed various novel STs indicating genetic variation. Multidrug-resistant (MDR) strains were observed and conferred resistance to ampicillin, azithromycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim and harboured variants of the SXT elements. For the first time since 1986, the presence of V. cholerae O1 Classical was reported causing cholera outbreaks in Thailand. In addition, we found that V. cholerae O1 El Tor variant and O139 were pre-dominating the pathogenic strains in Thailand. Using WGS and bioinformatic tools to analyze both historical and contemporary V. cholerae circulating in Thailand provided a more detailed understanding of the V. cholerae epidemiology, which ultimately could be applied for control measures and management of cholera in Thailand

O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139

A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups. A panel of molecular marker-based TMPCR method generates reproducible profiles of V. cholerae-specific (588 bp) amplicons derived from ompW gene encoding the outer membrane protein and serogroup-specific amplicons, 364 bp for the O1 and 256 bp for the O139, authentically copied from rfb genes responsible for the lipopolysaccharide biosynthesis. The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae. The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs. In addition, the O serogroup-specific TMPCR method had 100% agreement with the serotyping method when examined for the serotyped V. cholerae reference strains and those recovered from clinical samples. The potential benefit of using this TMPCR tool would augment the serotyping method used in epidemiological surveillance and monitoring of V. cholerae serogroups, O1, O139, and non-O1/non-O139 present in clinical and environmental samples

O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139

A novel, sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR), which is based on two-stage amplification pertaining to multiplex PCR and conditional touchdown strategy, was used in detecting and differentiating Vibrio cholerae serogroups. A panel of molecular marker-based TMPCR method generates reproducible profiles of V. cholerae-specific (588 bp) amplicons derived from ompW gene encoding the outer membrane protein and serogroup-specific amplicons, 364 bp for the O1 and 256 bp for the O139, authentically copied from rfb genes responsible for the lipopolysaccharide biosynthesis. The TMPCR amplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588 and 256 bp) or a DNA fragment of non-O1/non-O139 (588 bp) while providing no false positive identifications using the genomic DNA templates of the other vibrios and Enterobacteriaceae. The reciprocal analysis of two-template combinations demonstrated that, using V. cholerae O1, O139, or equally mixed O1 and O139, the TMPCR had a detection limit of as low as 100 pg of the O1, O139, or non-O1/non-O139 in reactions containing unequally or equally mixed gDNAs. In addition, the O serogroup-specific TMPCR method had 100% agreement with the serotyping method when examined for the serotyped V. cholerae reference strains and those recovered from clinical samples. The potential benefit of using this TMPCR tool would augment the serotyping method used in epidemiological surveillance and monitoring of V. cholerae serogroups, O1, O139, and non-O1/non-O139 present in clinical and environmental samples

Antimicrobial Activity of the Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate (EGCG) against Clinical Isolates of Multidrug-Resistant <i>Vibrio cholerae</i>

The spread of multidrug-resistant (MDR) Vibrio cholerae necessitates the development of novel prevention and treatment strategies. This study aims to evaluate the in vitro antibacterial activity of green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) against MDR V. cholerae. First, MIC and MBC values were evaluated by broth microdilution techniques against 45 V. cholerae strains. The checkerboard assay was then used to determine the synergistic effect of EGCG and tetracycline. The pharmaceutical mode of action of EGCG was clarified by time-killing kinetics and membrane disruption assay. Our results revealed that all of the 45 clinical isolates were susceptible to EGCG, with MIC and MBC values in the range of 62.5–250 µg/mL and 125–500 µg/mL, respectively. Furthermore, the combination of EGCG and tetracycline was greater than either treatment alone, with a fractional inhibitory concentration index (FICI) of 0.009 and 0.018 in the O1 and O139 representative serotypes, respectively. Time-killing kinetics analysis suggested that EGCG had bactericidal activity for MDR V. cholerae after exposure to at least 62.5 µg/mL EGCG within 1 h. The mode of action of EGCG might be associated with membrane disrupting permeability, as confirmed by scanning electron microscopy. This is the first indication that EGCG is a viable anti-MDR V. cholerae treatment

Genetic variation of SXT element in <i>Vibrio cholerae</i>.

<p>The SXT structures of among 43 <i>V</i>. <i>cholerae</i> strains from Thailand were compared. Reads were mapped to genes of ICE<i>Vc</i>Hai1 (accession no. JN648379) and <i>dfr</i>A18 gene in SXTMO10 element (accession no. AY034138).</p

Frequency of resistance of <i>Vibrio cholerae</i> strains in Thailand.

<p>Frequency of resistance of <i>Vibrio cholerae</i> strains in Thailand.</p

Frequency of SXT element and antimicrobial resistance genes in <i>Vibrio cholerae</i> strains, Thailand.

<p>Frequency of SXT element and antimicrobial resistance genes in <i>Vibrio cholerae</i> strains, Thailand.</p

<i>In silico</i> MLST tree of <i>Vibrio cholerae</i> strains related with virulence gene profiles.

<p>The <i>V</i>. <i>cholerae</i> clinical (*) and environmental (•) strains in Thailand were related to pandemic and epidemic strains. Seven housekeeping genes were extracted from <i>V</i>. <i>cholerae</i> genomes. The phylogenetic tree was generated by FigTree.</p