Molecular Typing of Vibrio cholerae O1 Isolates from Thailand by Pulsed-field Gel Electrophoresis

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999–April 2000 and December 2001–February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)—PF-I and PF-II—predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern—PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future

Molecular Typing of Vibrio cholerae O1 Isolates from Thailand by Pulsed-field Gel Electrophoresis

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)-PF-I and PF-II-predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern-PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future

Sequence and Apoptotic Activity of VacA Cytotoxin Cloned from a Helicobacter pylori Thai Clinical Isolate

The vacuolating cytotoxin VacA produced by Helicobacter pylori induces the formation of large cytoplasmic vacuoles in host gastric epithelial cells as well as a release of cytochrome C from mitochondria resulting in cell apoptosis. Considerable sequence diversity in VacA relating to different degrees of disease severity is observed with clinical samples from a multitude of geographic places. In this study we describe expression in Escherichia coli, purification to homogeneity and in vitro assay of its apoptotic activity of a VacA toxin from a H. pylori isolate of a Thai patient with gastrointestinal lymphoma. Sequencing revealed that the deduced amino acid sequence of the cloned Thai isolate VacA is similar to H. pylori s1/m2 type strains. The percent sequence similarity to the model strain 60190 was lower due to the presence of extra amino acids in the mid (m) region. The purified VacA toxin exhibited significant apoptotic activity on both T84 and MDCK epithelial cell lines, as revealed by DAPI staining, whereby the observed activity was significantly higher on MDCK cells. These findings could relate to a modulation of VacA activity on host cells in the Thai isolate-VacA toxin that may differ from those of the model strain

Virulence genes of clinical Vibrio cholerae O1 isolates in Thailand and their ribotypes

Objective: To determine virulence associated-genes and ribotypes of Vibrio cholerae epidemic strains isolated from cholera patients in Thailand. Method: A total of 240 V. cholerae El Tor, O1 strains, isolated from patients with cholera in Thailand during two different periods, i.e. 1999-2000 (200 strains; 193 Ogawa and 7 Inaba) and 2001-2002 (40 strains; all Inaba), were analyzed for the presence of virulence genes, namely ctxA, ctxB, zot, ace, toxR, tcpA, hlyA, nanH and ninT by PCR. For ribotyping, genomic DNA segments of the 240 strains and 10 reference V. cholerae strains isolated before 1999 from Thailand and elsewhere were digested with BglI endonuclease, subjected to a 0.8% agarose gel electrophoresis, blotted onto a nylon membrane and probed with enzyme-labeled Escherichia coli rRNA. The DNA bands were visualized by autoradiography. Results: Genes encoding the A and B subunits of CT, Zot, Ace, ToxR, TcpA, HlyA, NanH and NinT could be amplified from all of the 10 V. cholerae O1 reference strains and from 239 of the 240 studied isolates. One Inaba isolate of 2001-2002 gave only amplicons of toxR and hlyA. For ribotyping, the 10 reference strains revealed six different patterns designated A to F. None of the 240 strains isolated in Thailand during the two periods had the A-C, E and F ribotypes. The isolates of 1999-2000 revealed ribotype D and three other ribotypes, designated G, H and I. The majority of the isolates of 2001-2002 showed ribotype G. The remaining showed other new ribotypes, J and K. Conclusions: The clinical V. cholerae isolates of two epidemics from Thailand showed a sustained appearance of one epidemic V. cholerae clone, and a constant, but gradual and minor change in the genetic constituent of the other V. cholerae strains as indicated by the change of the ribotypes of the strains in the two study periods. Moreover, we found that a V. cholerae strain which cannot produce CT, Zot, Ace, TcpA, NanH and NinT can still cause symptomatic cholera

Comparative Genomic Characterization of a Thailand–Myanmar Isolate, MS6, of <i>Vibrio cholerae</i> O1 El Tor, Which Is Phylogenetically Related to a “US Gulf Coast” Clone

<div><p>Background</p><p>The cholera outbreaks in Thailand during 2007–2010 were exclusively caused by the <i>Vibrio cholerae</i> O1 El Tor variant carrying the cholera toxin gene of the classical biotype. We previously isolated a <i>V. cholerae</i> O1 El Tor strain from a patient with diarrhea and designated it MS6. Multilocus sequence-typing analysis revealed that MS6 is most closely related to the U. S. Gulf Coast clone with the exception of two novel housekeeping genes.</p><p>Methodology/Principal Findings</p><p>The nucleotide sequence of the genome of MS6 was determined and compared with those of 26 <i>V. cholerae</i> strains isolated from clinical and environmental sources worldwide. We show here that the MS6 isolate is distantly related to the ongoing seventh pandemic <i>V. cholerae</i> O1 El Tor strains. These strains differ with respect to polymorphisms in housekeeping genes, seventh pandemic group-specific markers, CTX phages, two genes encoding predicted transmembrane proteins, the presence of <i>metY</i> (MS6_A0927) or <i>hchA/luxR</i> in a highly conserved region of the <i>V. cholerae</i> O1 serogroup, and a superintegron (SI). We found that <i>V. cholerae</i> species carry either <i>hchA/luxR</i> or <i>metY</i> and that the <i>V. cholerae</i> O1 clade commonly possesses <i>hchA/luxR,</i> except for MS6 and U. S. Gulf Coast strains. These findings illuminate the evolutionary relationships among <i>V. cholerae</i> O1 strains. Moreover, the MS6 SI carries a quinolone-resistance gene cassette, which was closely related with those present in plasmid-borne integrons of other gram-negative bacteria.</p><p>Conclusions/Significance</p><p>Phylogenetic analysis reveals that MS6 is most closely related to a U. S. Gulf Coast clone, indicating their divergence before that of the El Tor biotype strains from a common <i>V. cholerae</i> O1 ancestor. We propose that MS6 serves as an environmental aquatic reservoir of <i>V. cholerae</i> O1.</p></div

<i>Vibrio cholerae</i> O1 genomes can be divided into two clusters that possess either <i>hchA/luxR</i> or <i>metY</i> in a conserved syntenic region of the small chromosome.

<p>Dendrograms were constructed based on <i>hchA</i>/<i>luxR</i> or <i>metY</i> using the method described in the legend to Fig. 3. Strains highlighted in blue belong to serogroup O1. However, four strains enclosed in the purple square may have undergone lateral gene exchange of O-antigen gene clusters; thus, strains V52 and MO10 were converted into O37 and O139 serogroups, respectively, while strains 12129(1) and TM11079-80 gained the O1-antigen gene cluster <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098120#pone.0098120-Chun1" target="_blank">[25]</a>.</p

MS6, but not U. S. Gulf Coast strain 2740-80, carries the <i>Vibrio</i> seventh pandemic island-1 (VSP-1) between <i>VC0174</i> and <i>VC0186</i>.

<p>Dendrograms were constructed based on the genes flanking VSP-1 (<i>VC0174</i> and <i>VC0186</i>) using the neighbor-joining method using MEGA version 5.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098120#pone.0098120-Tamura1" target="_blank">[37]</a>. VSP-1 was identified in the eight <i>V. cholerae</i> strains shown in red. MS6 as well as the seventh pandemic strains carry the full VSP-1 sequence between <i>VC0174</i> and <i>VC0186</i>, which is closely related to that of strain 2740-80. Scale bars indicate nucleotide substitutions per site.</p

Hypothetical evolutionary relationship among clades of <i>Vibrio cholerae</i> with reference to strain MS6.

<p>Hypothetical ancestral <i>Vibrio</i> organisms are indicated by open circles. Although <i>V. cholerae</i> O1 possesses the partially overlapping <i>hchA/luxR</i>, they are replaced by <i>metY</i> in strains MS6 and 2740-80.</p