Flagella-related gene mutations in Vibrio cholerae during extended cultivation in nutrient-limited media impair cell motility and prolong culturability

Additional supplemental data for the article "Flagella-related gene mutations in Vibrio cholerae during extended cultivation in nutrient-limited media impair cell motility and prolong culturability"</p

A Cholera Outbreak of the Vibrio cholerae O1 El Tor Variant Carrying Classical CtxB in Northeastern Thailand in 2007

Cholera outbreaks occurred in Thailand in 2007. Isolates from the northeastern regions were analyzed. Interestingly, the outbreak strain was identified as biotype El Tor; serotype Ogawa with cholera toxin B subunit gene (ctxB) of the classical type and CTX prophage repressor gene of the El Tor type. The clone was genetically closely related to pulsotype H, which is predominantly found in India. It was probably introduced into Thailand recently

Distribution of major MLVA types of <i>V. cholerae</i> O1 isolates during the 2007–2010 cholera outbreaks in Thailand.

<p>The distribution percentage of predominant MLVA type (s) is displayed in relatively scaled pie charts. The size of each chart and the number in parentheses indicate the ratio of each major MLVA type (corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030863#pone-0030863-t003" target="_blank">Table 3</a>) to the other types. The color of each slice indicates the year of isolation: 2007 (orange), 2008 (gray), 2009 (light blue), and 2010 (green). The color of the circle band denotes serotype: Ogawa (black) and Inaba (blue).</p

Ribotyping and virulence-related gene analyses of arbitrarily selected <i>V. cholerae</i> O1 isolates from each PFGE pulsotype.

<p>*Not Determined.</p>†<p>+, positive; −, negative.</p>‡<p>Data from Raychoudhuri <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030863#pone.0030863-Raychoudhuri3" target="_blank">[40]</a>.</p

Genotypic and PFGE/MLVA Analyses of <em>Vibrio cholerae</em> O1: Geographical Spread and Temporal Changes during the 2007–2010 Cholera Outbreaks in Thailand

<div><h3>Background</h3><p><em>Vibrio cholerae</em> O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of <em>V. cholerae</em> O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize <em>V. cholerae</em> O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.</p> <h3>Methods/Findings</h3><p>A total of 343 isolates of <em>V. cholerae</em> O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (<em>ctxB</em>) and El Tor <em>rstR</em> genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect <em>Vibrio</em> seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.</p> <h3>Conclusions</h3><p>MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of <em>V. cholerae</em> O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.</p> </div

Dendrogram showing genetic similarity between 343 isolates of <i>V. cholerae</i> O1 derived from MLVA.

<p>Sequence data of repeats on the five loci for each isolate were counted and imported into BioNumerics software version 6.1. Clustering analysis was performed using the unweighted pair group with arithmetic averaging (UPGMA) with a categorical similarity coefficient. <i>V. cholerae</i> O1 isolate MLVA types of the El Tor variant were grouped into 2 major clusters, I and II.</p

Growth curves of GAS under standard culture conditions (THY, 37°C).

<p>Growth curves of (A) SSI-1 wild-type and the corresponding mutant strains, and (B) JRS4 wild-type and the corresponding mutant strains. Data were expressed as the mean and standard deviation from 3 independent experiments.</p