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

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

YvqEC and CovRS have a strain-specific role in SSI-1 and JRS4.

<p>(A) Hyaluronic acid (HA) production (femtogram; fg/cfu) in SSI-1 and JRS4 wild-type, and their corresponding mutant strains were quantified by Stains-All assay. Data were expressed as the mean and standard deviation from 3 independent experiments. (B) Internalization of SSI-1 and JRS4 wild-type, and their corresponding mutant strains. HeLa cells were infected by SSI-1 and JRS4 wild-type, and their corresponding mutant strains at a multiplicity of infection (MOI) of 100. All data were expressed as the mean and standard deviation from 3 independent experiments in triplicate wells. (C) Biofilm formation of SSI-1 and JRS4 wild-type, and their corresponding mutant strains in C medium under static conditions at 37°C for 24 h. Images show biofilms attaching to the bottom of polystyrene plate after crystal violet staining. Data were expressed as the mean and standard deviation from 3 independent experiments in quadruplicate wells. (D) Lancefield assay of GAS survival in human blood. Multiplication factor is calculated by dividing the number of colony forming units (CFUs) after 3 h of incubation by CFUs in the original inoculum. Horizontal dotted line indicates starting inoculum number. Each strain was tested in 3 individually collected blood samples. Data were expressed as the mean and standard deviation from 3 independent experiments. Asterisks indicate statistically significant differences at <i>P</i> < 0.05 (*), <i>P</i> < 0.01 (**), and <i>P</i> < 0.001 (***) as determined by <i>t</i>-test.</p

The minimal inhibitory concentration (MIC) of SSI-1 and JRS4 wild-type and the corresponding mutant strains.

<p>The minimal inhibitory concentration (MIC) of SSI-1 and JRS4 wild-type and the corresponding mutant strains.</p

Survival of GAS after exposure to stress conditions.

<p>SSI-1 and JRS4 wild-type strains, and their corresponding mutant strains from overnight cultures were inoculated into fresh THY containing various stress-generating agents. An aliquot from each bacterial culture was used for the determination of the number of cfu/ml by colony counting on THY agar plates. The results were normalized to the OD₆₀₀ at time zero, and percent survival at the indicated time was calculated. All results were shown as the mean with standard deviation of 3 independent experiments performed in triplicate. Asterisks indicate statistically significant differences compared to the results for the wild-type strain. *<i>P</i> < 0.05. Sharps indicate statistically significant differences between Δ<i>yvqEC</i> or Δ<i>covRS</i> strain, and Δ<i>yvqEC</i>Δ<i>covRS</i> strain: <b>#</b><i>P</i> < 0.05, as determined by <i>t</i>-test.</p

The summary of synergistic effects.

<p>The interaction of YvqEC and CovRS TCSs to produce a combined effect is greater than the sum of their individual effects. Red colour (+) indicates synergistic effect of Δ<i>yvqEC</i>Δ<i>covRS</i>.</p