Table 1.
Frequency of switching of smooth phenotype of V. cholerae strains to rugose phenotype.
Table 2.
Conversion of smooth V. cholerae strains to rugose phenotype by years and sources of isolation.
Table 3.
Effect of temperature on the conversion of smooth V. cholerae strains to the rugose phenotype.
Table 4.
Bacterial strains and plasmids used in this study.
Figure 1.
Effect of temperatures on the colony phenotype of rugose variant of V. cholerae strains grown on L-agar.
Figure 2.
Colony morphology and associated biofilms (measured quantitatively) produced by V. cholerae wild-type and mutant strains.
(A) Colony morphology at 24 h and 48 h: each V. cholerae strain was subcultured on L-agar for isolated colonies, and the culture plate was incubated overnight at 37°C for 48 h. The images of bacterial colony were taken at 24 and 48 h of incubations; (B) Quantitative measurement of biofilm produced by each V. cholerae strain in nutrient-rich L-broth. All the values are expressed as means ± standard deviation (SD) from at least triplicate experiments.
Figure 3.
Topography and architecture of V. cholerae biofilms.
Each strain was grown in a 4-well cell culture plate containing 500 µl L-broth. A glass cover slip was dipped into each culture well and incubated overnight statically at room temperature. The glass cover slips were stained with SYTO 9 and the images were obtained using a laser scanning confocal microscopy with an excitation and emission wavelengths of 484- and 500 nm, respectively, as described previously [18]. (A) Images of x-y sections (top portions of panels) and x-z projections of the same biofilms (bottom portions of panels) were analyzed with DAIME software; magnification, ×200. (B) Average biofilm heights (µm) for each strain measured across five random x-z sections. (C) Total biomass of biofilm (µm3µm−2) for each strain calculated by x-y and x-z projections.
Figure 4.
Susceptibility of V. cholerae strains to diverse environmental stresses: (A) survival of the strains in the presence of 20 mM H2O2; (B) 2.5 M NaCl; and (C) 3 mg free chlorine/L.
The strains were grown (ca. 108 cfu/ml) in L-broth, washed and reconstituted with phosphate buffered saline (PBS). Stress assays were conducted in PBS supplemented with stress ingredients. The cultures were examined at different time intervals for the presence of bacteria as determined by standard plate count. Error bars indicate means ± standard deviation (SD) from triplicate experiments.
Figure 5.
Phylogenies of rugose and non-rugose producing O1 V. cholerae strains isolated in Haiti.
(A) Maximum likelihood phylogenetic tree of rugose (green) and non-rugose (red) producing clinical and environmental strains. Phylogeny was constructed with MEGA5 using the Kimura 2-parameter nucleotide substitution model with 1,000 bootstrap replicates. Clades with bootstrap support greater than 70 are indicated with an asterisk. Clinical strains are prefixed with AA or HC and environmental strains are prefixed with Env., (B) Neighbor-joining phylogeny of Haitian strains displaying the global relationship to the N16961 and O396 reference strains.