Figure 1.
Genetic relatedness of nine environmental isolates of V. cholerae from Gujarat.
PFGE (1%) analysis of SfiI-digested genomic DNA (A) Yeast Marker: CHEF DNA size standards (Bio-Rad) from S. cerevisiae. Positions of the marker bands have been indicated on the left. Agarose gel (1%) analysis of RAPD using 1281 primer (B) and 1283 primer (C). In B and C, 1 kb DNA ladder (Fermentas) with fragment sizes is indicated on the left side. For all the three panels, the identity of the sample used has been indicated at the top of each lane.
Figure 2.
Cytotoxic effect of environmental isolates on HT29 cells.
HT29 cells not treated with supernatant from any of the isolates (A); Cell rounding in HT29 cells treated with the supernatant from P1(B); Clumping of HT29 cells treated with the supernatant from Z2 (C); Bar diagram showing cytotoxic effect of culture supernatants from environmental isolates and the controls V. cholerae N16961 and E. coli DH5α in terms of percentage of dead cell (D). The experiment was independently performed three times. The error bars indicate standard deviation.
Figure 3.
Analysis of protease activity of the environmental isolates.
SDS PAGE (10%) analysis of culture supernatants (A). Lane 1: Protein markers (Pageruler, Fermentas) with molecular mass in kDa indicated on the left; Lanes 2 to 8, culture supernatants of Z2, Z3, P2, W1, W3, V. cholerae N16961 and E. coli DH5α, respectively. Protease activity of culture supernatants on 1.5% Skim milk agar plate (B). Sample identity has been indicated near each well.
Figure 4.
Rabbit ileal loop assay to assess entrotoxigenic activity.
Pictorial view of rabbit ileal loop of different clinical and environmental strains (A). Analysis of fluid accumulation of different V. cholerae strains (B). Rabbit ileal loops were inoculated with 108 CFU of each strain N16961 (O1 El tor, clinical), IDH02365(O1 El tor, clinical), Z2 (non-O1/non-O139, Environmental) and W3(non-O1/non-O139, Environmental) in PBS and incubated for 18 h. Results are expressed as fluid accumulation (FA) (in milliliters) per loop length (in centimeters). Shown are means ± standard deviations; n = 3.