Competition assay in vivo

The streptomycin-treated adult mouse model was used to assess ROS resistance in vivo for cbs deletion and complement strain, compared with C6706 wild-type, as previously described [7] with the following modifications. Five-week-old CD-1 mice were supplied with drinking water with or without 10 g/L of the antioxidant N-acetyl cysteine (NAC) for one week. Afterwards, 5 g/L streptomycin and 0.05 g/L aspartame were added to the drinking water for the rest of the experiment. One day after streptomycin treatment, mice were administrated with 100 μl of 10% (wt/vol) NaHCO₃ by gavage, then intragastrically administered with 100 μl of a 1:1 mixture of wild-type and mutant V. cholerae (approximately 10⁹ CFU for each strain per mouse). Fecal pellets were collected from each mouse at 1, 3, and 5 days post gavage, homogenized by bead beating, and resuspended in PBS buffer, serially diluted, and plated on LB agar containing streptomycin and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) for quantification of bacterial loads. Competition index was calculated as the ratio of mutant to wild-type colonies normalized with the input ratio. Competition assay was also conducted for ΔcbsΔdps and Δcbs in streptomycin-treated adult mouse model to examine the correlation between cbs-dependent ROS resistance and iron.

Five-day-old CD-1 mice were transferred to the 30°C incubator 2 hrs before inoculation. Mice were intragastrically administrated with 50 μl of 1:1 mixture of wild-type and mutant V. cholerae (approximately 10⁶ CFU for each strain per mouse), then put back into the 30°C incubators. Infant mice were sacrificed 18 hrs post gavage. Samples of the small intestine of each mouse were removed and homogenized in 1.5 ml of PBS buffer, serially diluted, and then plated on LB agar containing streptomycin and X-gal for quantification of bacterial loads. Competition index was calculated as the ratio of mutant to wild-type colonies normalized with the input ratio.