Fig. 6
Survival of female mice challenged with E. coli O111 or O157 strains after immunization with OMV delivering E. coli O111, O157, or mixed O-antigens. The immunized female mice underwent an intraperitoneal challenge with 5.6 × 107 CFU/100 µl of wild-type E. coli O111 strain (A) or 5.4 × 106 CFU/100 µl of wild-type E. coli O157 strain (B) at week 8 after the initial immunization, respectively. Following the challenge, mortality was recorded for 12 days. Data from two independent experiments were summarized (n = 8). Mouse survival differences were analyzed using the log-rank test, with the Kaplan-Meier survival curve used for monitoring; superscript letters a and b indicate P < 0.05 for comparisons with the PBS and OMV vector groups, respectively, while ns means no significant difference. (C. D) Female mice were provided unrestricted access to water added with streptomycin for three days preceding infection to clean the intestinal flora and were orally infected with 20 µl of a bacterial suspension comprising about 1 × 109 CFU of the E. coli O111 (C) or O157 (D), which is a non-lethal dose. Following the infection, water with added streptomycin was given back and fecal shedding was collected on days 3, 6, 9, and 12. Fecal pellets were weighed and homogenized in PBS (0.1 g/ml). The quantification of bacteria per 1 g of feces was performed by dropping serially diluted or undiluted fecal homogenate on the MacConkey agar plate containing 25 µg/ml streptomycin. Following the challenge, mortality was recorded for 12 days. Data from two independent experiments were summarized (n = 6). The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM. Statistical differences were assessed by a two-way ANOVA test followed by Tukey’s multiple comparison test; superscript letters a indicate P < 0.05 for comparison with the PBS