Fig 5. Assessment of the protective efficacy of SinH-3 against the bacteremia of multiple ExPEC sequence types (STs).

(A) Sequence alignment of sinH in different sequence types of ExPEC. The alignment was exported from Geneious and annotated using BioRender. (B) The vaccination scheme was used in this experiment. BALB/cJ, 6 weeks old, unvaccinated female mice (N = 4) and female mice were subcutaneously immunized with SinH-3 (N = 8), were both injected with an intraperitoneal (IP) injection of 5 × 10⁷ CFU of multiple ExPEC sequence type strains (ST73-mixture, ST95-mixture). Mice were monitored twice a day for 5 days, and moribund animals were euthanized/necropsied to determine bacterial levels in the kidneys, spleen, and liver. Organs were harvested and plated to determine bacteria levels. The schematic diagram was made in BioRender. The survival rate curve of (C) ST73-mixture or (D) ST95-mixture was determined using the Gehan-Breslow-Wilcoxon comparison. (E) Box-and-whisker plots of the bacterial levels (CFU/ml) of the counts from all organs following necropsy. Error bars indicate the median with 95% confidence interval (CI). Significant was determined by the Kruskal-Wallis analysis of variance (ANOVA) with Dunn’s multiple comparisons correction. Symbols represent data of individual mice. One star (*) P < 0.05, two stars (**) P < 0.01, three stars (***) P < 0.001, four stars (****) P < 0.0001. The Box-and-whisker plots and Kaplan Meier survival curves were exported from Graphpad Prism 9 and annotated using BioRender.