Fig. 5: Fitness costs of phage-resistant mutants.
a Biofilm formation of SCNJ1 and representative strains of phage-resistant mutants CM8, CR5, ZM10, and CM22 in a 96-well polystyrene plate at 12 h, 24 h, and 48 h, measured by OD595nm after crystal-violet staining. Data are presented as the mean ± SD (n = 3) from at least three independent experiments. Statistical analyses were performed using two-way ANOVA, accompanied by Dunnett’s multiple comparisons post hoc test [Interaction: F (8, 146) = 12.94, p < 0.0001; Row Factor: F (2, 146) = 102.8, p < 0.0001; Column Factor: F (4, 146) = 46.62, p < 0.0001]. b Time-dependent bactericidal effect of 75% normal human serum for 3 h at 37 °C. Data are presented as the mean ± SD (n = 3) from at least three independent experiments. Statistical analyses were performed using two-way ANOVA and Dunnett’s multiple comparisons post hoc test [Interaction: F (12, 40) = 9.362, p < 0.0001; Row Factor: F (3, 40) = 80.88, p < 0.0001; Column Factor: F (4, 40) = 34.24, p < 0.0001]. c In vivo phagocytosis assays. BALB/c mice (n = 6 for each strain) were infected intranasally with a dose of 1 × 108 CFU/mouse of SCNJ1 or phage-resistant mutants CM8, CR5, ZM10, or CM22. Bronchoalveolar lavage was performed at 90 min after inoculation before the number of bacteria engulfed by alveolar macrophages was counted. The changes of phage-resistant mutants in phagocytosis were normalized to the parental SCNJ1, which was set to the baseline value of 1. Each symbol represents 1 animal and error bars represent the standard error of the mean. Statistical analyses were performed using one-way ANOVA and Dunnett’s multiple comparisons test [F (4, 21) = 38.28, p < 0.0001].
