Fig. 5: Influence of bacterial evolution on the outcomes of internationally spreading high-risk multidrug-resistant non-HMV-Kp strains.

We used the non-HMV-Kp strain BIDMC1 as a representative for the internationally spreading high-risk clone ST258. a Antimicrobial susceptibility of BIDMC1. S and R indicate susceptible and resistant, respectively. b Gene mutation frequency of BIDMC1 and the mutS nonsense mutable mutant (BIDMC1 MutS_Tyr37Stop). A rifampicin assay was performed to determine gene mutation frequency. The floating bars represent the maximum and minimum values, and the lines represent the geometric means (n = 3 biologically independent experiments). A two-sided Student’s t test was used for the statistical analysis. c Serial passaging experiments with BIDMC1 and the mutS mutant in the presence of human serum. BIDMC1 (wild-type, WT) and BIDMC1 MutS_Tyr37Stop were used (the accumulated mutations are listed in Supplemental Data Dataset 6). The geometric means and geometric standard deviations for biologically independent triplicate experiments are given (d–g). Bacterial growth of the BIDMC1-derived mutants during the serial passaging experiment in (c). We examined three clones of each of the WT and MutS_Tyr37Stop strains. Bacterial growth was evaluated as turbidity (OD600) after 16 h of cultivation in MHBII in (d). Bacterial growth of the mutants derived after 20 days of the serial passaging experiment in the presence of human serum was also evaluated by counting the viable bacterial number as colony formation units (CFU) at 0, 1, 3, 6, and 16 h in (e–g). The means and standard deviations for biologically independent triplicate experiments are given. A two-sided one-way ANOVA test was used for statistical analysis with multiple comparisons vs WT Day 0 (* indicates p < 0.05). The significant dh, Number of accumulated gene mutations of BIDMC_1 and BIDMC_1 MutS_Tyr37Stop in (a). i Survival rates of immunosuppressed mice intrabronchially infected with BIDMC_1, MutS_Tyr37Stop, and serum-resistant MutS_Tyr37Stop (each n = 6 biologically independent experiments). The mouse infection model was the same as that described in Fig. 4c. The log-rank test was used for statistical analysis.