Fig. 5
Pre-antibiotic xylose level is associated with susceptibility to K. pneumoniae outgrowth. a Schematic showing passage of K. pneumoniae under antibiotic selection. Mice were pre-treated with vancomycin for 7 days, orally gavaged with 105 CFU of K. pneumoniae and maintained on vancomycin for 7 more days to allow K. pneumoniae growth. Stool was diluted 1000-fold to achieve ~105 K. pneumoniae CFU and gavaged into another vancomycin pre-treated mice. Such transfer of stool microbiota was performed for 8 iterations into singly housed mice to avoid any cross-contamination. K. pneumoniae colonies were cultured from the stool of the 8th iteration mouse. Colonies from independent lineages were whole-genome sequenced. Plate swipes containing aggregate pools of K. pneumoniae cultured directly from stool were also sequenced for each lineage. b Summary of XylR mutations derived from 5 separate lineages together with their allelic frequencies obtained from plate swipes. c In vitro anaerobic growth assays of evolved strains xylR-L and transposon strains xylR-Tn in minimal media containing xylose or glucose after 24 h. N=4. d Levels of xylose in stool before and after vancomycin or ampicillin treatments. N=10. e, f In vivo competition between e the wild type and xylR-Tn1 transposon strain, or f the wild type and xylR-L4 evolved strain in mice undergoing vancomycin or ampicillin treatment. Mice were orally gavaged with 105 CFU of each K. pneumoniae strain at day 0 (triangle). Strain levels at days 3 and 7 were quantified by qPCR using specific primers to each xylR allele. N=5. g Levels of xylose in stool at day 0, 7, or 14. Stool was obtained from the mice shown in Fig. 4a. N=13–14. For c, one-way ANOVA was performed followed by post hoc Dunnett’s test comparing each evolved or Tn strain to the wild type on each sugar. For d and g, a two-tailed t test was used. For c, d, and g, error bars show SEM. For e and f, a Mann-Whitney U test was used to compare evolved or mutant strains to the wild type at each time-point. *p value < 0.05, ***p value < 0.001