Comparative analysis of the in vitro and in vivo growth of IVp-I/IVp-II-locked mutants.

<p>(A) Competitive growth assay of ON/ON <i>B</i>. <i>fragilis</i> mutant and its parental YCH46 stain <i>in vitro</i>. Equivalent cell mixture of wild and mutant <i>B</i>. <i>fragilis</i> cells was inoculated into GAM broth. Closed circles indicate the periodical change of total viable cell number in GAM broth. Columns indicate the relative abundance of the wild and mutant <i>B</i>. <i>fragilis</i> cell in GAM broth. (B) IVp-I/IVp-II ON/OFF prevalence during <i>in vivo</i> gut colonization IVp-I/IVp-II under <i>in vivo</i> gut colonization by <i>B</i>. <i>fragilis</i>. A cell suspension (7.0 x10⁷ CFU in total) of wild-type <i>B</i>. <i>fragilis</i> was inoculated into three 8-week-old male BALB/cA germ-free mice via gavage. At 3, 7, 10, and 14 days after inoculation, feces samples were collected. DNA was extracted from the initial inoculum and the collected feces samples, and the ON/OFF ratio of IVp-I/IVp-II in each sample was determined by qPCR. The data are expressed as means ± standard deviation. The bars labeled with different letters indicate significant differences at <i>p</i> < 0.05. (C) Competitive growth assay of IVp-I/Vp-II-locked <i>B</i>. <i>fragilis</i> mutant and its parental YCH46 stain. Equivalent cell mixture of wild and the OFF/ON mutant cells was inoculated into BALB/c germ-free mice. Colony PCR was performed on at least 96 colonies per sample with primer pair encompassing the deletion site in BF2766 to compare population levels of mutants (closed triangles) with wild type (closed circles) cells in the mouse intestine. In this experiment, mice were kept in a vinyl isolator to maintain their gnotobiotic conditions.