Table 1.
Quantitative stool cultures from mice (n = 4–15 per group) that were inoculated with Smr S. aureus strain Newman on day zero.
Table 2.
Quantitative cultures of GI segments from germ-free mice inoculated with S. aureus and evaluated on days 7 or 14.
Fig 1.
S. aureus factors that impact colonization of the murine GI tract.
A) The Newman serotype 5 capsule-negative mutant showed a competitive fitness advantage relative to the wild type Newman capsule-positive strain. B) A sortase A mutant and C) a clumping factor A (ClfA) mutant showed a competitive defect compared to the parental strain Newman. The competitive index (CI) was defined as the log10 output ratio/ input ratio. A CI <0 indicates a mutant with a colonization advantage over the WT strain, and a CI >0 indicates a mutant with a colonization disadvantage compared to the WT. Horizontal bars represent the median competitive index for groups of 5–15 mice. P-values were determined by Wilcoxon signed-rank test. *P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 2.
Effects of WTA on S. aureus GI colonization and the influence of surface antigens on in vitro adherence of S. aureus to T84 cells.
A) Fecal cultures were performed 7 d after bacterial inoculation with 109 CFU S. aureus. Each dot indicates the CFU S. aureus/g stool for a single mouse, and the median of each group of animals is indicated by a horizontal line. The lower limit of detection by culture was ~2.5 log CFU/g stool. P-values were determined by Kruskal-Wallis test with Dunn’s multiple comparison test. B) At an MOI of 100, Newman ΔtagO and ΔclfA were less adherent in vitro to T84 intestinal epithelial cells than WT strain Newman, as measured by the number of adherent S. aureus per field of view (FOV). * P < 0.05; ** P < 0.01; *** P < 0.001. C) The relative adherence of strain Newman and its isogenic mutants lacking WTA (ΔtagO), ClfA, or capsular polysaccharide (Δcap5G) to T84 cells in vitro was viewed by confocal microscopy. Immunostaining was performed with mouse anti-ZO-1 (red) to visualize the T-84 apical tight junctions and S. aureus rabbit antiserum to identify the bacteria (green). Scale bar, 10 microns.
Fig 3.
The reduced GI fitness of the ΔtagO mutant correlates with its susceptibility to bile, defensins, and proteases.
A) Mice were inoculated with a 1:1 mixture of the WT and ΔtagO mutant. After 1 h segments of the gut were homogenized and cultured quantitatively. The average S. aureus bacterial burden in the stomach was 3.2 x 106 CFU/g, and the first three segments of the small intestine yielded S. aureus concentrations of 2.6 x 105, 5.3 x 105 and 3.4 x 107 CFU/g, respectively. Horizontal bars represent the median competitive index for groups of 9–10 mice. P values were determined by Wilcoxon signed-rank test. B) S. aureus suspensions were exposed in vitro to bile salts (0.075% sodium deoxycholate), and quantitative cultures were performed to evaluate bacterial survival compared with samples lacking deoxycholate. Data are representative of 3 separate determinations. C) S. aureus were exposed for 2 h to increasing concentrations of α-defensin 5. The data are means ± SEM of 3 experiments. D) S. aureus at 1 x 106 CFU/ml were incubated at 37°C in the presence of increasing concentrations of proteinase K, and bacterial viability was measured after 24 h. The data are means ± SEM of 3 independent experiments. P-values were determined by the Student t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4.
Culture supernatants of WT S. aureus were bactericidal for Newman ΔtagO.
A) Newman supernatants (untreated), boiled, or supplemented with a protease inhibitor cocktail (PIC) were inoculated with Newman or Newman ΔtagO and incubated at 37°C. Bactericidal activity was measured by quantitative cultures at the indicated time points. B) Newman culture supernatants were supplemented with 0, 2, or 4 mg peptidoglycan and inoculated with either Newman or Newman ΔtagO. CFU/ml determinations were performed to evaluate bacterial viability. The data represent means ± SEM of 3 independent experiments. C) Zymogram of Newman and ΔtagO culture supernatants. The samples were concentrated and adjusted to equivalent protein concentrations before electrophoresis. D) Newman mutants were inoculated into filter-sterilized Newman culture supernatants, and bactericidal activity was measured after 3 h. ΔtagO C’ carried pRBtagO, which restored WTA production to the tagO mutant. E) Newman or Newman ΔtagO was added to culture supernatants of S. aureus Newman, ΔtagO, ST80, or Sanger 252, and bactericidal activity was measured at 3 h. The data represent means ± SEM of 3–4 experiments.
Fig 5.
The relative susceptibilities of S. aureus strains and their peptidoglycan preparations to autolysis.
A) Triton X-100 induced autolysis of S. aureus Newman, ΔtagO, Δatl, and ΔtagOΔatl mutants. The results shown are representative of 3 independent determinations. B) An S. aureus Newman autolysin extract (25 μg protein/ml) was prepared and incubated with peptidoglycan isolated from Newman and Newman ΔtagO. The data represent means ± SEM of 3 independent experiments. Lysis was expressed as a percentage of the OD580 nm at time zero.
Fig 6.
Both Newman ΔtagO and Newman Δatl ΔtagO fail to persistently colonize the mouse GI tract.
Mice were inoculated on day 0 with Newman, Newman ΔtagO, or Newman Δatl ΔtagO, and stool samples were cultured quantitatively 1, 2, 5, and 7 days after inoculation. Each symbol represents a stool culture from an individual mouse. P values were determined by Wilcoxon signed-rank test. **P < 0.01.
Table 3.
Sm-resistant S. aureus strains used in this study.