Fig 1.
A) Two subcolonies of Smad3-/- mice were maintained concurrently. SFB+ mice were generated by gastric gavage of Smad3-/- breeders with pure SFB inoculum or sham, and subsequent breeding to generate experimental mice. At weaning (21 days old), mice from the SFB- and SFB+ colonies were inoculated via gastric gavage with two doses of Helicobacter hepaticus and Helicobacter bilis at approximately 1×108 CFUs per inoculum. Fecal samples were collected prior to inoculation (Pre), between the two inoculations (mid), then Day 1 (D1), D4, D7, 2W (two weeks), 3W, 5W, 8W, and 14W post-inoculation (PI). Mice were euthanized at 14 weeks, cecal contents collected, and colon collected for histopathological examination and lesion scoring. B) Representative Haemotoxylin and Eosin (H&E) stained colons of Smad3-/- mice sacrificed 14W post-inoculation showing i) severe inflammation of tissue and ii) adenocarcinoma invading into the lamina muscularis, exhibiting branched structure and cell hyperplasia. C) The lesion scoring system for colons in this study, taking into account epithelial changes, inflammation, and tumor scores to generate an overall score. Drawings are property of Annie E. Wolfe, an author of this paper.
Fig 2.
Sex and SFB interactions influence incidence and severity of CRC.
A) CRC incidence of SFB- and SFB+ male and female Helicobacter spp.-inoculated Smad3-/- mice. Fisher’s Exact Tests within SFB- and SFB+ category (to narrow down an overall chi square test p = 0.0226) revealed significant differences in incidence of CRC between males and females in SFB+ group (p = 0.01) but not within the SFB- group (p = 0.16). B) Overall Disease Scores C) Tumor Scores, and D) Inflammation Scores of male and female mice within the SFB- and SFB+ categories, analyzed via Two-Way ANOVA (post hoc Holm-Sidak). Significance and corresponding p values indicated by red lines between different groups. N values for each group indicated in panel A.
Fig 3.
SFB stabilizes the GM and prevents large shifts in GM structure following Helicobacter spp. inoculation.
A) Relative abundance of OTUs within CRC+ and CRC- mice in SFB- and SFB+ groups, shown as group means at pre-inoculation (P), after the first inoculation (M), and at day 1 (D1), D4, D7, and two weeks (2W) PI. Yellow-orange represents Helicobacter spp. (including Helicobacter ambiguous taxa and Helicobacter uncultured bacterium) and light blue represents Family Enterobacteriaceae (more specifically denoted as Escherichia-Shigella Escherichia sp.). These are indicated by arrows. B) Principal Component Analysis (PCA) of Pre and D4 samples of each mouse in SFB+ (green) and SFB- (purple) groups. Length of line denotes overall change in composition from Pre to D4 in each mouse. C) Mean (±SD) shifts in GM community shown in B, using Bray-Curtis Dissimilarity Index. Mann-Whitney Rank Sum test p = 0.021.
Table 1.
Spearman’s ranked correlation of bacterial families at Pre, D4, and D7 in SFB+ and SFB- mice.
Table 2.
Spearman’s ranked correlation of bacterial OTUs at Pre, D4, and D7 in SFB+ and SFB- mice.
Fig 4.
Relative abundance of Helicobacter spp. at D4 PI predictive of CRC development in SFB- but not SFB+ mice.
Family Enterobacteriaceae and SFB interact in CRC development. Relative abundance of (A) Helicobacter spp. and (B) family Enterobacteriaceae (Escherichia-Shigella Escherichia sp.) from Pre-inoculation to 3W PI in SFB- and SFB+ Helicobacter spp.-inoculated mice. Two-way RM ANOVA performed for SFB- and SFB+ mice separately: like letters denote significant (p < 0.05) differences. P values are as follows: A) SFB- group a p = 0.042; b p = 0.005; c p = 0.019; *p = 0.016; **p = 0.024 and SFB+ group a p = 0.012; b p = 0.046; c p = 0.031. B) SFB- group ns and SFB+ group *p = 0.048. SFB- group CRC+ (n = 19) and CRC- (n = 38) and SFB+ group CRC+ (n = 7) and CRC- (n = 22).
Fig 5.
Isolation of family Enterobacteriaceae.
A) MALDI-ToF identification of D4 PI fecal samples. B) Blood Agar and MacConkey Agar plates comparing colony morphology, hemolytic properties, and lactose fermentation of lab strain ATCC 21972 E. coli and unknown D4 isolated E.coli strain.