Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice

Abstract

The microbiota confers colonization resistance, which blocks Salmonella gut colonization¹. As diet affects microbiota composition, we studied whether food composition shifts enhance susceptibility to infection. Shifting mice to diets with reduced fibre or elevated fat content for 24 h boosted Salmonella Typhimurium or Escherichia coli gut colonization and plasmid transfer. Here, we studied the effect of dietary fat. Colonization resistance was restored within 48 h of return to maintenance diet. Salmonella gut colonization was also boosted by two oral doses of oleic acid or bile salts. These pathogen blooms required Salmonella's AcrAB/TolC-dependent bile resistance. Our data indicate that fat-elicited bile promoted Salmonella gut colonization. Both E. coli and Salmonella show much higher bile resistance than the microbiota. Correspondingly, competitive E. coli can be protective in the fat-challenged gut. Diet shifts and fat-elicited bile promote S. Typhimurium gut infections in mice lacking E. coli in their microbiota. This mouse model may be useful for studying pathogen-microbiota-host interactions, the protective effect of E. coli, to analyse the spread of resistance plasmids and assess the impact of food components on the infection process.

Figure 1. A shift to WD and oleic acid gavage promote S.Tm blooms, enteropathy and plasmid transfer.

(a) Experimental protocol. (b-d) Pathogen loads and cecum tissue histopathology of CON^E mice infected with 5x10⁷ CFU S.Tm by gavage (N=6,10,11). Non-infected controls 96h after diet-shift or oleic acid gavage (grey symbols; N=6; no CFU detected). See 'Histological procedures' in Methods for details of pathological score. H&E images represent median sample from corresponding treatment. (e) In vivo transconjugation was determined by stool plating (N=7,8; Kruskal-Wallis test, multiple comparison correction; see figure S6). (f) Protocol of back-and-forth diet-shifts in CON^E mice. (g) Fecal pathogen loads were analyzed 24h p.i. with 5x10⁷ CFU S.Tm (by gavage; N=5,6,8,9 mice). Bars: median; Two-way ANOVA on log-normalized data with Dunnett's multiple comparison test (for b,c,g), two-tailed Mann-Whitney-U (for d). Dotted lines = detection limit. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. MD: maintenance diet, WD: Western-type diet.

Figure 2. Primary bile salts can explain S.Tm blooms.

(a) Quantitative cholate mass spectrometry analysis in CON^E mice (N=5) 9h after the indicated intervention. (b) Cholate titration experiment. CON^E mice (N=5,6,7) were gavaged as indicated (100μl) 1h before and 4h after S.Tm infection (5x10⁷ CFU). (c) Effect of the indicated interventions on gut luminal S.Tm densities (N=8,9). Control: CON^E mice pre-treated with streptomycin (20 mg by gavage; 24h before infection; grey). (d) Cholate sensitivity of gut-luminal microbiota from ileum (N=2), cecum (N=3) and colon (N=2) microbiota (isolated from CON^E mice) and of wt S.Tm (black circles; N=3) or indicated S.Tm mutants (N=3). Analysis was performed by SYTOXgreen-exclusion and flow cytometry (exemplary gating for S. Tm^tolC is shown). The mean value of all experiments is shown (whiskers = range). (e) Cholate-sensitivity of individual microbiota strains as analyzed in MGAM medium (2% H2, 12% CO₂, 86% N₂; table S3; N=3, analysis vs. growth without inhibitor). Controls: Wt E. coli ED1a, indicated S.Tm strains. Bars: median; Two-way ANOVA on log-normalized data with Dunnett's multiple comparison test. Dotted lines = detection limit. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 3. Modeling and experimental data validate that bile-resistance promotes S.Tm growth in the fat-exposed gut.

(a,b) Mathematical model testing if bile-mediated growth inhibition can explain S.Tm vs. S.Tm^arcAB blooms after 24h of growth in the fat-, oleic acid- or cholate exposed gut (as detailed in Supplementary Information). (c,d) Competitive infections. CON^E mice (N=7,8,9) were treated as above and infected with S.Tm^wt and S.Tm^acrAB (1:1; 5x10⁷ CFU total, by gavage). S.Tm loads were quantified by plating. (d) The competitive index of S.Tm^wt vs. S.Tm^acrAB as analysed by WITS-qPCR. (e) Total S.Tm^wt or S.Tm^acrAB loads in CON^E mice (C57BL/6, N=5) infected as in figure 1a and analysed by plating 24h p.i. Bars: median; Two-way ANOVA on log-normalized data with Dunnett's multiple comparison test. Dotted lines = detection limit. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 4. E. coli limits the S.Tm infection after WD-shift or oleic acid gavage.

(a-c) Competitive infection with the E. coli mix. CON^E mice (N=7,8,9,10,17) were treated as in figure 1a and infected with wt S.Tm and the E. coli 8178, CFT073 and Z1324 mix, as indicated (5x10⁷ CFU by gavage). (a,b) E. coli and S.Tm loads were determined by plating. (c) Cecum histopathology 96h p.i.. H&E images represent median sample from corresponding treatment. (d) Competitive infection with E. coli 8178. CON^E mice (N=9,15) were treated as above and gavaged with E. coli 8178 and S.Tm, as indicated (5x10⁷ cfu). Triangles: control mice re-plotted from (b). #: none detected; Bars: median; ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, Two-way ANOVA on log-normalized data with Dunnett's multiple comparison test (for b,d), two-tailed Mann-Whitney U-test (for c). Dotted lines: detection limit.