Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition

Abstract

Neonates are highly susceptible to infection with enteric pathogens, but the underlying mechanisms are not resolved. We show that neonatal chick colonization with Salmonella enterica serovar Enteritidis requires a virulence-factor-dependent increase in epithelial oxygenation, which drives pathogen expansion by aerobic respiration. Co-infection experiments with an Escherichia coli strain carrying an oxygen-sensitive reporter suggest that S. Enteritidis competes with commensal Enterobacteriaceae for oxygen. A combination of Enterobacteriaceae and spore-forming bacteria, but not colonization with either community alone, confers colonization resistance against S. Enteritidis in neonatal chicks, phenocopying germ-free mice associated with adult chicken microbiota. Combining spore-forming bacteria with a probiotic E. coli isolate protects germ-free mice from pathogen colonization, but the protection is lost when the ability to respire oxygen under micro-aerophilic conditions is genetically ablated in E. coli. These results suggest that commensal Enterobacteriaceae contribute to colonization resistance by competing with S. Enteritidis for oxygen, a resource critical for pathogen expansion.

Keywords: Enterobacteriaceae; Salmonella; colonization resistance; microbiota; neonate; oxygen.

Figure 1:. S. Enteritidis virulence factors promote colonization of the neonatal gut.

1-day old chicks were infected with the 1×10⁹ CFU of the indicated S. Enteritidis strains. (A) S. Enteritidis colonization levels were determined in cecal contents (n = 5 for each time point) at the indicated time points. Statistically significant differences to chicks infected with the S. Enteritidis wild type (wt) are indicated. (B-G) Relative changes in transcript levels of inflammatory markers, including NOS2 (B), CXCL8 (C), IL1B (D), IL6 (E), IFNG (F), and IL22 (G), were determined by quantitative real-time PCR using RNA isolated from cecal tonsils 3 days after infection and were expressed as fold-change over samples from mock-infected animals. (A-G) data represent geometric means ± standard error. (H) Representative images of haematoxylin and eosin-stained cecal sections of chicks 3 days after infection. Scale bars = 400 μm. (I) Histopathology score for the ceca collected 3 days after infection was determined by scoring blinded sections using criteria listed in table S1. Each bar represents data from one individual animal. (B-I) The number of animals in each group (n) is indicated in panel I. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not statistically significantly different.

Figure 2:. Virulence factors promote pathogen expansion through aerobic respiration.

(A) 1-day old chicks (n = 4–5) were infected with a 1:1 mixture of the indicated bacterial strains. Cecal content were collected 3 days after infection to determine the competitive index. See also Figure S2. (B-G) 1-day old chicks were mock-treated or infected with the indicated bacterial strains. (B) Composition of the gut microbiota at the phyla-level (relative abundance) based on 16S rRNA gene sequencing of DNA isolated from cecal contents at the indicated time points after infection. See also Figure S1. (C-E) Total colony-forming units (CFU) in cecal contents recovered on MacConkey agar (Enterobacteriaceae, black bars) or CFU of S. Enteritidis (red bars) were determined in groups of chicks that were mock-treated (C), infected with S. Enteritidis (wt) (D) or infected with a S. Enteritidis invA spiB mutant (invA spiB) (E). Bars represent geometric means ± standard error. (F-G) Normalized abundances of members of the family Lachnospiraceae (G) and Ruminococcaceae (H) in cecal contents were determined by microbiota profiling. Bars represent mean ± standard deviation. (H-I) Representative images of pimonidazole (red, panel H) or MitoTracker (Dark orange, panel I) staining detected in cecal sections counter stained with DAPI nuclear stain (blue). (B-G) The number of animals in each group (n) is indicated in Figure S1. Scale bars = 40 μm. *, P < 0.05; ****, P < 0.0001.

Figure 3:. Bioluminescence imaging of luminal E. coli requires a live host.

(A) Cecal contents of an adult bird were cultured on MacConkey and the identity of bacterial colonies determined using an EnteroPluri assay. (B) Rooted maximum likelihood tree, visualized as a cladogram, of select E. coli isolates indicating their relatedness to the avian commensal isolate YL178. Color-coding denotes members of the same phylogroup (phylogroups A, B1, B2, D1, D2 or E1). Information on the biovar for each isolate is given in parentheses: ETEC, enterotoxigenic E. coli; EAEC, enteroaggregative E. coli; EHEC, Enterohemorrhagic E. coli; UPEC, uropathogenic E. coli; AIEC, adherent-invasive E. coli; ExPEC, etraintestinal pathogenic E. coli. (C-L) Chicks were infected one day after hatch (day 1) with E. coli strain YL178 transformed with a plasmid carrying a CP25::luxCDBAE transcriptional fusion (pBR2TTS:CP25::luxCDBAE) and bioluminescence imaging was determined one day later (day 2) in anesthetized birds (C), after euthanasia (D and E) or in the gastrointestinal tract open longitudinally (F). A representative image is displayed for each sample (n=8). (E) Time course of bioluminescence imaging (n = 6) determined before (red dot) and after (black dots) euthanasia. (C, D and F) The number of animals in each group (n) is indicated in Fig. 4A. Data are shown as geometric means ± standard error (n=6).

Figure 4:. S. Enteritidis competes with E. coli for luminal oxygen.

Birds were inoculated one day after hatch (day 1) with E. coli strain YL178(pBR2TTS:CP25::luxCDBAE) carrying the luxCDBAE reporter genes (E. coli (lux)) or with a 1:1 mixture of E. coli (lux) and either wild-type S. Enteritidis (SE wt) or a S. Enteritidis cydA mutant (SE cydA). Alternatively, birds were inoculated at the day of hatch (day 0) with E. coli (lux) and infected the next day (day 1) with wild-type S. Enteritidis. (A) Bioluminescence imaging was performed one day after the last inoculation (day 2). Each dot represents data from one animal. Lines indicate geometric means ± standard deviation. (B) Numbers of E. coli (lux) recovered from cecal contents. (C) Numbers of S. Enteritidis recovered from cecal contents. (B-C) Data are shown as geometric means ± standard deviation. The number of animals in each group (n) is indicated in panel A. ***, P < 0.001; ****, P < 0.0001.

Figure 5:. Enterobacteriaceae and spore-forming bacteria are required to confer niche protection.

(A and E) Germ-free Swiss Webster mice were mock-treated or inoculated with microbiota from a neonate (one-day-old) or adult (three-months-old) bird (A) or received the indicated components of adult chicken microbiota (E). 5 days later mice were challenged with S. Enteritidis (10⁸ CFU/mouse). Colonization levels of S. Enteritidis were determined in the feces two days after infection. (B-D and F) Microbial representation at the family-level was determined by 16S rRNA gene sequencing of DNA isolated from adult chicken microbiota (B), neonate chick microbiota (C), bacteria recovered from MacConkey agar seeded with adult chicken microbiota (Enterobacteriaceae component) (D) or feces of ex-germ-free Swiss Webster mice inoculated with chloroform-treated adult chicken microbiota (spore component) (F). (G) Day-of-hatch chicks were inoculated with the indicated components of adult chick microbiota and challenged the next day with S. Enteritidis (10⁹ CFU/chick). Colonization levels of S. Enteritidis were determined in the cecal contents (n = 6) one days after challenge. (H and I) Germ-free Swiss Webster mice were inoculated with chloroform-treated adult chicken microbiota (spores) and the indicated E. coli strains. 5 days later, E. coli colonization levels were determined (I) and mice were challenged with S. Enteritidis (10² CFU/mouse). Colonization levels of S. Enteritidis were determined in the feces (n = 6) two days after infection (H). (A, E, G, H and I) Bars represent geometric means ± standard error. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significantly different.

Figure 6:. Model for cooperation between Enterobacteriaceae and Clostridia to confer colonization resistance against S. Enteritidis.

Clostridia in the large intestine break down complex carbohydrates from the diet (fiber) into fermentation products, such as butyrate. Butyrate helps to polarize the epithelial metabolism towards oxidative phosphorylation in the mitochondria, resulting in high oxygen (O₂) consumption. In turn, high oxygen consumption renders the epithelial surface hypoxic, thereby limiting the amount of oxygen diffusing from the mucosal surface into the intestinal lumen. The small amount of oxygen that emanates from the hypoxic epithelial surface is consumed by commensal Enterobacteriaceae through aerobic respiration. Epithelial hypoxia and aerobic respiration by Enterobacteriaceae cooperate to maintain anaerobiosis in the lumen. Anaerobiosis confers colonization resistance because it blocks the access to oxygen for the facultative anaerobic S. Enteritidis (Salmonella), thereby curbing pathogen growth. The resulting decline in pathogen numbers can lead to an extinction, an outcome that becomes more likely when the host is exposed to a low infectious dose.