Intestinal microbiota shifts towards elevated commensal Escherichia coli loads abrogate colonization resistance against Campylobacter jejuni in mice

Abstract

Background: The zoonotic pathogen Campylobacter jejuni is a leading cause of bacterial foodborne enterocolitis in humans worldwide. The understanding of immunopathology underlying human campylobacteriosis is hampered by the fact that mice display strong colonization resistance against the pathogen due to their host specific gut microbiota composition.

Methodology/principal findings: Since the microbiota composition changes significantly during intestinal inflammation we dissected factors contributing to colonization resistance against C. jejuni in murine ileitis, colitis and in infant mice. In contrast to healthy animals C. jejuni could stably colonize mice suffering from intestinal inflammation. Strikingly, in mice with Toxoplasma gondii-induced acute ileitis, C. jejuni disseminated to mesenteric lymphnodes, spleen, liver, kidney, and blood. In infant mice C. jejuni infection induced enterocolitis. Mice suffering from intestinal inflammation and C. jejuni susceptible infant mice displayed characteristical microbiota shifts dominated by increased numbers of commensal Escherichia coli. To further dissect the pivotal role of those distinct microbiota shifts in abrogating colonization resistance, we investigated C. jejuni infection in healthy adult mice in which the microbiota was artificially modified by feeding live commensal E. coli. Strikingly, in animals harboring supra-physiological intestinal E. coli loads, colonization resistance was significantly diminished and C. jejuni infection induced enterocolitis mimicking key features of human campylobacteriosis.

Conclusion/significance: Murine colonization resistance against C. jejuni is abrogated by changes in the microbiota composition towards elevated E. coli loads during intestinal inflammation as well as in infant mice. Intestinal inflammation and microbiota shifts thus represent potential risk factors for C. jejuni infection. Corresponding interplays between C. jejuni and microbiota might occur in human campylobacteriosis. Murine models introduced here mimick key features of human campylobacteriosis and allow for further analysis of immunological and molecular mechanisms of C. jejuni-host interactions.

Figure 1. C. jejuni colonization along the gastrointestinal tract of mice with T. gondii-induced acute ileitis.

Acute ileitis was induced in conventionally colonized 3-months-old C57BL/6 mice by oral infection with 100 cysts of Toxoplasma gondii ME49 strain. Four days following ileitis induction, mice were orally infected with C. jejuni strain ATCC 43431 as described in Methods. (A) At day 7 p.i. when T. gondii infected mice display severe ileitis (ILE) C. jejuni densities in distinct compartments of the gastrointestinal tract were determined by quantification of live C. jejuni in luminal samples taken from stomach, duodenum, ileum, and colon by cultural analysis (CFU, colony forming units; solid circles) and compared to control mice without ileitis (Con). (B) E. coli loads in luminal ileum samples of mice with T. gondii-induced acute ileitis (day 7 p.i.; open circles) were compared to naïve controls (open diamonds). (C) Relative translocation frequencies (%) of live C. jejuni 43431 ATCC (Cj, black bars) and E. coli (Ec, open bars) were determined in ex vivo biopsies of mesenteric lymphnodes (MLN), spleen, liver, and blood derived from mice with T. gondii-induced acute ileitis at day 7 p.i. by culture in enrichment broths. (D) A complex molecular analysis of the ileal microbiota composition was assessed in mice with T. gondii-induced acute ileitis at day 7 p.i. (ILE; solid circles) and compared to naïve control animals (N; open circles). Main gut bacterial groups were quantified by Real-Time-PCR analyses amplifying bacterial 16S rRNA variable regions and 16S rRNA gene numbers/ng DNA derived from luminal ileal samples: Enterobacteriaceae (EB), Enterococci (EC), Lactic acid bacteria (LB), Bifidobacteria (BIF), Bacteroides/Prevotella spp. (BP), Clostridium leptum group (CLEP), Clostridium coccoides group (CLOCC), Mouse intestinal bacteroidetes (MIB), and total eubacterial load (TL). Numbers of animals harboring C. jejuni or E. coli out of the total number of analyzed animals are given in parentheses. Medians (black bars), standard deviations and significance levels (P-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from at least three independent experiments.

Figure 2. C. jejuni colonization along the gastrointestinal tract of IL-10^−/− mice with chronic colitis.

Conventionally colonized 6-months-old IL-10^{− /−} mice with chronic colitis were orally infected with C. jejuni strain ATCC 43431 as described in Methods. (A) At day 14 p.i. C. jejuni densities in distinct compartments of the gastrointestinal tract were determined by quantification of live C. jejuni in luminal samples (CFU, colony forming units; solid circles) taken from stomach, duodenum, ileum, and colon by cultural analysis and compared to wildtype controls (WT, open circles). (B) Furthermore, E. coli loads in luminal colonic samples of C. jejuni infected IL-10^{− /−} mice (IL-10, open circles) were compared to healthy wildtype control animals (WT, open diamonds). (C) A complex molecular analysis of the colonic microbiota composition was assessed in IL-10^{− /−} mice with chronic colitis (IL-10; solid circles) and compared to wildtype control animals (WT; open circles). Main gut bacterial groups were quantified by Real-Time-PCR analyses amplifying bacterial 16S rRNA variable regions and 16S rRNA gene numbers/ng DNA derived from luminal colonic samples: Enterobacteriaceae (EB), Enterococci (EC), Lactic acid bacteria (LB), Bifidobacteria (BIF), Bacteroides/Prevotella spp. (BP), Clostridium leptum group (CLEP), Clostridium coccoides group (CLOCC), Mouse intestinal bacteroidetes (MIB), and total eubacterial load (TL). Numbers of animals harboring C. jejuni or E. coli out of the total number of analyzed animals are given in parentheses. Medians (black bars) and significance levels (P-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from at least three independent experiments.

Figure 3. Kinetic analyses of C. jejuni colonization in fecal samples of infant mice.

Conventionally colonized 3-weeks-old infant (3W; open circles) and 3-months-old adult (3M; solid circles) mice were orally infected with C. jejuni strain ATCC 43431 (A) or B2 (B) as described in Methods. Kinetic analyses of pathogen densities were performed by quantification of live C. jejuni in fecal samples by cultural analysis (CFU, colony forming units) at day (d) 6 p.i. ** indicates significant differences (p<0.01) of C. jejuni 43431 loads in infected infant mice at d1 versus d3, d5, and d6 p.i. (C) E. coli loads in colonic samples taken from naïve 3-weeks-old infant mice (3W, open diamonds) were compared to conventionally colonized 3-months-old adult mice (3M, open circles). (D) Kinetic analyses of disease activity of conventionally colonized 3-weeks-old infant mice following infection with C. jejuni strain ATCC 43431 (43431, open circles, n = 10) or B2 (solid circles, n = 12) were performed applying a standardized clinical score (see Methods). Numbers of animals harboring C. jejuni or E. coli out of the total number of analyzed animals are given in parentheses. Days past infection, medians (black bars) and significance levels (P-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from at least three independent experiments.

Figure 4. Inflammatory and immune cell responses following C. jejuni infection of infant mice.

Conventionally colonized 3-weeks-old infant mice were orally infected with C. jejuni strain ATCC 43431 (crossed circles) or B2 (solid circles) as described in Methods. The average numbers of apoptotic cells (positive for caspase-3, panel A), neutrophilic granulocytes (neutrophils, positive for MPO-7, panel B), macrophages (positive for F4/80, panel C), T-lymphocytes (positive for CD3, panel D), regulatory T-cells (Tregs, positive for FOXP3, panel E) and B-lymphocytes (positive for B220, panel F) from at least six high power fields (HPF, ×400 magnification) per animal were determined microscopically in immunohistochemically stained colon sections at day 6 p.i. Uninfected mice (Naïve, open circles) served as negative controls. Numbers of analyzed animals are given in parentheses. Means (black bars) and levels of significance (P-values) determined by the Student’s t-test are indicated. Data shown are representative for three independent experiments.

Figure 5. Pro-inflammatory cytokine responses in the colon of C. jejuni infected infant mice.

Conventionally colonized 3-weeks-old infant mice were orally infected with C. jejuni strain ATCC 43431 (43431, crossed circles) or B2 (solid circles) as described in Methods. Concentrations (pg per mg colon) of (A) nitric oxide, (B) TNF-α, (C) IL-6, (D) MCP-1, and (E) IFN-γ were determined in supernatants of ex vivo colon cultures at day 6 p.i. by Griess reaction or CBA-ELISA, respectively. Uninfected mice (Naïve, open circles) served as negative controls. Numbers of analyzed animals are given in parentheses. Means (black bars) and levels of significance (P-values) as compared to the respective group (determined by Student’s t-test) are indicated. Data shown are representative for three independent experiments.

Figure 6. E. coli loads of adult mice following application of E. coli via drinking water.

In conventionally colonized 3-months-old adult mice (n = 6) intestinal E. coli loads were raised by adding live E. coli into the drinking water (+ E. coli, until day 8) as described in Methods. At day 8, the E. coli suspension was withdrawn and replaced by regular tap water. Kinetic analyses of E. coli densities were performed by quantification of live bacteria in fecal samples by cultural analysis (CFU, colony forming units) until day 13 p.i. Medians (black bars) and levels of significance (P-values) as compared to the indicated groups (determined by Mann-Whitney-U test) are indicated. Data shown are representative for three independent experiments.

Figure 7. Time course of C. jejuni colonization in adult mice with increased E. coli loads.

In conventionally colonized 3-months-old adult mice intestinal E. coli loads were raised up to 10⁸ bacteria per gram feces by adding live E. coli into the drinking water (+ E. coli) as described in Methods. Mice were orally infected with C. jejuni strain ATCC 43431 (A) or B2 (B). Kinetic analyses of pathogen densities were performed by quantification of live C. jejuni in fecal samples derived from mice with elevated (Ec; solid circles) or normal (N; open circles) E. coli loads by cultural analysis (CFU, colony forming units) until day 12 p.i. (C) C. jejuni B2 densities in distinct compartments of the gastrointestinal tract were determined in luminal samples taken from stomach, duodenum, ileum, and colon of mice with elevated (Ec; solid circles) or normal (N; open circles) E. coli loads at day 12 p.i. Numbers of animals harboring C. jejuni out of the total number of analyzed animals are given in parentheses. Days post infection, medians (black bars) and levels of significance (P-values) as compared to the respective group (determined by Mann-Whitney-U test) are indicated. Data shown are pooled from at least three independent experiments.

Figure 8. Immune responses following C. jejuni infection of adult mice with increased E. coli loads.

In conventionally colonized 3-months-old adult mice intestinal E. coli loads were raised up to 10⁸ bacteria per gram feces by adding live E. coli into the drinking water (+ E. coli) as described in Methods. Then, mice were orally infected with C. jejuni strain ATCC 43431 (crossed circles) or B2 (solid circles). The average numbers of apoptotic cells (positive for caspase-3, panel A), neutrophilic granulocytes (Neutrophils, positive for MPO-7, panel B), macrophages (positive for F4/80, panel C), T-lymphocytes (positive for CD3, panel D), regulatory T-cells (Tregs, positive for FOXP3, panel E) and B-lymphocytes (positive for B220, panel F) from at least six high power fields (HPF, 400×magnification) per animal were determined microscopically in immunohistochemically stained colon sections at day 12 p.i. Uninfected mice (Naïve, open circles) served as negative controls. Numbers of analyzed animals are given in parentheses. Means (black bars) and levels of significance (P-values) as compared to uninfected (Naïve) control mice (determined by the Student’s t-test) are indicated. Data shown are pooled from at least three independent experiments.

Figure 9. Colonic pro-inflammatory cytokine responses of C. jejuni infected adult mice with increased E. coli loads.

In conventionally colonized 3-months-old adult mice intestinal E. coli loads were raised up to 10⁸ bacteria per gram feces by adding live E. coli into the drinking water (+ E. coli) as described in Methods. Then, mice were orally infected with C. jejuni strain ATCC 43431 (crossed circles) or B2 (solid circles). Concentrations (pg per mg colon) of (A) nitric oxide, (B) IL-6, (C) MCP-1, (D) IFN-γ, and (E) TNF-α were determined in supernatants of ex vivo colon cultures at day 12 p.i. by Griess reaction or CBA-ELISA, respectively. Uninfected mice (Naïve, open circles) served as negative controls. Numbers of analyzed animals are given in parentheses. Means (black bars) and levels of significance (P-values) as compared to the uninfected (Naïve) group determined by Student’s t-test are indicated. Data shown were pooled from at least three independent experiments.