Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits

Abstract

Infection with antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a dangerous and costly complication of broad-spectrum antibiotic therapy. How antibiotic-mediated elimination of commensal bacteria promotes infection by antibiotic-resistant bacteria is a fertile area for speculation with few defined mechanisms. Here we demonstrate that antibiotic treatment of mice notably downregulates intestinal expression of RegIIIgamma (also known as Reg3g), a secreted C-type lectin that kills Gram-positive bacteria, including VRE. Downregulation of RegIIIgamma markedly decreases in vivo killing of VRE in the intestine of antibiotic-treated mice. Stimulation of intestinal Toll-like receptor 4 by oral administration of lipopolysaccharide re-induces RegIIIgamma, thereby boosting innate immune resistance of antibiotic-treated mice against VRE. Compromised mucosal innate immune defence, as induced by broad-spectrum antibiotic therapy, can be corrected by selectively stimulating mucosal epithelial Toll-like receptors, providing a potential therapeutic approach to reduce colonization and infection by antibiotic-resistant microbes.

Figure 1. MyD88-mediated signalling in non-haematopoietic cells is required for RegIIIγ-mediated clearance of VRE

a, b, One-thousand VRE were injected into ileal loops of MyD88^+/+, MyD88^−/− (a) and MyD88 bone-marrow-chimaeric (b) mice. Two hours after injection the isolated section of the intestine was harvested and colony-forming units were determined; n=5–6 each group. Bacterial numbers are expressed as the percentage of recovered VRE. For a, *P=0.02, unpaired t-test; for b, **P < 0.01, one-way ANOVA with Bonferroni correction. WT, wild type. c, Antiserum against RegIIIγ or pre-immune serum was injected into ileal loops of wild-type mice before injection of 1,000 VRE. After 2 h, bacterial growth in the luminal fluid of the isolated section of the small intestine was determined, Bacterial numbers are expressed as the percentage of recovered VRE; n=5 each group. *P=0.049, unpaired t-test. Error bars denote s.e.m.

Figure 2. RegIIIγ expression is downregulated in antibiotic-treated mice, correlating with decreased clearance of VRE

a, Persistence and density of VRE intestinal colonization in mice. Mice were treated with MNV in drinking water starting 2 days before oral infection with 10⁹ VRE by gavage. Bacterial counts within the distal small intestine were determined 1, 3 and 5 days after VRE infection (n=5 each group and time point; **P=0.008, Mann–Whitney test). b, Messenger RNA was extracted from the terminal ileum of wild-type mice (first bar) and mice receiving antibiotics (MNV) for 1, 3 and 7 days. RegIIIγ expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh; n=4–10 mice per group; *P=0.02, **P=0.0011, ***P=0.0001, each compared to untreated, unpaired t-test). c, Protein extracts from the distal small intestine of untreated and MNV-treated mice for 1, 3 and 7 days were analysed by western blotting with RegIIIγ-specific antiserum. Tubulin was used as a loading control. d, Messenger RNA was extracted from the terminal ileum of untreated or enrofloxacin and clindamycin (EC)-treated mice, and RegIIIγ expression was determined (n=2 for wild-type and n=4 for EC-treated mice; **P=0.0017, unpaired t-test). e, Protein extracts from the distal small intestine of wild-type mice and mice receiving antibiotics (EC) for 7 days were analysed by western blotting with RegIIIγ-specific antiserum. f, Wild-type mice and mice administered antibiotics (MNV) in the drinking water for 7 days were infected with 10¹⁰ VRE by gastric gavage, and the number of bacteria in the blood was determined 24 h after infection (n = 10 each group; *P = 0.013, Mann–Whitney test). g, Ileal loops of wild-type mice and mice receiving antibiotics for 7 days (MNV) were injected with 1,000 VRE, and bacterial survival was measured 2 h later. Bacterial numbers are expressed as percentage of recovered VRE (n = 8 each group; *P = 0.04, unpaired t-test). h, RegIIIγ or control protein (β2-microglobulin) was inoculated into ileal loops, and VRE survival was assessed (n = 5 each group; ***P = 0.0004, unpaired t-test). Error bars denote s.e.m.

Figure 3. Administration of LPS to antibiotic-treated mice restores RegIIIγ levels and enhances luminal VRE killing

a, Mice were treated with MNV alone, MNV plus 2–4 µg µl⁻¹ LPS, MNV plus 0.25 µg µl⁻¹ LTA, or were left untreated, and RegIIIγ mRNA expression was determined and is expressed relative to untreated mice (n=5–12 each group; ***P < 0.001, one-way ANOVA with Bonferroni correction). b, Quantitative western blot analysis with RegIIIγ-specific antiserum was performed on protein extracts from the distal small intestine of untreated mice as well as mice receiving MNV,MNV plus LPS, or MNV plus LTA (n = 5–12; * P < 0.05, *** P < 0.001, one-way ANOVA with Bonferroni correction). c, VRE survival was determined in ileal loops of wild type, MyD88^−/− or TLR4^mut/mut mice treated with MNV, MNV plus LPS, MNV plus LTA, or left untreated. Bacterial numbers are expressed as the percentage of recovered VRE (n = 6–11 for wild type, n = 7–8 for MyD88^−/−, n = 6–8 for TLR4^mut/mut (C3HeJ); **P < 0.01, ***P < 0.001). d, Mice were treated for 7 days with MNV,MNV plus LPS, or left untreated, and then were orally infected with 10¹⁰ VRE. Twenty-four hours later, bacterial counts in the distal small intestine were determined (n = 14–15; **P < 0.01, one-way ANOVA with Bonferroni correction). e, Mice were treated as in d and, 24 h later, bacterial counts within MLNs were determined (n = 4–5;*P < 0.05, one-way ANOVA with Bonferroni correction). Error bars denote s.e.m.

Figure 4. Delayed LPS treatment of mice receiving antibiotics restores RegIIIγ levels and enhances luminal VRE killing

a, Mice were treated with MNV for 4 days before the addition of LPS (1–4 µg µl⁻¹) to the drinking water. RegIIIγ mRNA induction was determined after 7 days LPS treatment and is expressed relative to untreated mice (n = 9–15 in each group; **P < 0.01, one-way ANOVA with Bonferroni correction). b, RegIIIγ protein levels in the distal small intestine of mice treated as in a were determined (n = 5–8; **P < 0.01, one-way ANOVA with Bonferroni correction). c, Immunohistochemical detection of RegIIIγ in paraffin-embedded distal small intestine sections in untreated mice, mice receiving MNV and mice receiving MNV plus LPS (magnification 400-fold, inset 1,000-fold). Representative samples from one of three mice is shown. d, VRE killing in ileal loops of mice treated as in a was determined and expressed as percentage of recovered VRE (n = 5–6 each group; *P < 0.05, ***P < 0.001, one-way ANOVA with Bonferroni correction). Error bars denote s.e.m.