Intestinal translocation of enterococci requires a threshold level of enterococcal overgrowth in the lumen

Abstract

Enterococci are subdominant members of the human gastrointestinal microbiota. Enterococcus faecalis is generally harmless for healthy individuals, but it can cause a diverse range of infections in immunodeficient or elderly patients with severe underlying diseases. In this study, we analysed the levels of intestinal translocation of indigenous enterococci in C57BL/6, CF-1 and CX3CR1^-/- mice upon clindamycin antibiotic-induced dysbiosis. We found that C57BL/6 was the most permissive model for enterococcal translocation and that initiation of E. faecalis translocation coincided with a threshold of enterococcal colonisation in the gut lumen, which once reached, triggered E. faecalis dissemination to deeper organs. We showed that the extent to which E. faecalis clinical strain VE14821 competed with indigenous enterococci differed between the C57BL/6 and CX3CR1^-/- models. Finally, using a simplified gnotobiotic model, we observed E. faecalis crossing an intact intestinal tract using intestinal epithelial cells as one route to reach the lamina propria. Our study opens new perspectives for assessing the effect of various immunodeficiencies and for investigating mechanisms underlying enterococcal translocation.

Figure 1

Effect of clindamycin on the levels of indigenous enterococci in the intestinal lumen and tissues, and translocation to organs in C57BL/6 mice. Each dot represents one mouse (n ≥ 7) and indicates the number of colony-forming units (CFUs) per gram of content (g of content) or per organ. The dotted line represents the detection limit. Horizontal bars represent the median values from two independent experiments. Enterococcal counts in the gut lumen (A) were conducted on luminal content from the small intestine (cSi), the caecum (cCA) and the colon (cCO) of C57BL/6J and C57BL/6N conventional mice that were untreated (◯) or treated for three consecutive days with clindamycin (●). Enterococcal counts in the gut tissues (B) were conducted on the small intestine (SI), the caecum (CA) and the colon (CO) from clindamycin-treated C57BL/6J and C57BL/6N conventional mice. Enterococcal counts in peripheral organs (C) were conducted on the mesenteric lymph nodes (MLNs), the mesenteric adipose tissue (MAT), the spleen (SP), the kidneys (KDs), the liver (LV) and the heart (HR) from clindamycin-treated C57BL/6J and C57BL/6N conventional mice. Statistical analysis was performed using the Mann–Whitney test. The asterisk (*) and hash (#) symbols indicate statistically significant differences between counts (*P < 0.05, **P < 0.01, ***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001); ns, non-significant difference.

Figure 2

Translocation of indigenous enterococci in CX3CR1^−/− mice. Each dot (●) represents one CX3CR1^−/− mouse (n = 8) treated for three consecutive days with clindamycin and indicates the number of colony-forming units (CFUs) per gram of content (g of content) or per organ. The dotted line represents the detection limit. Horizontal bars represent the median values from two independent experiments. Enterococcal counts in the gut lumen (A) were conducted on luminal content from the small intestine (cSi), the caecum (cCA) and the colon (cCO). Enterococcal counts in the gut tissues (B) were conducted on the small intestine (SI), the caecum (CA) and the colon (CO). Enterococcal counts in the peripheral organs (C) were conducted on the mesenteric lymph nodes (MLNs), the mesenteric adipose tissue (MAT), the spleen (SP), the kidneys (KDs), the liver (LV) and the heart (HR).

Figure 3

Correlations between enterococcal levels in the gut lumen and translocation to the liver. Spearman correlation analysis was used to evaluate the association between enterococcal levels in luminal content (X axis) from the small intestine (cSi), the caecum (cCA) and the colon (cCO), and in the liver (Y axis). Total enterococcal counts from CF-1 (▲), C57BL/6 (●) – including both the C57BL/6J and C57BL/6N substrains – and CX3CR1^−/− (■) mice are plotted. r indicates the Spearman’s rank correlation coefficient and P the p-value.

Figure 4

Translocation of E. faecalis VE14821_cm in clindamycin-treated C57BL/6 and CX3CR1^−/− mice. Total enterococcal counts () and VE14821_cm counts () were evaluated in clindamycin-treated C57BL/6J (B6J), C57BL/6N (B6N) and CX3CR1^−/− (CX) mice 13 hours after oral inoculation. Each dot represents one mouse (n ≥ 7) and indicates the number of colony-forming units (CFUs) per gram of content (g of content) or per organ. The dotted line represents the detection limit. Horizontal bars represent the median value from two independent experiments. Enterococcal counts in the gut lumen (A) were conducted on luminal content from the small intestine (cSi), the caecum (cCA) and the colon (cCO). Enterococcal counts in the gut tissues (B) were conducted on the small intestine (SI), the caecum (CA) and the colon (CO). Enterococcal counts in the peripheral organs (C) were conducted on the mesenteric lymph nodes (MLNs), the mesenteric adipose tissue (MAT), the spleen (SP), the kidneys (KDs), the liver (LV) and the heart (HR). Statistical analysis was performed using the Mann–Whitney test. Asterisks (*) indicate statistically significant differences between counts (*P < 0.05, **P < 0.01, ***P < 0.001); ns, non-significant difference.

Figure 5

Intestinal crossing of E. faecalis VE14821_GFP in gnotobiotic C57BL/6J mice. (A) Immunofluorescence microscopy showing claudin-1 expression (red) and GFP-expressing E. faecalis (green) on a colonic section. Cell nuclei were stained with Hoechst. Bacteria, either from the epithelial villi (left panel) or from the colonic patch (right panel), are shown at higher magnification in boxed areas (1 to 4). The white triangles indicate additional VE14821_GFP. (B) Transmission electron microscopy of the colon. Dotted windows indicate a region interest. Close-up images of these regions are shown in panels on the right of the original picture, with the correspondence between sets of images being indicated by a letter and a number. L: lumen; BB: brush border; Ef: E. faecalis VE14821_GFP; IEC: intestinal epithelial cell; TJ: tight junction; n: nucleus; ECM: extracellular matrix; LP: lamina propria; M: mitochondria; BM: basal membrane. Scale bars are provided for each image.