Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant <i>Enterococcus faecium</i> and Carbapenem-Resistant <i>Klebsiella pneumoniae</i>

<div><p>Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant <i>Enterococcus faecium</i> (VRE) does not reduce <i>in vivo</i> growth of carbapenem-resistant <i>Klebsiella pneumoniae</i>. Reciprocally, <i>K</i>. <i>pneumoniae</i> does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and <i>K</i>. <i>pneumoniae</i> from the gut. Fluorescence <i>in situ</i> hybridization demonstrates that VRE and <i>K</i>. <i>pneumoniae</i> localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and <i>K</i>. <i>pneumoniae</i> occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific <i>in vivo</i> metabolic needs.</p></div

<i>K</i>. <i>pneumoniae</i> and VRE reside within the same intestinal regions but occupy distinct metabolic niches.

<p>(A-D) Spatial localization of <i>K</i>. <i>pneumoniae</i> and VRE in the colon. Colon sections from ampicillin-treated mice colonized for 21 days with <i>K</i>. <i>pneumoniae</i> alone (A), VRE alone (B) and <i>K</i>. <i>pneumoniae</i> together with VRE (C, D) were hybridized with probes specific for <i>K</i>. <i>pneumoniae</i> and Enterococcus. (D) VRE and <i>K</i>. <i>pneumoniae</i> islands (dashed circles and square) in the colonic lumen of co-colonized mice. (A-D) All sections were counterstained with Hoechst dye to visualize nuclei. Scale bars, 10 μm. Insets, 63X oil objective plus 4X digital zoom. Images are representative of at least 5 mice per group. (E) Minimum distance between neighboring bacteria determined by confocal microscopy. ns = non-significant; ****<i>P</i><0.0001, by the Mann-Whitney test.</p

<i>K</i>. <i>pneumoniae</i> and VRE achieve similar densities in the large intestine of co-colonized mice.

<p>Ampicillin-treated mice were inoculated with <i>K</i>. <i>pneumoniae</i> by oral gavage or left uninfected. Three days later, half of the <i>K</i>. <i>pneumoniae</i>-infected mice and the uninfected group were challenged with VRE. Microbiota composition of mice colonized with VRE alone (V), <i>K</i>. <i>pneumoniae</i> alone (K) or both (VK) was determined by sequencing of the V4-V5 region of the 16S rRNA genes. (A) Fecal microbiota composition at different time points post VRE challenge. (B) Ileal and cecal microbiota composition at day 21 of colonization. (A,B) Each stacked bar represents the average of five individually-housed mice per time point.</p

Pre-colonization with VRE does not prevent colonization by <i>K</i>. <i>pneumoniae</i>.

<p>(A) Experimental design. Mice were treated with ampicillin for 29 days. On day 5 of ampicillin treatment, mice were inoculated with 5x10⁴ colony-forming units (CFU) of VRE by oral gavage or left uninfected. Three days later, half of the VRE-infected mice and the uninfected group were challenged with 5x10⁴ CFU of <i>K</i>. <i>pneumoniae</i> (Kpn). (B, C) CFU of <i>K</i>. <i>pneumoniae</i> (B) and VRE (C) were quantified in fecal pellets collected at different time points post <i>K</i>. <i>pneumoniae</i> inoculation. (D, E) Mice were sacrificed 21 days post <i>K</i>. <i>pneumoniae</i> challenge. <i>K</i>. <i>pneumoniae</i> (D) and VRE (E) burden was quantified in the luminal contents from the duodenum, ileum and cecum. L.O.D., limit of detection. Data were pooled from two independent experiments (n = 10 per group). (B-E) Data were analyzed by the Mann-Whitney test.</p

<i>K</i>. <i>pneumoniae</i> and VRE occupy a fraction of the total available space in the colon.

<p>(A-E) Visualization of bacterial localization by FISH. Entire colon cross-sections from untreated mice (A) and mice treated with ampicillin for 3 weeks (B) were stained with a universal probe that targets the 16S rRNA gene of all bacteria. Cross-sections from ampicillin-treated mice colonized with <i>K</i>. <i>pneumoniae</i> (C) or VRE (D) for 21 days were hybridized with probes specific for <i>K</i>. <i>pneumoniae</i> (Kpn) and Enterococcus, respectively. Sections were counterstained with Hoechst dye to visualize nuclei. Images are representative of 5 mice per group. Scale bar, 500 μm. (E) Number of bacteria per unit area of whole colon cross-sections. n = 3 per group. ND = non-detectable. Error bars (mean ± SEM). **<i>P</i><0.005, ***<i>P</i><0.0005 by the Mann-Whitney test.</p

Pre-colonization with <i>K</i>. <i>pneumoniae</i> does not prevent colonization by VRE.

<p>(A) Experimental design. Mice were treated with ampicillin for 29 days. On day 5 of ampicillin treatment, mice were inoculated with 5x10⁴ colony-forming units (CFU) of <i>K</i>. <i>pneumoniae</i> (Kpn) by oral gavage o left uninfected. Three days later, half of the <i>K</i>. <i>pneumoniae</i>-infected mice and the uninfected group were challenged with 5x10⁴ CFU of VRE. (B, C) CFU of VRE (B) and <i>K</i>. <i>pneumoniae</i> (C) were quantified in fecal pellets collected at different time points post VRE inoculation. (D, E) Mice were sacrificed 21 days post VRE challenge. VRE (D) and <i>K</i>. <i>pneumoniae</i> (E) burden was quantified in the luminal contents from the duodenum, ileum and cecum. L.O.D., limit of detection. Data were pooled from two independent experiments (n = 10 per group). (B-E) ****<i>P</i><0.0001, by the the Mann-Whitney test.</p