Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis

Abstract

Antibiotic-induced changes in the intestinal microbiota predispose mammalian hosts to infection with antibiotic-resistant pathogens. Clostridium difficile is a Gram-positive intestinal pathogen that causes colitis and diarrhea in patients following antibiotic treatment. Clindamycin predisposes patients to C. difficile colitis. Here, we have used Roche-454 16S rRNA gene pyrosequencing to longitudinally characterize the intestinal microbiota of mice following clindamycin treatment in the presence or absence of C. difficile infection. We show that a single dose of clindamycin markedly reduces the diversity of the intestinal microbiota for at least 28 days, with an enduring loss of ca. 90% of normal microbial taxa from the cecum. Loss of microbial complexity results in dramatic sequential expansion and contraction of a subset of bacterial taxa that are minor contributors to the microbial consortium prior to antibiotic treatment. Inoculation of clindamycin-treated mice with C. difficile (VPI 10463) spores results in rapid development of diarrhea and colitis, with a 4- to 5-day period of profound weight loss and an associated 40 to 50% mortality rate. Recovering mice resolve diarrhea and regain weight but remain highly infected with toxin-producing vegetative C. difficile bacteria and, in comparison to the acute stage of infection, have persistent, albeit ameliorated cecal and colonic inflammation. The microbiota of "recovered" mice remains highly restricted, and mice remain susceptible to C. difficile infection at least 10 days following clindamycin, suggesting that resolution of diarrhea and weight gain may result from the activation of mucosal immune defenses.

Fig 1

Clindamycin predisposes mice to C. difficile infection and associated disease. Mice (n = 10 per group) received 10⁵ C. difficile spores (strain VPI 10463) without clindamycin (untreated), a single dose of clindamycin (200 μg) intraperitoneally, or a single dose of clindamycin, followed by 10³ CFU of C. difficile spores 24 h later. Survival (A) and weight loss (B) were monitored over the subsequent 4-week period. (C) The cytotoxicity of cecal content derived from clindamycin-treated, C. difficile-infected mice was quantified using a cell-based assay. Total (black) and spore (gray) CFU in the cecum (D) and ileum (E) were quantified by culture. Each bar represents the average for three mice. Panels A and B and panels D and E, respectively, share keys.

Fig 2

C. difficile infection following clindamycin treatment results in persistent intestinal inflammation. Mice were treated with clindamycin (200 μg) intraperitoneally on day −1 and infected with 10³ C. difficile spores on day 0. (A and B) Uninfected mice and mice infected 2, 4, 7, 10, and 30 days postinfection were sacrificed, and their ceca were isolated and fixed, and tissue sections were stained with hematoxylin and eosin. Cecum sections were scored for edema (thin arrows), inflammatory cell infiltration (white arrows), and epithelial cell loss (arrowheads). Scale bar, 200 μm (inset, 50 μm). *, P < 0.05 (Mann-Whitney test).

Fig 3

Single-dose clindamycin treatment does not reduce 16S rRNA gene copy numbers in the ileum and cecum. The quantity of 16S rRNA gene copies was determined by qPCR of ileal (A, B, and C) and cecal (D, E, and F) samples from mice that received clindamycin (200 μg) intraperitoneally on day −1 and/or inoculation of 10³ C. difficile spores on day 0. Samples were obtained on days (postinfection) indicated. Each bar represents the average of three mice.

Fig 4

Clindamycin induces profound, long-lasting decreases in intestinal microbial diversity. Rarefaction analyses of microbial communities in the ilea (A, B, and C) and ceca (E, F, and G) of mice that received C. difficile spores (A and E), clindamycin (B and F), or clindamycin and spores (C and G). Clindamycin was administered on day −1, and C. difficile spores were administered on day 0. The Shannon biodiversity index values of microbial communities in the ileum (D) and cecum (H) were also calculated at the time points indicated. Untreated, C. difficile-inoculated mice were only measured on days 1, 7, 14, and 21. Panels A, B, C, E, F, and G and panels D and H, respectively, share keys.

Fig 5

Clindamycin treatment depletes a broad range of microbial populations. Heat map showing the relative abundance of phylotypes (OTUs defined with 97% similarity) in the ileum (A, B, and C) and cecum (D, E, and F) over time following inoculation with 10³ C. difficile spores (A and D), clindamycin administration (B and E), or clindamycin administration plus C. difficile inoculation (C and F).

Fig 6

Characteristics of the microbiota of the ileum and cecum of untreated mice. Phylogenetic classification of the 16S rRNA gene of the most predominant bacterial taxa in the ilea and ceca of untreated mice. (A) Each bar represents the microbiota of an individual mouse. (B) The relative abundances of major bacterial OTUs of the ilea and ceca of untreated mice administered 10⁵ CFU of C. difficile spores on day 0 over the subsequent 21-day period are shown. (C) The relative abundance of major bacterial OTUs of untreated mice are given with reference to a phylogenetic tree. The thickness of branches corresponds to the relative abundance of the respective phylotypes. The number following phylotype name distinguishes different OTUs within the same genus. (D) PCA of ileal and cecal microbiota samples. Each point represents an individual mouse.

Fig 7

Clindamycin treatment alters the composition of the microbiota. Mice from three separately housed colonies were administered clindamycin (200 μg) on day −1, and samples of cecal and ileal content were obtained for analysis on the days indicated. Each bar represents the microbiota of an individual mouse.

Fig 8

Clindamycin and C. difficile infection alter intestinal microbiota composition. Mice were administered clindamycin (200 μg) intraperitoneally on day −1 and/or inoculated with C. difficile spores on day 0, and samples of cecal and ileal content were obtained from a representative mouse from three separately housed colonies on the days indicated. Each bar represents the pooled microbiota of three separately housed mice on the indicated day. The most predominant bacterial phylotypes are color coded as indicated.

Fig 9

PCA of the effect of clindamycin and C. difficile infection on the intestinal microbiota composition. (A) Principal components generated to explain maximal variance of samples from all treatment arms. The thickness of phylogenetic tree branches indicates the relative weight of that phylotype on the principal component. The color of branches indicates the sign of the phylotype (red, positive; green, negative). (B and C) Longitudinal principal coordinate analysis of cecal and ileal microbiota from mice treated with clindamycin (B) or clindamycin plus C. difficile spores (C). Untreated mice are represented as circles. Clindamycin was administered on day −1, and C. difficile spores were given on day 0. Each point represents the average of three separately housed mice. Number labels indicate the day (postinfection) on which the samples were harvested.

Fig 10

Single-dose clindamycin treatment confers long-term susceptibility to C. difficile infection. Mice received 10⁵ spores of C. difficile (without clindamycin) or 200 μg of clindamycin intraperitoneally 1, 4, or 10 days before inoculation with 10³ spores of C. difficile. One day following inoculation, C. difficile CFU in the fecal content were quantified. Figure 10 represents pooled results of two independent experiments. Each bar represents the average of six mice. ***, P < 0.001; **, P < 0.003.