Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains

Abstract

The toxin-producing bacterium C. difficile is the leading cause of antibiotic-associated colitis, with an estimated 500,000 cases C. difficile infection (CDI) each year in the US with a cost approaching 3 billion dollars. Despite the significance of CDI, the pathogenesis of this infection is still being defined. The recent development of tractable murine models of CDI will help define the determinants of C. difficile pathogenesis in vivo. To determine if cefoperazone-treated mice could be utilized to reveal differential pathogenicity of C. difficile strains, 5-8 week old C57BL/6 mice were pretreated with a 10 d course of cefoperazone administered in the drinking water. Following a 2-d recovery period without antibiotics, the animals were orally challenged with C. difficile strains chosen to represent the potential range of virulence of this organism from rapidly fatal to nonpathogenic. Animals were monitored for loss of weight and clinical signs of colitis. At the time of harvest, C. difficile strains were isolated from cecal contents and the severity of colitis was determined by histopathologic examination of the cecum and colon. Cefoperazone treated mice challenged with C. difficile strains VPI 10463 and BI1 exhibited signs of severe colitis while infection with 630 and F200 was subclinical. This increased clinical severity was correlated with more severe histopathology with significantly more edema, inflammation and epithelial damage encountered in the colons of animals infected with VPI 10463 and BI1. Disease severity also correlated with levels of C. difficile cytotoxic activity in intestinal tissues and elevated blood neutrophil counts. Cefoperazone treated mice represent a useful model of C. difficile infection that will help us better understand the pathogenesis and virulence of this re-emerging pathogen.

Figure 1.C. difficile strains exhibit different clinical outcomes in cefoperazone treated mice. Body weight was measured daily starting from Day 0 or the day of infection. Animals were challenged with C. difficile strains (A) VPI 10463 (2 × 10⁵ CFU closed circles and 4 × 10⁴ CFU open circles) (B) BI1 (6 × 10⁴ CFU closed squares and 8 × 10³ CFU open squares) (C) 630 (2 × 10⁵ CFU closed triangles and 8 × 10⁴ CFU open triangles) and (D) F200 (4 × 10⁵ CFU closed diamonds and 5 × 10³ CFU open diamonds). Black lines represent mean percentage of the baseline weight for animals in each group.

Figure 2. Early murine infection with different C. difficile strains. Cefoperazone treated C57BL/6 WT mice challenged with C. difficile strains, VPI 10463 (n = 4), BI1 (n = 3), 630 (n = 5), F200 (n = 3) and a mock (n = 3) infected were all sacrificed early during infection, or 48 h post infection. (A) Average body weight was measured daily starting from Day 0 or the day of infection (B) Colonization levels in CFU per gram of cecal content at the time of necropsy (C) Vero cell cytotoxicity assay from cecal content of each mouse in log₁₀ reciprocal dilution toxin per gram of cecal content at the time of harvest (VPI 10463 vs. F200, p < 0.05; VPI 10463 vs. mock, p < 0.05 Kruskal-Wallis 1way ANOVA).

Figure 3. Histopathology and biomarkers of disease severity during early infection with different C. difficile strains. (A) Rank order analysis of histology slides from the murine colon showing disease severity in order from 1, most severe histopathological changes, to 18, least severe histopathological changes. (B) Total white blood cell count in thousand (K)/μl. The black shaded boxes indicate the number of neutrophils present out of the total number of white blood cells present (in white) in the blood of infected C. difficile mice (VPI vs. 630, p < 0.05 Kruskal-Wallis 1way ANOVA).

Figure 4. Histopathology in the colon of C. difficile-infected animals during early infection. (A) Colon of a mouse with clinically severe CDI infected with VPI 10463. There is severe submucosal edema accompanied by inflammatory cells within the mucosa and invading the submucosal lymphoid tissue. The epithelial surface is also irregular and eroded. (B) Colon of a mouse infected with BI1 that shows significant submucosal edema and scattered inflammatory cells. (C) Colon of a mouse infected with the 630 strain of C. difficile shows minimal to no histopathological changes. (D) Colon of a mouse colonized with F200 which shows some inflammation but otherwise no significant histopathological changes. HE. All images were the same magnification (x200). Scale bar = 100 µm.

Figure 5.C. difficile strains exhibit different clinical outcomes in cefoperazone treated mice when infected with spores. Average body weight was measured daily from Day 0 or the day of infection. Animals were challenged with C. difficile strains (A) VPI 10463 circles (2 × 10⁵ spores) (B) BI1 squares (2 × 10⁵ spores). All animals infected with VPI 10463 met their clinical endpoint by day 2 post infection and had to euthanized. Animals infected with BI1 never met the clinical endpoint and were euthanized when the experiment was terminated on day 7 post infection.