Infection of hamsters with the UK Clostridium difficile ribotype 027 outbreak strain R20291

Abstract

Clostridium difficile is the main cause of antibiotic-associated disease, a disease of high socio-economical importance that has recently been compounded by the global spread of the 027 (BI/NAP1/027) ribotype. C. difficile cases attributed to ribotype 027 strains have high recurrence rates (up to 36 %) and increased disease severity. The hamster model of infection is widely accepted as an appropriate model for studying aspects of C. difficile host–pathogen interactions. Using this model we characterized the infection kinetics of the UK 2006 outbreak strain, R20291. Hamsters were orally given a dose of clindamycin, followed 5 days later with 10 000 C. difficile spores. All 100 % of the hamsters succumbed to infection with a mean time to the clinical end point of 46.7 h. Colonization of the caecum and colon were observed 12 h post-infection reaching a maximum of approximately 3×104 c.f.u. per organ, but spores were not detected until 24 h post-infection. At 36 h post-infection C. difficile numbers increased significantly to approximately 6×107 c.f.u. per organ where numbers remained high until the clinical end point. Increasing levels of in vivo toxin production coincided with increases in C. difficile numbers in organs reaching a maximum at 36 h post-infection in the caecum. Epithelial destruction and polymorphonuclear leukocyte (PMN) recruitment occurred early on during infection (24 h) accumulating as gross microvilli damage, luminal PMN influx, and blood associated with mucosal muscle and microvilli. These data describe the fatal infection kinetics of the clinical UK epidemic C. difficile strain R20291 in the hamster infection model.

Analysis of Bacterial Communities during Clostridium difficile Infection in the Mouse

“…Rather, we found that C. difficile forms mucin-associated communities in the cecum and colon up to at least 8 days p.i. We did not detect any C. difficile cells in direct association with the epithelium or within crypts, as was shown previously (16). This difference could be explained by differences in our infection models and/or properties of the strain we used for infection.…”

“…For example, transmission electron microscopy revealed that during infection in the mouse, a mat of rod-shaped bacterial cells is found overlaying damaged microvilli (5). Scanning electron microscopic analysis of cecum and colon samples of infected hamsters showed C. difficile-like bacteria associated with mucus, over both the tissue surface and crypt crevasses, forming aggregates (16).…”

The Clostridium difficile Exosporium Cysteine (CdeC)-Rich Protein Is Required for Exosporium Morphogenesis and Coat Assembly

“…The C. difficile R20291 strain used in this study belongs to ribotype 027, is positive for tcdA (tcdA ϩ ), tcdB ϩ , tcdC ϩ , and cdtB ϩ , and lacks ctdA (19). Escherichia coli strains BL21 (DE3)-RIL and HB101 harboring the conjugative plasmid pRK24 (Tra ϩ , Mob ϩ ; bla, tet r ) are described elsewhere (20)(21)(22).…”

Protective Efficacy Induced by Recombinant Clostridium difficile Toxin Fragments

“…Production of C. difficile toxins was detected in vitro by using filtered cecum contents taken postmortem as described previously (53). Briefly, monolayers of Vero cells (kidney epithelial cells) or HT29 cells were washed with preheated sterile PBS before addition of serially diluted filtered gut content in supplemented EMEM and then were incubated for 18 h at 37°C with 5% CO 2 .…”