Susceptibility of hamsters to clostridium difficile isolates of differing toxinotype

Clostridium difficile is the most commonly associated cause of antibiotic associated disease (AAD), which caused ~21,000 cases of AAD in 2011 in the U.K. alone. The golden Syrian hamster model of CDI is an acute model displaying many of the clinical features of C. difficile disease. Using this model we characterised three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) and BI-7 (toxinotype III). The naturally occurring non-toxic strain colonised all hamsters within 1-day post challenge (d.p.c.) with high-levels of spores being shed in the faeces of animals that appeared well throughout the entire experiment. However, some changes including increased neutrophil influx and unclotted red blood cells were observed at early time points despite the fact that the known C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. In contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonised. It is currently unclear why some hamsters survive infection, as bacterial and toxin levels and histology scores were similar to those culled at a similar time-point. Hamsters challenged with strain BI-7 resulted in a rapid fatal infection in 100% of the hamsters approximately 26 hr post challenge. Severe caecal pathology, including transmural neutrophil infiltrates and extensive submucosal damage correlated with high levels of toxin measured in gut filtrates ex vivo. These data describes the infection kinetics and disease outcomes of 3 clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression

Survival of hamsters challenged with <i>C. difficile</i> strains.

<p>(A) Survival graph of hamsters challenged with either <i>C. difficile</i> CD1342 (full line), M68 (long dashed line), BI-7 (dash/dot line) or R20291 [dotted line (R20291 data from 24]. (B) Typical body temperature kinetics of a hamster challenged with CD1342 following the usual diurnal pattern. (C) Body temperature kinetics of a surviving hamster challenged with M68, (D) a hamster that succumbed to M68 challenge & (E) a hamster challenged with BI-7. Top bar represents when symptoms, typically ‘wet tail’, manifest. A typical febrile response (maximum temperature of 39.3°C) was seen prior to onset of observed symptoms.</p

Colonisation kinetics of <i>C. difficile</i> CD1342 in hamsters.

<p>To monitor colonisation throughout the infection process hamsters were culled at 1- (A), 3- (B) and 11 (C) days post challenge. <i>C. difficile</i> was recovered from the caecum (CAE) and the colon (COL) either associated with the lumen (LA) or the tissue (TA). Filled bars represent vegetative bacteria whilst empty bars indicate bacteria in spore form. Bacterial recoveries represent the geometric mean plus the standard error of the mean (SEM) of 2 biological replicates, where a total of at least 5 animals were used per time interval.</p

Mean histology scores from hamsters challenged with <i>C. difficile</i> strains.

<p><i>C. difficile</i> CD1342 (filled columns), M68 (open columns) or BI-7 (checked column) at either 1-, 3- & 11-d.p.c (A) or if the hamsters succumbed to infection (B). Caecal pathology was graded by neutrophil margination, haemorrhagic congestion, hyperplasia and percent barrier involvement from at least four animals. Typical caecal histology from hamsters challenged with either CD1342 (at 1-d.p.c. - C), M68 (at 3-d.p.c. - D) or BI-7 (at ∼26 h - E). Red arrows denotes unclotted red blood cells within the villus structure; black arrows denotes epithelial barrier destruction & green arrows denotes transmural neutrophil infiltrate.</p

Colonisation kinetics of <i>C. difficile</i> BI-7 in hamsters.

<p>Bacteria recovered from hamsters challenged with BI-7 at time of cull (∼26 h). <i>C. difficile</i> was recovered from the caecum (CAE) and the colon (COL) either associated with the lumen (LA) or the tissue (TA). Filled bars represent vegetative bacteria whilst empty bars indicate bacteria in spore form. Bacterial recoveries represent the geometric mean plus the standard error of the mean (SEM), where a total of at least 5 animals were used.</p

Colonisation kinetics of <i>C. difficile</i> M68 in hamsters.

<p>To monitor colonisation throughout the infection process hamsters were culled at 1- (A), 3- (B) and 11 (D) days post challenge and if animals succumbed to infection (C). <i>C. difficile</i> was recovered from the caecum (CAE) and the colon (COL) either associated with the lumen (LA) or the tissue (TA). Filled bars represent vegetative bacteria whilst empty bars indicate bacteria in spore form. Bacterial recoveries represent the geometric mean plus the standard error of the mean (SEM) of 2 biological replicates, where a total of at least 5 animals were used per time interval.</p

Protective efficacy induced by recombinant Clostridium difficile toxin fragments

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection