BackgroundClostridium difficile infections (CDI) in humans range from asymptomatic carriage to life-threatening intestinal disease. Findings on C. difficile in various animal species and an overlap in ribotypes (RTs) suggest potential zoonotic transmission. However, the impact of animals for human CDI remains unclear.MethodsIn a large-scale survey we collected 1,447 fecal samples to determine the occurrence of C. difficile in small companion animals (dogs and cats) and their owners and to assess potential epidemiological links within the community. The Germany-wide survey was conducted from July 2012-August 2013. PCR ribotyping, Multilocus VNTR Analysis (MLVA) and PCR detection of toxin genes were used to characterize isolated C. difficile strains. A database was defined and logistic regression used to identify putative factors associated with fecal shedding of C. difficile.ResultsIn total, 1,418 samples met the inclusion criteria. The isolation rates for small companion animals and their owners within the community were similarly low with 3.0% (25/840) and 2.9% (17/578), respectively. PCR ribotyping revealed eight and twelve different RTs in animals and humans, respectively, whereas three RTs were isolated in both, humans and animals. RT 014/0, a well-known human hospital-associated lineage, was predominantly detected in animal samples. Moreover, the potentially highly pathogenic RTs 027 and 078 were isolated from dogs. Even though, C. difficile did not occur simultaneously in animals and humans sharing the same household. The results of the epidemiological analysis of factors associated with fecal shedding of C. difficile support the hypothesis of a zoonotic potential.ConclusionsMolecular characterization and epidemiological analysis revealed that the zoonotic risk for C. difficile associated with dogs and cats within the community is low but cannot be excluded.

This study provides data on the distribution and relationship of C. difficile PCR ribotypes in diarrhoeic calves in Germany. C. difficile was isolated from 176 of 999 (17.6 %) faecal samples or swabs of diarrhoeic calves from 603 farms collected between January 2010 and August 2012 by eight federal laboratories of six states. Strains were assigned to 17 PCR ribotypes. PCR ribotypes 033 (57 %), 078 (17 %) and 045/FLI01 (closest match to 045 in the WEBRIBO database; 9 %) were found the most frequently. Nine per cent of all culture-positive tested animals shed more than one multiple locus variable number tandem repeat analysis (MLVA) or PCR ribotype. Eight PCR ribotypes with related profiles (including 033, 078 and 045/FLI01) representing 92 % of all isolates were grouped into three clusters. Molecular relatedness was supported by the absence of the MLVA locus A6 Cd only in clustered strains and identical toxin gene profiles for strains within each cluster. Previously reported mulitilocus sequence typing analysis for PCR ribotypes that were also recovered in this study found identical sequence types and a tcdC deletion (D39 bp) for 033, 045, 078 and 126 (ST-11), confirming this clustering. A different geographical occurrence of PCR ribotypes was shown for cluster 033 (found more frequently in southern Germany) and 045 (found more frequently in northern Germany). This study showed that clusters of C. difficile PCR ribotypes related to 033, 078 and 045 are predominant in diarrhoeic calves in Germany. The high number of strains belonging to PCR ribotype 078 demonstrated that diarrhoeic calves are also potential reservoirs for human pathogenic C. difficile strains. INTRODUCTIONClostridium difficile is an anaerobic, Gram-positive, sporeforming and rod-shaped bacterium that causes gastrointestinal infections [C. difficile infection (CDI)] in humans with symptoms ranging from mild diarrhoea to severe pseudomembranous colitis (Rupnik et al., 2009). The major virulence factors are two high-molecular-mass toxins, the enterotoxin TcdA and the cytotoxin TcdB (Thelestam & Chaves-Olarte, 2000;Rupnik & Just, 2006;Jank et al., 2007;Kuehne et al., 2011). Some strains additionally produce the binary toxin CDT (Stubbs et al., 2000). Human disease is closely associated with hospitalization, but community-associated CDI is increasingly being reported (Wilcox et al., 2008;Khanna et al., 2012). Companion animals such as dogs and cats, but also farm animals such as pigs and cattle, are possible reservoirs for human pathogenic C. difficile strains (Debast et al., 2009;Weese et al., 2010;Koene et al., 2012;Rodriguez et al., 2012). Foods (meat, salads, fish and seafood) are often contaminated with virulent C. difficile strains (Rodriguez-Palacios et al., 2009;Metcalf et al., 2010; Metcalf et al., 2011). However, transmission of C. difficile from animals or foods to humans has so far not been demonstrated, and its zoonotic potential is subject to discussion .C. difficile is pathogenic also for various animal species, including hamsters, guin...

dClostridium difficile was isolated from 147 of 201 (73%) rectal swabs of piglets from 15 farms of Lower Saxony and North Rhine-Westphalia. In 14 farms, 14 to 100% (mean, 78%) of the animals tested were culture positive. The rate of isolation was 68% postpartum, increased to 94% in animals 2 to 14 days of age, and declined to 0% for animals 49 days of age and older. There was no link between isolation and antibiotic treatment or diarrhea of piglets. Strains were assigned to 10 PCR ribotypes, and up to 4 PCR ribotypes were found to be present at the same time on a farm. The closely related PCR ribotypes 078 (55%) and 126 (20%) were most frequently recovered and were present in 13 of the 14 positive farms. The comparison of multilocus VNTR (variable number of tandem repeats) analysis (MLVA) data from this study and previously published data on human, porcine, and bovine PCR ribotype 078 isolates from 5 European countries revealed genetic differences between strains of different geographic origin and confirmed the relatedness of human and porcine C. difficile isolates. This study demonstrated that the human-pathogenic PCR ribotypes 078 and 126 are predominant in piglets in Germany. The results suggest that presence of C. difficile is correlated with animal age but not with antibiotic treatment or clinical disease. MLVA indicated that strains of the same geographical origin are often genetically related and corroborated the hypothesis of a close epidemiological connection between human and porcine C. difficile isolates.

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