Presence of Clostridium difficile PCR ribotype clusters related to 033, 078 and 045 in diarrhoeic calves in Germany

Abstract

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...

Clostridium difficile Genotypes in Piglet Populations in Germany

“…Some PCR ribotypes recovered during this survey (078, 126, 002, and 598) were previously found in bovine, equine, and/or porcine isolates (7,10,12,19,28,29,30). As shown in previous studies, PCR ribotype 078 was the predominating PCR ribotype in pigs (9,10,23,25,29).…”

“…3). Therefore, data recovered in this work (including 5 strains isolated from human CDI patients of the University Hospital of Heidelberg, Germany) and data obtained in previous studies (12,13) were analyzed. C. difficile strains investigated by Bakker et al (13) were isolated mainly between 2006 and 2009 (149 of 158 strains; 7 isolation dates were not available, and one strain each was isolated in 1953 and 2005).…”

“…C. difficile strains investigated by Bakker et al (13) were isolated mainly between 2006 and 2009 (149 of 158 strains; 7 isolation dates were not available, and one strain each was isolated in 1953 and 2005). PCR ribotype 078 strains typed by Schneeberg et al (12) and this study were isolated in 2007 (5 strains of human origin only) and between 2010 and 2012 (bovine and porcine strains). Clonal or related MLVA types were present within geographical regions over several years: e.g., branches B and C contain clonal/related types of human, bovine, and porcine origin which were isolated in Germany between 2007 and 2012; four strains of identical MLVA type were isolated from humans in United Kingdom and Ireland in 1953 and 2008 (see Fig.…”

“…Clonal clusters were defined by a STRD of Յ2, and genetically related clusters were defined by STRD of Ͼ2 and Յ10 (13,18,19), provided that isolates were single-or double-locus variants. For MST analysis using MLVA data from previous surveys (12,13) and this study, interlaboratory comparability of MLVA data was assumed.…”

Clostridium difficile infection: Early history, diagnosis and molecular strain typing methods

“…In addition, MLST is a relatively costly and laborious technique that requires specific technical skills. Multilocus variable-number tandem-repeat analysis (MLVA) has been suggested to have higher discriminatory power than other typing methods for investigating the relatedness between C. difficile strains [201]. It has been widely used in medical microbiology as an alternative or complement to other typing techniques such as PFGE, rep-PCR or MLST [202].…”