Clin Microbiol Infect 2012; 18: 635–645 Abstract Clostridium difficile infections (CDIs) are traditionally seen in elderly and hospitalized patients who have used antibiotic therapy. In the community, CDIs requiring a visit to a general practitioner are increasingly occurring among young and relatively healthy individuals without known predisposing factors. C. difficile is also found as a commensal or pathogen in the intestinal tracts of most mammals, and various birds and reptiles. In the environment, including soil and water, C. difficile may be ubiquitous; however, this is based on limited evidence. Food products such as (processed) meat, fish and vegetables can also contain C. difficile, but studies conducted in Europe report lower prevalence rates than in North America. Absolute counts of toxigenic C. difficile in the environment and food are low, however the exact infectious dose is unknown. To date, direct transmission of C. difficile from animals, food or the environment to humans has not been proven, although similar PCR ribotypes are found. We therefore believe that the overall epidemiology of human CDI is not driven by amplification in animals or other sources. As no outbreaks of CDI have been reported among humans in the community, host factors that increase vulnerability to CDI might be of more importance than increased exposure to C. difficile. Conversely, emerging C. difficile ribotype 078 is found in high numbers in piglets, calves, and their immediate environment. Although there is no direct evidence proving transmission to humans, circumstantial evidence points towards a zoonotic potential of this type. In future emerging PCR ribotypes, zoonotic potential needs to be considered.

ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

The presence and characteristics of Clostridium difficile were investigated in 839 faecal samples from seven different animal species in the Netherlands. The number of positive samples ranged from 3.4% (cattle) to 25.0% (dogs). Twenty-two different PCR ribotypes were identified. Among 96 isolates, 53% harboured toxin genes. All C. difficile isolates from pigs, cattle and poultry were toxinogenic, whereas the majority of isolates from pet animals consisted of non-toxinogenic PCR ribotypes 010 and 039. Ribotype 012 was most prevalent in cattle and ribotype 078 in pigs. No predominant ribotypes were present in horse and poultry samples. Overall, PCR ribotypes 012, 014 and 078 were the most frequently recovered toxinogenic ribotypes from animal samples. Comparison with human isolates from the Dutch Reference Laboratory for C. difficile at Leiden University Medical Centre (LUMC) showed that these types were also recovered from human hospitalized patients in 2009/2010, encompassing 0.8%, 11.4% and 9.8% of all isolates, respectively. Application of multiple-locus variable-number tandem-repeat analysis indicated a genotypic relation of animal and human ribotype 078 strains, but a clear genotypic distinction for ribotypes 012 and 014. We conclude that toxinogenic C. difficile PCR ribotypes found in animals correspond to PCR ribotypes associated with human disease in hospitalized patients in the Netherlands. Contrary to PCR ribotype 078, significant genetic differences were observed between animal and human PCR ribotype 012 and 014 isolates.

We compared Campylobacter jejuni/coli multilocus sequence types (STs) from pets (dogs/cats) and their owners and investigated risk factors for pet-associated human campylobacteriosis using a combined source-attribution and case-control analysis. In total, 132/687 pet stools were Campylobacter-positive, resulting in 499 strains isolated (320 C. upsaliensis/helveticus, 100 C. jejuni, 33 C. hyointestinalis/fetus, 10 C. lari, 4 C. coli, 32 unidentified). There were 737 human and 104 pet C. jejuni/coli strains assigned to 154 and 49 STs, respectively. Dog, particularly puppy, owners were at increased risk of infection with pet-associated STs. In 2/68 cases vs. 0.134/68 expected by chance, a pet and its owner were infected with an identical ST (ST45, ST658). Although common sources of infection and directionality of transmission between pets and humans were unknown, dog ownership significantly increased the risk for pet-associated human C. jejuni/coli infection and isolation of identical strains in humans and their pets occurred significantly more often than expected.

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