Possible Seasonality of Clostridium difficile in Retail Meat, Canada

We previously reported Clostridium difficile in 20% of retail meat in Canada, which raised concerns about potential foodborne transmissibility. Here, we studied the genetic diversity of C. difficile in retail meats, using a broad Canadian sampling infrastructure and 3 culture methods. We found 6.1% prevalence and indications of possible seasonality (highest prevalence in winter).

Culture binary data were analyzed by using a randomized block design approach with a conditional logistic regression analysis (SAS Institute, Cary, NC, USA) and p value estimations with Monte Carlo simulations. Exact tests for pairwise comparisons were based on LogXact 7 and a Fortran program (Cytel Inc, Cambridge, MA, USA). Kappa, χ 2 , and Fisher exact tests were also used. Significance was held at p<0.05.
Combining the results from 4 cultures, we found the prevalence of C. diffi cile was 6.7% (10/149) in ground beef and 4.6% (3/65) in veal chops from milk-fed calves.
The combined prevalence was 6.1% (13/214). The prevalence of C. diffi cile recovery determined by using different culture methods varied from 1.4% to 2.3%, but no culture agreement or reproducibility was observed (p>0.1). Overall, the individual diagnostic sensitivity of each method was low (<39%; Table 1).
When month-to-month variability was considered, C. diffi cile was more commonly isolated from meat in January and February (11.5%, 7/61) than during the remaining 5 months of the study (4%; 6/150; p = 0.041). This fi nding indicates possible seasonality, although further studies are needed.

Conclusions
In contrast to our fi rst study (4), this study evaluated the genetic diversity of C. diffi cile in retail meats in a large area of Canada and tested 1-2 samples per store to prevent clustering. Thus, the overall prevalence observed (6.1%) was lower than that of previous studies in Canada (20%) (5) and the United States (42%) (13). Although different sampling and culture methods may account for the different prevalences, taken altogether, these studies support recent concerns regarding food safety.  (4). #No statistical differences were found between ground beef and veal in any culture replicate (p>0.1). Duplicate cultures, irrespective of method, could yield higher rates of C. diffi cile recovery from meat. However, the sensitivity of duplicate testing of meat is still suboptimal (46.2%-61.5%) compared with the sensitivity reached by one of our methods (4) in human stool samples (>95%) (6). Suboptimal performance might be due to reduced culture selectivity and nonhomogeneous distribution or a low number of spores.
In addition to cross-contamination at slaughter and during processing, it is possible that contamination of muscle tissue with C. diffi cile spores occurs preharvest. In horses, Clostridium spores have been recovered from muscle tissue in healthy horses (14), and a recent muscle sample yielded C. diffi cile in a healthy cow (unpub. data). Translocation from the intestines and deposition of dormant spores in muscle are reasonable assumptions that need investigation.
The increased recovery of C. diffi cile from meat in winter suggests that a seasonal component might exist. This component is currently uncertain, but a possible epidemiologic link between this observation and the seasonality observed in human disease (15) and the high rate of C. diffi cile toxins in calves in winter (11) requires further elucidation.
The C. diffi cile genotypes identifi ed in this and other studies (especially the NAP1 clone and PCR ribotypes 077 and 014) (3,4,11) provide further molecular evidence that spore dissemination through foods should be considered. Although ingestion of spores does not necessarily imply infection, this study supports the potential for foodborne transmissibility and raises questions about possible seasonality.    (4,11). PCR ribotype designations are described in Table 2. Note the genetic similarity (94.1%-100%) and antimicrobial resistance profi les between human and meat isolates, especially PCR ribotypes 014 and J. Also note the genetic similarity (81.8%-100%) between meat isolates from 2005 and 2006 for multidrug-resistant epidemic PCR ribotype 077, clindamycinvariable, PCR ribotype 014, and nontoxigenic PCR ribotype M26. Resistance to all 4 antimicrobial drugs was observed in meat isolates of ribotypes 077 and F, which also yielded the highest level of clindamycin resistance (>256 μL/mL; breakpoint: >6 μL/mL). The breakpoints for moxifl oxacin (12) were also used for levofl oxacin and gatifl oxacin. R (resistant), S (susceptible), and I (intermediate) represent antimicrobial profi les. CDC, Centers for Disease Control and Prevention; NAP, North America PFGE type; NAP1-r, NAPrelated strain; Tox, toxinotyping nomenclature (M. Rupnik, Maribor, Slovenia); U, unnamed.