Possible Seasonality ofClostridium difficilein Retail Meat, Canada

We previously reported Clostridium diffi cile in 20% of retail meat in Canada, which raised concerns about potential foodborne transmissibility.Here, we studied the genetic diversity of C. diffi cile 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). C lostridium diffi cile infection (CDI) has been associ-ated with increased illness and death in Canada since 2000 (1,2).Although multiple genotypes with higher levels of virulence and antimicrobial resistance have been recognized (1,3), little is known about risk factors for CDI acquisition outside healthcare facilities.In a 2005 study, we found C. diffi cile in 20% of retail meats sampled in Canada (4).Limitations to that study included limited geographic representation, nonsystematic sampling, and the use of a nonvalidated culture method.These sampling limitations prevent valid extrapolations.Broader sampling and a better understanding of the culture methods were thus required to reassess the prevalence of retail meat contamination with C. diffi cile.Here, we determined the prevalence of C. diffi cile in retail meat by using a broad-based government sampling infrastructure, compared 3 culture methods, characterized recovered isolates, and evaluated month-to-month variability in C. diffi cile recovery. The StudyRetail meats were obtained from 2 randomly selected census divisions per week from various retailers across Canada as part of the active retail surveillance component of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) (5).We tested random packages of ground beef as well as veal chops from milkfed calves; the packages were purchased by CIPARS in Ontario, Québec, and Saskatchewan, Canada, from January through August 2006.Purchased packages were sent to the Laboratory of Foodborne Zoonoses, Québec (ground beef), and to the Canadian Research Institute for Food Safety, Ontario (veal chops), where 35-g composite samples were made.Rinsates were prepared by mixing 25 g of meat and 225 mL of buffered peptone water (placed in a stomacher for 15 min).Rinsates (12 mL) and the remains of the composite samples (10 g) were then sent to the University of Guelph for C. diffi cile testing.Sample size estimations indicated that 211 packages were adequate to verify a prevalence of 20% ± 8% (α = 0.05, power = 0.8; Stata sampsi command [Stata Corp., College Station, TX, USA]).A total of 214 meat samples were cultured by using 3 methods.One method, used in an earlier study (4), was tested in duplicate to assess reproducibility.All protocols had an enrichment phase of 7 days (Table 1), followed by ethanol treatment of culture sediments (96%, 1:2 [vol/ vol], 30 min), and inoculation onto solid agar for colony identifi cation (4,6).Suspected colonies (swarming, nonhemolytic) were subcultured onto 5% sheep blood agar.C. diffi cile was preliminarily identifi ed with L-proline aminopeptidase activity (Pro Disc; Remel, Lenexa, KS, USA) but confi rmed by PCR detection of the triose phosphate isomerase gene (7).PCR ribotyping and detection of genes for toxins A (tcdA), B (tcdB), binary toxin (cdtB), and toxin regulator (tcdC) were performed as previously described (4,8,9).Isolates having either tcdA, tcdB, or cdtB were classifi ed as toxigenic (10).Resulting PCR ribotypes were visually compared to representative PCR ribotypes previously identifi ed in cattle (n = 8, 2004), retail meats (n = 4, 2005), and humans (n = 39, 2004-2006) in Ontario and Québec, Canada (2,4,11).The fi rst meat-derived isolate of each PCR ribotype and 1 matching human isolate were submitted to the Centers for Disease Control and Prevention, Atlanta, Georgia, USA, for SmaI pulsed-fi eld gel electrophoresis (PFGE) and toxinotyping (1).We tested selected isolates to determine the MICs of clindamycin, levofl oxacin, moxifl oxacin, and gatifl oxacin by using the Etest (AB Biodisk, Solna, Sweden) and interpreted the results after the isolates were incubated for 48 h on Brucella agar (12).Controls included C. diffi cile strain ATCC 700057.