Prevalence of Clostridium difficile in retail meats from Minnesota and comparison of growth and survival of human and animal isolates

Abstract

Clostridium difficile is a Gram positive, anaerobic, spore-forming, rod-shaped bacterium that causes antibiotic-associated diarrhea in hospitalized and community-dwelling patients. Recent findings have suggested that this organism may be transmitted from animals to humans through the consumption of contaminated foods. Genotypic similarities have been found among C. difficile strains isolated from animals and humans. However, comparisons of the behavior of these two groups of strains at the physiological level have not been conducted in detail. This study sought to determine the prevalence of C. difficile in retail meats from Minnesota and to compare human and animal isolates by measuring the growth rate and their survival in meats during cold storage. Meat samples were obtained from retail stores from Minnesota and consisted mainly of raw beef, pork, and poultry. These samples were analyzed for the presence of C. difficile by initial enrichment in Clostridium difficile moxalactam-norfloxacin (CDMN) broth, followed by ethanol shock, plating onto CDMN agar, and anaerobic incubation for 48 h at 37°C. Suspicious colonies were subjected to confirmation by colony morphology, Gram staining, and production of L-proline aminopeptidase. Growth rate parameters were determined for a total of 35 C. difficile strains isolated from humans and animals. Optical density was measured during exponential growth on brain hearth infusion broth at 37°C anoxically. The average growth rates of the two groups were then compared. A subgroup of 5 human and 5 animal strains was selected to assess the survival of C. difficile in meats during cold storage. C. difficile strains were inoculated onto ground meats to a level of approximately 105 CFU/g. Inoculated meats were stored at 4°C and -15°C for 5 and 20 days, respectively. C. difficile counts were determined at different times during storage by plating onto CDMN agar. A total of 342 raw meat and poultry samples were collected from 5 different counties in central Minnesota from 25 retail stores. Twenty nine samples had CDMN-presumptive colonies, but none of them were confirmed as C. difficile C.I. 0-0.013% of meats positive for C. difficile at a 95% level. The average growth rate of 22 human strains at 37°C was 0.52± 0.25 h-1, which was not significantly different from the value for 13 animal strains (0.62± 0.15 h-1) (p>0.05). Counts of C. difficile strains inoculated on ground beef and chicken diminished during storage at 4°C and -15°C. Overall reduction in bacterial counts was not significantly different between human and animal strains. These findings suggested that C. difficile was not commonly present in retail meats from Minnesota. C. difficile strains isolated from animals had similar growth rates at 37°C as the strains from human origin. C. difficile viable counts diminished over time during refrigeration and freezing of inoculated meats. Additionally, the methodology used in the present study was effective in recovering C. difficile from artificially inoculated meats.