A Comprehensive Study Investigating the Potential Health Risk of Clostridium Difficile as a Foodborne Pathogen

Non-Technical Summary: Clostridium difficle is a major cause of intestinal disease in humans, and has been isolated from a variety of food animals, including beef, pork and chicken. Further, identical C. difficile strains implicated in human infections have been isolated from food animals and foods, thereby further strengthening the role of food in the transmission of C. difficle to humans. However, no information currently exists on the survival characteristics of C. difficile in foods, or the effect of various food processing methods on C. difficile. Therefore, before C. difficile becomes a major foodborne pathogen, it is important to understand its occurrence in the food supply, identify and develop appropriate control strategies, and spread science-based information to professionals, regulatory agencies and public. This multistate study will determine the 1.Occurrence of C. difficile in ground beef, pork and chicken in retail stores in CT, PA, MN and AL. 2.The characteristics of C. difficile isolated from food and compare them with those of human isolates from the respective states. 3.Effect of chilling, freezing, cooking, salt, and acidity on C. difficile. This study is expected to provide a comprehensive understanding on the mode of transmission, viability characteristics in foods, and susceptibility of C. difficile to the common antimicrobial steps applied in foods. This information is important for developing appropriate control measures before C. difficile emerges as a significant food safety threat. <P> Approach: To determine the prevalence of C. difficile in ground beef, ground pork and chicken wings, fifty grams each meat will be separately added to 50 ml of C. difficile moxalactam-norfloxacin (CDMN)broth with 0.1% sodium taurocholate, and massaged thoroughly. One hundred microliters of the broth will be inoculated onto duplicate C. difficile moxalactam-norfloxacin (CDMN) agar, while the remaining broth-sample mixture will be incubated anaerobically at 37C for 48 h. An aliquot of the broth will be subjected to alcohol shock by adding anhydrous ethanol for 1 h to eliminate competing vegetative bacteria. Then, this mixture will be centrifuged at 4000 x g for 10 min and the pellet will be streaked on CDMN agar. The agar plates will be incubated anaerobically at 37C for 48 h. The suspected C. difficile colonies will be subcultured onto 5% horse blood agar and presumptive confirmation of C. difficle will be done based by Gram's staining reaction, colony morphology, yellow-green fluorescence under ultraviolet, characteristic odor and positive L-proline aminopeptidase reaction. In objective 2, the antibiotic resistance profiles of C. difficile isolates will be determined using methods approved by the Clinical and Laboratory Standards Institute. The resistance of the isolates to ampicillin, oxytetracycline, tetracycline, neomycin, sulfamethazine, clindamycin, metronidazole), enrofloxacin, vancomycin, erythromycin), florfenicol, cefoxitin, and ciprofloxacin will be determined. Additionally, each C. difficile isolate will be screened for the presence of TcdA/B toxin using a commercially available enzyme immunoassay kit. In the genotypic characterization, a real-time PCR to detect the presence of genes encoding toxins TcdA, TcdB, and CDT (binary toxin encoded in 2 subunit genes cdtA and cdtB) in the C. difficile isolates, and the genotypic diversity among the various isolates using pulsed field gel electrophoresis (PFGE) will be performed. The general protocol used for the characterization of C. perfringens isolates by Co-PD, Dr. Smyth will be followed. The relatedness of PFGE profiles will be determined using the unweighted pair group method with arithmetic mean clustering. All clustering will be based on the Dice correlation coefficient. In objective 3, The effect of chilling (4C) and freezing (-18C) on the viability of C. difficile in ground beef, pork and chicken will be determined in irradiated and non-irradiated meat to determine if the meat endogenous bacteria have any effect on pathogen survival. Additionally, the effect of sodium chloride (0, 2.5, 5, 7.5, and 10%) and pH (5.5, 5, 4.5 and 4.0) on the viability of C. difficile cells and spores will be determined. The heat resistance of C. difficile in ground meat at different temperatures (60, 65, 70, 75, 80, 85, 90, 95C) will be investigated. The D-values and z-values of C. difficile in meat will be reported.