Development and Validation of a New Protocol for Detecting and Recovering Clostridium difficile from Meat Samples

Abstract

There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.

Clostridioides difficile in national food surveillance, Slovenia, 2015 to 2017

“…In previous small sporadic pilot studies performed by NLZOH where 124 meat samples of pork, beef, sheep and poultry were tested, all were negative for C. difficile (Tkalec and Rupnik; data not shown). Therefore, it seems that the systematic sampling and optimised methodology [23] used in national surveillance contributed to better detection. Early studies about possible presence of C. difficile in food of animal origin from North America reported high contamination rates of meat and meat products (20% in beef and veal ground meat in Canada [27]; 14.3% to 62.5% depending on the meat sample type, in the US [28]).…”

“…Retail meat samples and seafood were analysed by the microbiological laboratory of the Institute of Food Safety, Feed and Environment, which is a part of the National Veterinary Institute. The isolation protocol for meat samples was previously established and evaluated by Biasizzo et al, 2018 [23], where samples spiked with 1,000, 100 and 10 CFU per sample were tested and 100% sensitivity was determined for all spiked samples.…”

“…The samples were then incubated under anaerobic conditions at 37 °C for 7 days. The enriched samples were tested for C. difficile by quantitative real-time PCR and plated after spore selection by ethanol treatment onto blood agar plates (blood agar supplemented with 5% defibrinated bovine blood), C. difficile agar base with added C. difficile selective supplement and 7% (v/v) defibrinated horse blood ( cycloserine cefoxitin fructose agar (CCFA); all from Oxoid, Hampshire, United Kingdom (UK)) as previously described [23] and onto chromID C. difficile agar (bioMerieux, Lyon, France).…”

Clostridioides (Clostridium) difficile

“…8,18 Although there is no consensus on the optimal method for C. difficile culture from stool, a heat shock or alcohol treatment before direct plating on a selective media (namely, cycloserine-cefoxitin-fructose agar or its variant) is recommended by many investigators. 18,[81][82][83] This is mainly to inhibit or minimize the growth of stool organisms and enhance C. difficile spores. 18 However, other studies revealed that using an enrichment broth containing antimicrobials, carbohydrates, lysozyme, and taurocholate can increase the recovery of C. difficile without requiring heat shock or ethanol pretreatment.…”