Determining the Fate of Clostridium difficile in Animal Manure-based Compost

Introduction: Clostridium difficile is one of the most important nosocomial human pathogens causing antibiotic-associated diarrhea and pseudomembranous colitis. Studies have reported the prevalence of this pathogen in a variety of animal manure.  However, there is a lack of research on the fate of C. difficile during composting.

Purpose: The purpose of this study was to develop a sensitive method for detecting C difficilefrom animal manure-based compost.

Methods: Vegetative cell and spore suspensions of C. difficile ATCC 43593 were artificially inoculated into the autoclaved and unautoclaved dairy compost. Inoculated cells were recovered on cycloserine cefoxitin fructose agar (CCFA) enriched with 7% horse blood and 1% sodium taurocholate (HT-CCFA), CCFA enriched with 7% horse blood (H-CCFA), brain hear infusion agar with yeast extract (BHIA-YE), BHIA-YE  with antibiotic supplement (BHIA-YE-CC), CCFA enriched with 1% sodium taurocholate (T-CCFA), and Clostridium difficile agar with horse blood (H-CDA). Inoculated spores were recovered on HT-CCFA and BHIA-YE with taurocolate (BHIA-YE-T). The recovery studies were performed at selected intervals after anaerobic incubation at 37 ºC for 2~3 days. Commercial compost samples were enriched in CCFB for 7 days and analyzed for C. difficile spores on HT-CCFA supplemented with cycloheximide (HTC-CCFA). Suspected colonies were isolated as pure cultures and tested for Gram’s reaction, endospores, PRO disc method, PCR for housekeeping gene tpi, and latex agglutination test.

Results: Our plate count data revealed that HT-CCFA was the best recovery medium among 7 media tested for both C. difficile cells and spores. The recovery rate of cell suspension (containing 1~5% of spores) for day 0 was 105.2% and 102.1% for autoclaved and unautoclaved compost, respectively, as compared with 76.4% and 69.1% for spore suspension, respectively. There was a slight reduction of spore population in both types of compost during room temperature storage up to 3 days.  Using the optimized C. difficile detection method, we analyzed 29 commercial compost samples and 3 samples were positive for the presence of C.difficile.

Significance: Our data suggests that CCFA supplemented with horse blood and taurocholate enhances the recovery of C. difficile spores, which can be used for analyzing commercial composts for the presence of C. difficile.