Evaluation of Animal Manure-based Compost as an Environmental Source of Clostridium difficil

The number of Clostridium difficile infection (CDI) cases associated with healthcare and community is increasing. Increased isolation of this pathogen from environmental samples of soil, water, livestock animals and their feces, and produce suggests its dissemination in the environment. Identification, molecular characterization, and revealing the relationship of those isolates with human pathogenicity would be very helpful in understanding the epidemiology of CDI. Livestock manure is a rich source of nutrients for the growth of many crops, and composted manure is widely used as a fertilizer in organic farming. Microbial activities increase the temperature of composting materials, providing sufficient heat to kill vegetative cells of most pathogenic bacteria. C. difficile, is an endospore-forming pathogen present in livestock manure and its fate during composting is not well-understood. However, the survival of C. difficile in compost poses a microbial safety threat to the post-harvest environments of crops. The objectives of this study were to 1) to improve the culture media for isolation of C. difficile endospores and vegetative cells from compost, 2) to isolate and characterize C. difficile and Clostridium perfringens from commercially available compost and manure, 3) to study the thermal resistance of C. difficile endospores on wet and dry heat exposure in composted dairy cattle manure, and 4) to study the survival of C. difficile in composted dairy cattle manure under controlled storage conditions.;The detection methods for the isolation of C. difficile from the animal manure-based composts were optimized. Both autoclaved and unautoclaved dairy composts were inoculated with a 12-h old suspension of a non-toxigenic C. difficile strain (ATCC 43593), and then plated on selected agar for vegetative cells and endospores separately. Six types of enrichment broths supplemented with taurocholate and L-cysteine were assessed for detecting a low level of artificially inoculated C. difficile (ca. 5 spores/g) from dairy composts. The efficacy of selected enrichment broths was further evaluated by isolating C. difficile from 29 commercial compost samples. Our results revealed that using heat-shock was more effective than using ethanol-shock for inducing endospore germination, yielding the the highest endospore count at 60°C for 25 min treatment. C. difficile agar base, supplemented with 0.1% L-cysteine, 7% defibrinated horse blood, and cycloserine-cefoxitin (CDA-CYS-H-CC agar) was the best medium (p < 0.05) for recovering vegetative cells from compost. C. difficile endospore populations from both types of composts enumerated on both CDA-CYS-H-CC agar supplemented with 0.1% sodium taurocholate (CDA-CYS-H-CC-T agar) and brain heart infusion agar supplemented with 0.5% yeast extract, 0.1% L-cysteine, cycloserine-cefoxitin, and 0.1% sodium taurocholate (BHIA-YE-CYS-CC-T agar) media were not significantly different from each other (p > 0.05). Overall, enrichment of inoculated compost samples in broths containing moxalactum-norfloxacin (MN) produced significantly higher (p < 0.05) spore counts than in non-selective broths or broths supplemented with CC. Enrichment in BHIB-YE-CYS-MN-T broth followed by culturing on an agar containing 7% horse blood and 0.1% taurocholate provided a more sensitive and selective combination of media for detecting a low population of C. difficile from environmental samples with high background microflora.;As live-stock animals carry and shed C. difficile and C. perfringens in the feces, animal manure-based composts may play an important role in disseminating both of these toxigenic clostridia species into agricultural environment. A survey study was performed to isolate C. difficile and C. perfringens contaminations from commercially available composts and livestock animal manure (n=142) using the optimized media. Presumptive C. difficile and C. perfringens isolates were confirmed by testing for the tpi house-keeping gene and 16S rRNA gene, respectively, in addition to staining methods. The confirmed C. difficile isolates were further tested for toxigenicity and PCR ribotyping. Both clostridia isolates were tested for susceptibility to selected antibiotics. A total of 58 C. difficile strains and 11 C. perfringens strains were identified from 142 compost/manure samples and the majority of C. difficile isolates were toxigenic with 63.8% isolates (n=37) positive for toxin A (tcdA) while 67.2% isolates (n=39) positive for toxin B (tcdB). Only 3 isolates (5.17%) were positive for the binary toxins. (Abstract shortened by ProQuest.).