Purpose: The objective of this study was to isolate C. difficle from animal manure and commercial compost samples and characterize their toxigenicity.
Methods: Commercial compost and fresh manure samples (n=142) were enriched in brain heart infusion broth supplemented with 0.5% yeast extract, 0.1% L-cysteine, 0.1% sodium taurocholate, and antibiotics (moxalactam and norfloxacin) (BHIB-YE-CYS-T-MN) for 7 days, anaerobically, and recovered on C. difficile agar base supplemented with 7% horse blood, 0.1% L-cysteine, 0.1% sodium taurocholate, and antibiotics (moxalactam and norfloxacin). Suspected colonies were confirmed as C. difficile by PRO disc method, PCR for tpi, and latex agglutination tests. Isolates were tested for toxigenic genes, tcdA, tcdB, cdtA, and cdtB, and further identified by capillary gel electrophoresis.
Results: Samples were categorized as cow manure-based, poultry litter-based, mix manure-based, biosolid waste-based, plant-based, and other. A total of 58 C. difficile isolates were detected from 51 samples (47 finished compost and 4 manure). The majority of the isolates were toxigenic (67%), but binary toxins were observed only in 3 (5%) isolates. There were 45 different PCR-ribotypes among the 58 C. difficile isolates. The highest diversity in ribotypes was observed among the C. difficile isolates from biosolid waste-based composts. The most common toxigenic ribotype was RT500, and other more prevalent ribotypes were RT020, RT412, and RT251. RT009 was found as the most prevalent non-toxigenic ribotype.
Significance: The presence of toxigenic C. difficile ribotypes in finished compost suggests the contaminated compost/manure-amended soil could serve as a potential reservoir for C. difficile that may contaminate fresh produce, water, or field workers on the farm.