Q-263. QPCR Quantification and Rep-PCR Characterization of Clostridium perfringens Populations in Composted Biosolids

E. Karpowicz1, A. Novinscak1, F. Baerlocher2, M. Filion1;
1Univ. de Moncton, Moncton, NB, CANADA, 2Mount Allison Univ., Sackville, NB, CANADA.

Background: Disposal of human biosolids is becoming more challenging as the human population increases and safety regulations become more demanding. Composting biosolids is an inexpensive natural alternative. Present guidelines in North America and Europe ensure safety of composted biosolids by controlling for Salmonella and/or fecal indicators only. Many studies have suggested the suitability of Clostridium perfringens as a better indicator of sanitation processes and its recognition as an important human and animal pathogen. We therefore decided to investigate the presence and diversity of this ubiquitous microorganism in composted biosolids. Methods: A TaqMan qPCR assay targeting the chaperonin (cpn60) gene was adapted to enumerate the pathogen directly from composted biosolids samples and rep-PCR was utilized to characterize the genetic diversity of C. perfringens at different composting stages. A total of 30 samples were collected from six open windrow piles aged 1, 4, 8, 13, 18 and 24 months. Total genomic DNA was extracted from the samples for qPCR assays and Iron Milk and Tryptose Sulfate Clostridium Agar medium were employed to isolate C. perfringens for rep-PCR analysis. Results: The limit of detection for qPCR was established to be between 83.1 and 12.9 copy numbers of cpn60 gene, equivalent to 120 and 12 cells per gram, respectively. The method yielded 100% efficiency of recovery and high reproducibility of the results was obtained. Significantly higher copy numbers were detected in 24 months composts as compared to younger composts. Also, genetically more diverse C. perfringens populations were found after 24 months of composting, suggesting a possible external environmental contamination of composted biosolids by C. perfringens strains. Conclusion: This study provides useful, robust and rapid tools for Clostridium perfringens enumeration and source tracking in environmental samples.