Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

Abstract

ABSTRACTWe first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of aClostridium perfringens-specific real-time PCR (rtPCR) assay based on thecpagene (cpartPCR) by using a bacterial strain panel composed ofC. perfringensand non-C. perfringens Clostridiumstrains. All non-C. perfringens Clostridiumstrains tested negative, whereas allC. perfringensstrains tested positive with thecpartPCR, for an analytical specificity and ubiquity of 100%. ThecpartPCR assay was then used to confirm the identity of 116 putativeC. perfringensisolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar andcpartPCR were identified by sequencing the 16S rRNA andcpagenes. Four mCP−/rtPCR+colonies were identified asC. perfringens, whereas 3 mCP+/rtPCR−colonies were identified as non-C. perfringens. ThecpartPCR was negative with all 51 non-C. perfringensstrains and positive with 64 of 65C. perfringensstrains. Finally, we compared mCP agar and a CRENAME (concentration andrecovery of microbial particles,extraction ofnucleicacids, andmolecularenrichment) procedure pluscpartPCR (CRENAME +cpartPCR) for their abilities to detectC. perfringensspores in drinking water. CRENAME +cpartPCR detected as few as oneC. perfringensCFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME +cpartPCR also allows the simultaneous and sensitive detection ofEscherichia coliandC. perfringensfrom the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.