Comparision of MALDI-TOF MS, RAPID ID 32 A, and multiplex real time-PCR for identification of Clostridioides difficile in mussels (Mytilus galloprovincialis) and seawater

The fact that Clostridioides difficile, known as a nosocomial pathogen, causes infections in individuals lacking traditional risk factors raises the possibility that its source may not be confined to hospital environments. In this context, investigating food matrices and accurately identifying presumptive isolates are important for understanding the possible foodborne relevance of this bacterium. This study comparatively evaluated two selective supplement systems, D-cycloserine–cefoxitin (CC) and moxalactam–norfloxacin (MN), for the recovery of presumptive C. difficile isolates from 536 mussels collected from nine sites in Çanakkale, Turkey, together with seawater samples obtained from the same locations. Presumptive isolates were further examined using MALDI-TOF MS (Bruker and VITEK MS), RAPID ID 32 A, and multiplex real-time PCR. Considerable discrepancies were observed among the identification methods. In particular, one isolate recovered from mussels was identified as C. bifermentans by MALDI-TOF MS but as C. difficile by RAPID ID 32 A; however, this result was not confirmed by PCR. Overall, none of the presumptive isolates from mussel or seawater samples were confirmed as C. difficile by multiplex real-time PCR. These findings demonstrate that detection outcomes are strongly influenced by both the isolation strategy and the identification method used. They also indicate that presumptive recovery and preliminary identification do not necessarily correspond to confirmed C. difficile detection in complex food and environmental matrices. Therefore, molecular confirmation remains essential, and further methodological improvements are needed to improve the reliable detection of C. difficile in food samples.