Identification of Clostridioides difficile and Bacillus cereus surrogates to assess the sporicidal activity of industrial laundering processes.

Industrial laundry processes are a critical control point for microbiological risk management on reusable textiles, particularly in healthcare and other high-risk settings. Spore-forming bacteria such as Clostridioides difficile and Bacillus cereus present a challenge to laundering efficacy due to their resistance to thermal and chemical stresses. Validation of sporicidal performance is constrained by the absence of standardized test methods and the biohazard risks associated with using pathogenic spores at industrial scale. This study aimed to identify a safe, technically representative surrogate organism for use in bioindicator-based validation of sporicidal efficacy in industrial laundering processes. Spores of selected candidate organisms (Metaclostrioides mangenotii, Clostridium sporogenes, and Bacillus licheniformis) were produced and purified alongside C. difficile and B. cereus. Chemical resistance was assessed using in-solution carrier tests and full in-wash bioindicator testing under representative commercial laundry conditions (60 °C, 10 min), using a commercial detergent system and peracetic acid. Minimum inhibitory concentrations (MICs) for peracetic acid were determined to compare intrinsic resistance profiles. All species were inactivated at the in-wash peracetic acid concentration (131 ppm), with substantial differences in resistance observed at lower concentrations; C. difficile and B. cereus exhibited the highest MICs (131 ppm). Bacillus licheniformis demonstrated resistance profiles comparable to the target pathogens during detergent and in-wash testing, while offering a reduced biosafety risk (BSL 1). These findings support B. licheniformis as a practical, safer surrogate for sporicidal validation of industrial laundry processes, enabling robust process assurance while reducing operational and biohazard risks during routine testing.