Reliable delineation of Bacillus cytotoxicus from other members of the Bacillus cereus group by MALDI-TOF MS – An extensive validation study

Bacillus cytotoxicus is classified as a thermotolerant member of the Bacillus cereus group, producing the diarrhoea causing cytotoxin K1. This species came into the focus of food microbiologists when the type strain was isolated as the cause of a fatal food-borne outbreak. However, knowledge of the role of B. cytotoxicus as a food-borne microbe and data on its prevalence in food is still limited today. One reason for this is the lack of an easy to perform method of identification at the species level other than PCR, due to the highly similar phenotype of many B. cereus group representatives. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is widely used for the identification of microorganism species. This study describes the formal validation of a MALDI-TOF MS system for a reliable differentiation between B. cytotoxicus and other members of the B. cereus group. An extensive number of well-supported individual spectra from B. cytotoxicus , several other species of the B. cereus group, and 652 other microorganism species in a taxonomically wide range served as data sets. These were used to perform the validation for two database compilations: the commercial MALDI-Biotyper version K, and an extended compilation, including reference spectra from an open collection of spectra created by several users. Using both database combinations, the obtained values fulfil the formal criteria for the targeted identification of B. cytotoxicus in accordance with a national guideline for the validation of MALDI-TOF MS parameters in food analysis , with significant improvements due to the expanded database. By using 183 single spectra from 170 isolates, created in eleven different laboratories, the number of samples significantly exceeded the sample sizes for validation currently specified for ISO 16140–4:2020 for the validation of identification methods in food microbiology by single laboratories. Therefore, the method is suitable for routine application in an accredited laboratory context. • Bacillus cytotoxicus is accurately identified using MALDI-TOF MS. • Validation was performed using spectra from different institutions. • Obtained values fulfil formal criteria for the validation of identification methods.