Abstract
In this study, the performance of four alternative selective chromogenic B. cereus agar was compared to the reference mannitol-yolk polymyxin (MYP) agar (ISO 7932) using inclusion and exclusion test strains (n = 110) and by analyzing naturally contaminated milk and other food samples (n = 64). Subsequently, the panC group affiliation and toxin gene profile of Bacillus cereus senso lato (s.l.) isolates were determined. Our results corroborate that the overall best performing media CHROMagar™ B. cereus (93.6% inclusivity; 82.7% exclusivity) and BACARA® (98.2% inclusivity, 62.7% exclusivity) are more sensitive and specific compared to Brilliance™ B. cereus, MYP and ChromoSelect Bacillus Agar. Both media allow unequivocal detection of B. cereus with low risks of misidentification. Media containing ß-D-glucosidase for the detection of presumptive B. cereus may form atypical colony morphologies resulting in a false negative evaluation of the sample. Naturally contaminated samples presented high numbers of background flora, while numbers of presumptive B. cereus were below the detection limit (<10 CFU g−1 or mL−1). Recovery after freezing resulted in the highest detection of B. cereus s.l. on BACARA® (57.8%), CHROMagar™ B. cereus (56.3%) and MYP agar (54.7%). The panC/toxin profile combination IV/A was the most abundant (33.0%), followed by III/F (21.7%) and VI/C (10.4%). More panC and toxin combinations were present in 15.6% of samples when reanalyzed after freezing. In order to improve detection and confirmation of B. cereus s.l. in food samples, we recommend the parallel use of two complementary selective media followed by molecular characterization (e.g., panC typing combined with toxin gene profiling). When determining psychrotolerant or thermophilic members of the B. cereus group, the selective agar media should additionally be incubated at appropriate temperatures (5 °C, ≥45 °C). If high-risk toxin genes (e.g., ces or cytK-1) are detected, the strain-specific ability to produce toxin should be examined to decisively assess risk.
Highlights
Bacillus senso latu (s.l.) consists of Gram-positive, rod-shaped, aerobic or facultative anaerobic spore-forming bacteria that are widespread in the environment and commonly found in soil, plant material and in the gut of insects [1,2,3]
Chromogenic reactions rely on enzymatic cleavage of a particular substrate and the release of a chromogen, which is more specific than conventional microbiological growth media
This study focused on the comparison of alternative chromogenic selective nutrient media to identify the best performer for B. cereus s.l. detection and enumeration
Summary
Bacillus senso latu (s.l.) consists of Gram-positive, rod-shaped, aerobic or facultative anaerobic spore-forming bacteria that are widespread in the environment and commonly found in soil, plant material and in the gut of insects [1,2,3]. In order to protect consumer health, a process criterion for presumptive B. cereus in infant formulas (≤500 colony forming units [CFU] g−1 ) was set within the Commission. Warning values are available, for example, for dried herbs and spices; tofu and bakery products (4 log CFU g−1 ); or fruits and vegetables, cereals, pasta, mayonnaises, dressings, soups and ready-to-eat instant products (3 log CFU g−1 ) (https://www.dghmricht-warnwerte.de/de/dokumente; accessed on 17 December 2021). Presumptive B. cereus s.l. is detected and enumerated on classical culture media as for example mannitol egg yolk polymyxin (MYP) agar. Selectivity is achieved in both media types by the addition of antibiotic substances (e.g., polymyxin B or trimethoprim), which inhibit the growth of undesirable Gram-positive and Gram-negative bacteria [11,12]
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