Isolation and Enumeration of the Bacillus cereus Group Using a Chromogenic Substrate that Targets Phosphatidylcholine Phospholipase C Activity

Abstract

The Bacillus cereus group is comprised of diverse yet closely related species that are ubiquitous in nature. These Gram-positive, spore-forming bacteria are commonly isolated as potential pathogens in environmental and food samples, and they are also beneficially used in industrial applications such as probiotics or agricultural pesticides. Although phylogenetic and genomic analyses identified eight formally recognized species within the Bacillus cereus group, only five members are currently acknowledged using standardized isolation procedures. Therefore, current enumeration procedures may be inadequate and inaccurate in determining the clinical importance of the B. cereus group and their prevalence in food and environmental sources. In this investigation, the R & F® Bacillus cereus Group Chromogenic Plating Medium (BCG) was developed to target the enzyme phosphatidylcholine phospholipase C (PC-PLC) found widely distributed among the B. cereus group species. The performance evaluations of the plating medium demonstrated a 100% plating productivity ratio, inclusivity, and exclusivity when compared with Trypto Soy Agar (TSA). B. cereus group species, including B. anthracis, B. cereus sensu stricto (s.s.), B. cytotoxicus, B. pseudomycoides, B. mycoides, B. thuringiensis, B. toyonensis, B. weidmannii, and B. weihenstephanensis, formed turquoise-colored colonies on the BCG agar, indicating PC-PLC activity after 24–28 h of incubation at the selected optimal temperature of 35 °C. Furthermore, no significant difference was observed between BCG and TSA when enumerating B. cereus s.s. and B. cytotoxicus artificially inoculated in whey protein powder and ultra-high temperature milk after long-term storage.