Quantification methods for Bacillus cereus vegetative cells and spores in the gastrointestinal environment

Abstract

There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments.