Bacillus cereus spore damage recovery and diversity in spore germination and carbohydrate utilisation

Bacterial spores are extremely robust survival vehicles that are highly resistant towards environmental stress conditions including heat, UV radiation and other stresses commonly applied during food production and preservation. Spores, including those of the toxin-producing food-borne human pathogen Bacillus cereus, are ubiquitously present in a wide range of environmental niches such as soil, plant rhizosphere, intestinal tract of insects and animals, and it is virtually impossible to prevent contamination at the primary production level. Heat treatments are conventionally applied in food processing to reduce the microbial load of food products, however, to comply with consumer desire for products with higher sensory and nutritional values, the treatment intensity may become milder. Consequently, subpopulations of spores may emerge that are sublethally damaged rather than inactivated conceivably causing quality and safety issues following repair and outgrowth. In this thesis, a functional genomics approach was used in combination with subpopulation and single spore analysis to identify factors involved in recovery of heat damaged spores, and to link B. cereus genotypes to nutrient-induced germination capacity and carbohydrate utilisation capacity. Using comparative analysis of B. cereus ATCC 14579 wild type and targeted mutants, putative damage repair factors were identified such as putative transcriptional regulator CdnL, that supported recovery of spores in a range of conditions including model foods. The majority of identified genes encoding putative damage repair factors appeared to be unique for B. cereus group members. This novel information on spore recovery adds to further insights in versatility of survival strategies of B. cereus. Different types of foods may contain different types and levels of nutrients including amino acids and carbohydrates, that can affect spore germination capacity and subsequent outgrowth performance of vegetative B. cereus cells. Nutrient germinants present in food products can trigger specific germinant receptors (GRs) located in the spore inner membrane leading to spore germination, a critical step before growth resumes. Combined analysis of genotypes and nutrient-induced germination phenotypes using high throughput flow cytometry analysis at the level of individual spores, revealed substantial diversity in germination capacity with a subset of strains showing a very weak germination response even in nutrient-rich media containing high levels of amino acids. Phylogenetically, these B. cereus strains grouped in subgroup IIIA encompassing strains containing pseudogenes or variants of some of the Ger clusters and two strains containing the recently identified SpoVA2mob transposon, that induced heat resistance with concomitant reduced germination response in Bacillus subtilis spores. The same B. cereus isolates were also used to link genotypes with carbohydrate utilisation clusters present on the genomes, and this revealed representatives of subgroup IIIA to lack specific carbohydrate utilisation clusters (starch, glycogen, aryl beta-glucosides; salicin, arbutin and esculin) suggesting a reduced capacity to utilise plant-associated carbohydrates for growth. Since these B. cereus subgroup IIIA representatives contain host-associated carbohydrate utilisation gene clusters and a subset of unique Ger clusters, their qualification as poor germinators may require revision following assessment of spore germination efficacy using host-derived compounds as germinants. The research described in this thesis has added novel insights in B. cereus capacity to cope with spore damage and provided novel overviews of the distribution and putative functionality of (sub)clusters of GRs and carbohydrate utilisation clusters. Knowledge on spore damage repair, germination and metabolism capacity adds to further understanding of B. cereus ecology including niche occupation and transmission capacity.