Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains

Abstract

Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat activation of spores, germination in complex media (brain heart infusion [BHI] and tryptone soy broth [TSB]), and exposure to saturating concentrations of single amino acids and the combination of alanine and inosine. Whole-genome sequence comparison revealed a total of 11 clusters of operons encoding germinant receptors (GRs): GerK, GerI, and GerL were present in all strains, whereas GerR, GerS, GerG, GerQ, GerX, GerF, GerW, and GerZ (sub)clusters showed a more diverse presence/absence in different strains. The spores of tested strains displayed high diversity with regard to their sensitivity and responsiveness to selected germinants and heat activation. The two laboratory strains, B. cereus ATCC 14579 and ATCC 10987, and 11 food isolates showed a good germination response under a range of conditions, whereas four other strains (B. cereus B4085, B4086, B4116, and B4153) belonging to phylogenetic group IIIA showed a very weak germination response even in BHI and TSB media. Germination responses could not be linked to specific (combinations of) GRs, but it was noted that the four group IIIA strains contained pseudogenes or variants of subunit C in their gerL cluster. Additionally, two of those strains (B4086 and B4153) carried pseudogenes in the gerK and gerR I (sub)clusters that possibly affected the functionality of these GRs.IMPORTANCE Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

The Bacillus cereus Food Infection as Multifactorial Process

“…Nevertheless, the majority of studies investigating B. cereus spore germination target spores present in foods, to either trigger germination in foodstuffs to eliminate germinated spores/vegetative cells or to completely avoid germination and outgrowth in foods [10,[277][278][279][280][281]. B. cereus spores can be triggered by nutrient-rich media, by amino acids such as alanine, cysteine, threonine or glutamine, by the purine ribonucleosides inosine and adenosine, by sugars, by heat treatment, or by their combination [279,[282][283][284][285]. Only a small number of publications focus on germination of B. cereus spores under (simulated) gastrointestinal conditions.…”

Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

“…There are three GRs: GerA responding to L-alanine, and GerB and GerK that collaborate in responding to L-asparagine, D-glucose, D-fructose and potassium chloride [10]. In contrast, there are seven functional GRs-GerR, GerK, GerG, GerL, GerQ, GerI and GerS-in B. cereus ATCC 14579 spores [22]. In particular, GerR, with the order GerRA-GerRC-GerRB, has a critical role in spore germination in response to L-alanine, or inosine alone and in food products, for example meat broth and rice water [23].…”

Effect of sublethal heat treatment on the later stage of germination‐to‐outgrowth of Clostridium perfringens spores

“…Consistent with this, heat activation has not been observed with C. difficile spores, which use an atypical GR to initiate germination (18,19). Additionally, responsiveness to heat activation in some strains of Bacillus may correlate with the genetic characteristics of GRs (20,21). Taken together, these findings suggest that the effect of heat activation on Bacillus species is probably mediated by a structural change in GR proteins or the inner spore membrane that makes GRs more accessible or receptive to nutrient-induced germination (11).…”

“…Examination of the effect of sublethal heating on spore germination in a GerKC‐deficient isogenic mutant may therefore reveal additional mechanisms. Given that we have observed that responsiveness to heat treatment differs among several C. perfringens clinical isolates (our unpublished observation in 2017), better understanding of inter‐strain variations associated with different GerKC amino acid sequences would also assist in elucidating the mechanisms of heat activation .…”

“…c–f) because it is suspected that spores’ responses to sublethal heating differ when stimulated by different germination inducers. Differences between L‐cysteine and L‐glutamine as germinants are of interest for several reasons: (i) these polar uncharged amino acids are reportedly among the most effective germinants for C. perfringens strains linked to FBI ; (ii) Warda and colleagues have reported that BHI medium does not contain detectable amounts of cysteine or glutamine as free amino acid constituents ; and (iii) a previous study analyzing germination at single‐cell resolution only used KCl and L‐asparagine as germinants . Both amino acids stimulated germination of SM101 and NCTC8239 spores (Fig.…”