Germination and outgrowth of spores of Bacillus cereus group members: Diversity and role of germinant receptors

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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.

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confidence: 99%

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confidence: 99%

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

Warda

Xiao

Boekhorst

et al. 2017

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“…Over the last decades, research concerning this problematic microorganism has focused on various different research disciplines, including toxin production and virulence (1,2), resistance mechanisms against applied stresses (3,4), cell structure, metabolism (5,6), and cellular developments, such as cell division, biofilm formation (7), sporulation (8), and spore germination (9). In this way, B. cereus is becoming an increasingly studied model organism for Gram-positive pathogenic bacteria next to its well-known but fairly distant and nonpathogenic relative Bacillus subtilis.…”

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confidence: 99%

Live-Cell Imaging Tool Optimization To Study Gene Expression Levels and Dynamics in Single Cells of Bacillus cereus

Eijlander

Kuipers

2013

Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium Bacillus cereus specifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of single B. cereus cells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stageand cell compartment-specific gene expression in B. cereus. With a case study concerning cotD expression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show that cotD is not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.

“…Recovery is a complex phenomenon, comprising germination of spores, restoration of metabolic activity in suboptimal or favorable conditions and emergence of the first vegetative cell able to multiply. The incubation temperature during storage and food pH are among factors that will deeply influence the recovery of surviving spores (3).…”

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confidence: 99%

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

Trunet

Mtimet

Mathot

et al. 2015

eThe apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (␦ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.T he multiplication of spore-forming bacteria in foods can cause poisoning and/or spoilage. The heat process applied to foods (from mild in cooked and refrigerated foods to very intense in canned or ultrahigh-temperature foods) creates a positive selection of spore-forming species of bacteria because of the high resistance of their spores (1). Therefore, control of spore-forming bacteria in foods first of all requires inactivation of dormant spores by heat (or by any other appropriate inactivation treatment). The extent of inactivation depends on a number of factors, naturally including the inactivation process intensity and more importantly the spore resistance properties at the time of treatment, which may vary with the conditions and environment of sporulation (2). Respect for the organoleptic quality of food may limit the intensity of the process and therefore the extent of spore inactivation. Controlling the recovery of surviving spores in processed food strengthens the safety and stability level achieved after the inactivation process. Recovery is a complex phenomenon, comprising germination of spores, restoration of metabolic activity in suboptimal or favorable conditions and emergence of the first vegetative cell able to multiply. The incubation temperature during storage and food pH are among factors that will deeply influence the recovery of surviving spores (3).Spore recovery is influenced by multiple physical and (bio) chemical factors, such as temperature, pH, and water activity (a w ) and by the presence of germinants (such as amino acids, ribosides, and minerals) or enzymes, such as lysozyme (4,7,8,37). The...