Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: Phenotype and transcriptome analysis

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%

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

“…We previously investigated the impact of sorbic acid (SA) on germination and outgrowth of B. cereus spores for a population as a whole (18), but heterogeneity in outgrowth between individual spores in the population was not quantified and will affect the germination and outgrowth profile. Furthermore, it is well documented that heat treatments can trigger activation of spores and accelerate germination (7) and thereby can influence outgrowth dynamics and possibly also heterogeneity.…”

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confidence: 99%

“…Therefore, we assessed the impact of SA on germination and outgrowth kinetics at both the population level and single-cell level for spores that were heat shocked (HS) prior to initiation of germination. For that purpose, B. cereus ATCC 14579 spores were harvested from cultures grown in defined, minimal sporulation medium (4) and prepared as described previously (18) with the following modification: the Tween 80 concentrations were reduced from 0.1% to 0% in five daily washing steps. Pure spore crops devoid of vegetative cells and debris were stored in phosphate-buffered saline (PBS; pH 7) at 4°C for at least 2 weeks and not more than 8 weeks until use.…”

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confidence: 99%

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Besten

Melis

Sanders

et al. 2012

dPopulation heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth. Bacillus cereus is a food-spoiling and food-poisoning sporeforming organism that can be found in a large variety of foods and food ingredients (10). It has the ability to survive in harsh environments because it can form endospores that are resistant to heat, dehydration, and other physical and/or chemical stresses, and these spores may germinate and grow when conditions become more favorable. Thermal preservation methods that ensure complete inactivation of highly stress-resistant spores will negatively affect food quality, and the current trend toward milder preservation methods advocates application of mild food preservation factors, including mild heat treatments combined with addition of weak organic acids (11), to delay or inhibit germination and outgrowth of spores and cells.We previously investigated the impact of sorbic acid (SA) on germination and outgrowth of B. cereus spores for a population as a whole (18), but heterogeneity in outgrowth between individual spores in the population was not quantified and will affect the germination and outgrowth profile. Furthermore, it is well documented that heat treatments can trigger activation of spores and accelerate germination (7) and thereby can influence outgrowth dynamics and possibly also heterogeneity. To date, the impact of heat treatment on germination and outgrowth kinetics has been described at the population level (for example, see references 8 and 16), but heterogeneity at the single-cell level has not been studied in combination with mild (acidic) stress factors to control the germination and outgrowth of B. cereus spores. Therefore, we assessed the impact of SA on germination and outgrowth kinetics at both the population level and single-cell level for spores that were heat shocked (HS) prior to initiation of germination. For that purpose, B. cereus ATCC 14579 spores were harvested from cultures grown in defined, minimal sporulation medium (4) and prepared as described previously (18) with the following modification: the Tween 80 concentrations were reduced from 0.1% to 0% in five daily washing steps. Pure spore crops devoid of vegetative cells and debris were stored in phosphate-buffered saline (PBS; pH 7) at 4°C for at least 2 weeks and not more than 8 weeks until use. Spore germination was assessed under either unstressed conditions (brain heart infusion [BHI] buffered with 100 mM PBS [bBHI]; pH 7) or under mild acid stress (bBHI at pH 5.5 or bBHI at pH 5.5 with supplementation with 0.75 mM undissociated sorbic acid [HSA]). Spore germination was monitored by the transition of phase-bright spores turning phase dark, which is obs...

“…We previously assessed the impact of SA on germination and outgrowth of Bacillus cereus spores (16) and reported that at concentrations of 3 mM undissociated sorbic acid (HSA) and higher, amino acid-and inosineinduced spore germination was blocked, with spores remaining fully refractile. An earlier study on Clostridium botulinum and B. cereus spores suggested that the mode of action of sorbic acid was by competitive inhibition on the L-alanine/inosine receptor (13).…”

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confidence: 99%

“…Spore preparations of the B. cereus ATCC 14579 wild type and its gerR deletion mutant were prepared for germination assays as described previously (6,16). In short, heat-activated spores (70°C for 15 min) of both the wild type and gerR mutant were resuspended in morpholineethanesulfonic acid (MES) buffer corresponding to the test conditions prior to addition to a microtiter plate.…”

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confidence: 99%

Impact of Sorbic Acid on Germinant Receptor-Dependent and -Independent Germination Pathways in Bacillus cereus

Melis

Groot

Abee

2011

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