Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells

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...

supporting

confidence: 91%

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

Besten

Melis

Sanders

et al. 2012

“…If not stated otherwise, spores were heat activated at 80°C for 10 min or at 70°C for 30 min and subsequently cooled on ice and washed again with cold water. Heat-activated spores were diluted to a final optical density at 600 nm (OD 600 ) of 1 in 200 l of BHI or Luria-Bertani (LB) medium supplemented with vitamin B 12 (1 mg/liter) and chloramphenicol (7.5 mg/liter) to prevent outgrowth of vegetative cells (34,35). Alternatively, spores were diluted in mixtures of various nutrient-based germinants dissolved in 25 mM Tris-HCl, pH 7.4: (i) 100 mM L-alanine; (ii) L-asparagine, Dfructose, D-glucose, and KCl (all at 50 …”

Section: Methodsmentioning

confidence: 99%

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

Berendsen

Krawczyk

Klaus

et al. 2015

c High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrientinduced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D 120°C , 1.9 ؎ 0.2 and 1.3 ؎ 0.1 min; z value, 12.2 ؎ 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients. Bacillus endospores (spores) are widely present in nature and may contaminate food ingredients and food products. Due to the intrinsic stability of spores, which allows them to withstand environmental insults, sufficient inactivation of spores in commercially sterile food products is a major challenge for the food industry (1-4).Bacillus thermoamylovorans produces spores with high-level heat resistance (3), and the spores are known to survive industrial food sterilization processes. The organism is facultatively anaerobic and has the ability to grow at temperatures between 40°C and 58°C (5, 6). In our experience, strains of B. thermoamylovorans are able to grow at 37°C but not at 30°C. The organism was first described as a nonsporogenous species (5, 7), but in an amended species description the formation of spores was reported (8). The occurrence of B. thermoamylovorans in a gelatin production plant and at dairy farms has been reported (3, 9). The genome sequence of one non-food-related B. thermoamylovorans strain from a biogas plant was published recently (6). Overall, the species has not been well characterized, and little is known about the spore properties that are important for control in foods, including spore resistance to various processing conditions and germination of spores that survive.When spores exit dormancy via germination, food spoilage can occur upon outgrowth. These processes have been well studied in Bacillus subtil...

“…High spore wet heat resistance is acquired late in sporulation, largely in parallel with the uptake of DPA by the developing forespore and the final decrease in spore core water content. However, the precise time of acquisition of full spore heat resistance during sporulation is not completely clear because (i) precise measurements of wet heat resistance are usually carried out only on purified spores; (ii) analysis of acquisition of wet heat resistance can be carried out only with spore populations, and the lack of precise synchrony in sporulation of cell populations can complicate kinetic analysis of events in sporulation; and (iii) the specific conditions in sporulating cultures, including the composition of the medium and the environment in the mother cell surrounding the developing spore, may change during sporulation, and such changes could modify spore wet heat resistance compared to that of purified spores in water.It is also clear that the wet heat resistance of individuals in spore populations can vary significantly (1,3,9,13,19,27,34,36). Indeed, analyses of spore killing by wet heat as a function of time often suggest that there is a significant level of more wet-heat-resistant spores in populations (27).…”

mentioning

confidence: 99%

“…It is also clear that the wet heat resistance of individuals in spore populations can vary significantly (1,3,9,13,19,27,34,36). Indeed, analyses of spore killing by wet heat as a function of time often suggest that there is a significant level of more wet-heat-resistant spores in populations (27).…”

mentioning

confidence: 99%

Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation

Sánchez-Salas

Setlow

Zhang

et al. 2011

The first ϳ10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ϳ24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca 2؉ but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.Spores of Bacillus and Clostridium species are dormant and extremely resistant to a variety of agents, including high temperatures, radiation, and many chemicals (19,31). This extreme spore resistance has major applied implications, because spores of Bacillus and Clostridium species are vectors for food spoilage and a number of diseases, some of which are food borne. As a consequence, there is much interest in the mechanisms of spore resistance as well as methods for spore inactivation.The most commonly used method for spore inactivation is wet heat treatment, which is extremely effective, even though spores are generally resistant to temperatures ϳ40°C higher than their growing-cell counterparts (4,19,31). Factors involved in spores' extreme resistance to wet heat include (i) the protection of spore DNA by its saturation with the ␣/␤-type small, acid-soluble spore proteins (SASPs); (ii) the thick proteinaceous spore coat layer; (iii) the thick layer of cortex peptidoglycan surrounding the spore core; (iv) the high level of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and its associated divalent cations, mostly Ca 2ϩ , in the spore core; and, most importantly, (v) the low water content of the core of spores suspended in water (4,5,19,31,32). High spore wet heat resistance is acquired late in sporulation, largely in parallel with the uptake of DPA by the developing forespore and the final decrease in spore core water content. However, the precise time of acquisition of full spore heat resistance during sporulation is not completely clear because (i) precise measurements of wet heat resistance are usually carried out only on purified spores; (ii) analysis of acquisition of wet heat resistance can be carried out only with spore populations, and the lack of precise synchrony in sporulation of cell populations can complicate ...