Historical and Contemporary NaCl Concentrations Affect the Duration and Distribution of Lag Times from Individual Spores of Nonproteolytic
            <i>Clostridium botulinum</i>

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In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (t germ ), one mature cell (t C1 ), and two mature cells (t C2 ) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the t germ , the outgrowth time (t C1 ؊ t germ ), and the first doubling time (t C2 ؊ t C1 ). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (t C1 ) and outgrowth (t C1 ؊ t germ ) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.

Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic Clostridium botulinum Bacteria

Stringer

Webb

Peck

2009

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“…The observed heterogeneity is the sum of heterogeneity during germination, the first doubling phases, and further outgrowth and is likely to result from multiple sources (6,15). For C. botulinum, it has been shown that the first spore that germinated was not the first to develop into a vegetative cell, also under stress conditions, highlighting the lack of correlation between germination and outgrowth times (12)(13)(14)19). The most important source of variability in outgrowth might depend on the relative magnitude of the contributions of the different phases in outgrowth.…”

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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...

“…CO 2 concentration affected spore coat protein gene expression, especially that of CBO0114 (similar to Bacillus subtilis YtaA and Clostridium tetani spore coat protein (16). This marked effect may influence properties of the spore coat and thereby spore resistance and the time to emergence for germinated spores (31,32,35). Spore germinant receptor proteins are usually encoded by tricistronic operons, of which there are three in the ATCC 3502 genome (28).…”

Section: Resultsmentioning

confidence: 99%

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Artin

Mason

et al. 2010

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The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO 2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO 2 had little effect on any of these parameters. At all CO 2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.Proteolytic Clostridium botulinum and nonproteolytic C. botulinum are the two neurotoxigenic clostridia most frequently associated with food-borne botulism (22,23,24), an intoxication caused by consumption of contaminated food containing preformed botulinum neurotoxin. The botulinum neurotoxins are the most potent toxins known, with as little as 30 ng being potentially fatal (24, 25). There are seven botulinum neurotoxin subtypes (A to G) and also a number of subtypes, of which subtype A1 has received the most attention to date (25 Carbon dioxide is frequently used as an antimicrobial gas in modified atmosphere packaging (MAP) of food (14,22,23). However, strains of nonproteolytic C. botulinum types B and E, despite having increased lag time and reduced growth rate (13, 15), display an increase in both neurotoxin gene expression and neurotoxin formation with higher CO 2 concentrations (5, 20). The purpose of the present study was to quantify the effect of CO 2 concentration on the growth, relative gene expression, and neurotoxin production by proteolytic C. botulinum type A1 strain ATCC 3502. Relative expression of cntA/A1 and cntR was measured using a new quantitative reverse transcription-PCR (qRT-PCR) method, and a DNA microarray was used to study the transcriptome. MATERIALS AND METHODSCulture conditions and growth parameters. Overnight cultures of proteolytic C. botulinum type A1 strain ATCC 3502 were grown in TPY medium flushed with the appropriate gas mixture (10%, 35%, or 70% CO 2 ; 10% H 2 ; and the balance N 2 [by vol]) and used to inoculate flasks of similarly treated TPY-C broth (5). The initial pH values of the media were 6.8 to 6.4, reflecting the increasing CO 2 concentration. Cultures were grown at 37°C in an anaerobic workstation with the same atmosphere, each in triplicate. Samples for RNA and neurotoxin measurement were withdrawn at four to nine time points during the experiment. Maximum specific growth rates [(OD)] of the change in optical...