Lag time variability in individual spores of Clostridium botulinum

Stringer, Sandra C.; Webb, Martin D.; Peck, Michael W.

doi:10.1016/j.fm.2010.03.003

Cited by 72 publications

(58 citation statements)

References 19 publications

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“…In addition, heat treatment of spores indeed only reduced the variability when spores were subsequently exposed to 0.75 mM HSA at pH 5.5. This reduced heterogeneity in germination and outgrowth kinetics, introduced by a relatively mild heat pretreatment of spores, is in line with previous findings in other sporeformers where mild heat treatment of spores made the distributions smaller in Bacillus subtilis (9), whereas more severe lethal heat treatment widened the distribution not only for B. subtilis (9), but also for Clostridium botulinum (14). We demonstrated now for B. cereus that mild heat pretreatment only affected the heterogeneity in outgrowth when spores were exposed to rather stressful conditions, like 0.75 mM HSA at pH 5.5.…”

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confidence: 91%

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

Besten

Melis

Sanders

et al. 2012

Appl Environ Microbiol

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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...

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supporting

confidence: 91%

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

Besten

Melis

Sanders

et al. 2012

Appl Environ Microbiol

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“…In contrast, times for the actual release of Ն90% of a spore's CaDPA once its rapid release begins (⌬T release s) are relatively constant for spores of any particular strain/species (8)(9)(10). Although all reasons for the heterogeneity in spores' nutrient germination are not fully understood, an important factor in this heterogeneity is variation in the number of GRs between individual spores (11).…”

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Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Kong

Doona

Setlow

et al. 2014

Appl Environ Microbiol

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Germination of Bacillus spores with a high pressure (HP) of ϳ150 MPa is via activation of spores' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phasecontrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ϳ150 MPa and 37°C but individual spores' germination kinetics were heterogeneous; (ii) individual spores' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (T lag ) prior to a period of the rapid release (⌬T release ) of the spores' dipicolinic acid in a 1:1 chelate with Ca 2؉ (CaDPA); (iii) spore germination at 50 MPa had longer average T lag values than that at ϳ150 MPa, but the ⌬T release values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ⌬T release values ϳ15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average T lag values; and (v) the germination of wild-type spores given a >30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for >35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ϳ150 MPa for <30 s is sufficient to fully activate spores' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.

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“…1A). In contrast, times (⌬T release ) for the actual release of Ն90% of a spore's CaDPA once its rapid release begins are relatively constant for individual spores (18)(19)(20). It is not known precisely what causes the variability in the T lag times for individual spores in populations, although factors that affect the commitment times of spore populations and T lag times of individual spores include spore heat activation, germinant concentrations, and GR levels (15,19,21).…”

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Monitoring of Commitment, Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Zhang

Liang

et al. 2014

J Bacteriol

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bShort exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ϳ7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T release ) greater than the time of germinant removal (T b ) increased, while the time T lag ؊ T b , where T lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (⌬T release ‫؍‬ T release ؊ T lag ) were increased; Factors affecting average T release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.

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Lag time variability in individual spores of Clostridium botulinum

Cited by 72 publications

References 19 publications

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

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

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Monitoring of Commitment, Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

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