Historical and contemporary NaCl concentrations affect the duration and distribution of lag times from individual spores of nonproteolytic clostridium botulinum

In this study we determined the effect of NaCl concentration during sporulation (0 or 3.0% [wt/vol] added NaCl) and subsequent growth (0 or 2.0% [wt/vol] added NaCl) on the distributions of times associated with various stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B. The effects of NaCl on the probability of germination and the probability of subsequent growth were also determined. Spore populations exhibited considerable heterogeneity at all stages of lag phase for each condition tested. Germination time did not correlate strongly with the times for later stages in the lag phase, such as outgrowth and doubling time. Addition of NaCl to either the sporulation or growth media increased the mean times for, and variability of, all the measured stages of the lag phase (germination, emergence, time to one mature cell, and time to first doubling). There was a synergistic interaction between the inhibitory effects of NaCl in the sporulation medium and the inhibitory effects of NaCl in the subsequent growth medium on the total lag time and each of its stages. Addition of NaCl to either the sporulation medium or the growth medium reduced both the probability of germination and the probability of a germinated spore developing into a mature cell, but the interaction was not synergistic. Spores formed in medium with added NaCl were not better adapted to subsequent growth in suboptimal osmotic conditions than spores formed in medium with no added NaCl were. Knowledge of the distribution of lag times for individual spores and quantification of the biovariability within lag time distributions may provide insight into the underlying mechanisms and can be used to improve predictions of growth in food and to refine risk assessments.     

Heterogeneity of times required for germination and outgrowth from single spores of nonproteolytic Clostridium botulinum

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t(germ)), time to emergence (t(emerg)), and times to reach the lengths of one (t(C1)) and two (t(C2)) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t(germ)), emergence (t(emerg) - t(germ)), cell maturation (t(C1) - t(emerg)), and doubling (t(C2) - t(C1)) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.     

Effect of gramicidin C on the formation and germination of Bacillus brevis var. GB (P+-variant) spores]

The effect of gramicidin C added to the medium at various periods of cultivation in concentrations of 20, 40 and 100 gamma/ml on sporulation of P+-variant of Bac. brevis var. GB was studied. The most effective increase in the sporulation rate and percentage of the cells germinating into the spores was observed on addition of the antibiotic to the medium in amounts of 20 and 40 gamma/ml in 13 hours of the culture development. The amount of gramicidin C during sporulation decreased and partially passed into the spores which did not differ after germination from those of P+-variant grown on the synthetic medium with glucose and without preliminary addition of the antibiotic. Addition of gramicidin C in an amount of 100 gamma/ml at the end of the lag phase, i.e. 4 hours after the culture inoculation suppressed sporulation and had no effect on growth of the cells of its own producing organism.     

Heterogeneity of Times Required for Germination and Outgrowth from Single Spores of Nonproteolytic Clostridium botulinum

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t_germ), time to emergence (t_emerg), and times to reach the lengths of one (t_C1) and two (t_C2) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t_germ), emergence (t_emerg − t_germ), cell maturation (t_C1 − t_emerg), and doubling (t_C2 − t_C1) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.     

The rate of development of a microsporidan in moth cell culture.

A microsporidan parasite of the forest tent caterpillar Malacosoma disstria infected cells and replicated in vitro in a line from the moth Heliothis zea. After spore germination, the incidence of infected cells increased with time until leveling off with sporulation. During the first 24 hr, there was a static number of parasites, followed by a 2-day logarithmic growth phase during which the population doubled five to six times. The growth rate was 9 to 11 hr per population doubling. Sporulation commenced on day 3, and 40 to 50 spores were recovered from each infected cell. The life cycle was completed within 6 days, culminating in spores that were infectious for cultured cells. The antibiotic fumagillin at a dose of 1 ppm in the culture medium was microsporida-static.     

Influence of Carbohydrates on Growth and Sporulation of Clostridium perfringens Type A

Growth and sporulation of Clostridium perfringens type A in Duncan and Strong (DS) sporulation medium was investigated. A biphasic growth response was found to be dependent on starch concentration. Maximal levels of heat-resistant spores were formed at a starch concentration of 0.40%. Addition of glucose, maltose, or maltotriose to a sporulating culture resulted in an immediate turbidity increase, indicating that biphasic growth in DS medium may be due to such starch degradation products. Amylose and, to a lesser extent, amylopectin resulted in biphasic growth when each replaced starch in the sporulation medium. A levels of heat-resistant spores approximately equal to the control was produced with amylopectin but not amylose as the added carbohydrate. Addition of glucose or maltose to a DS medium without starch at stage II or III of sporulation did not alter the level of heat-resistant spores as compared with the level obtained in DS medium with starch. Omission of starch or glucose or maltose resulted in an approximately 100-fold decrease in the number of heat-resistant spores, although the percentage of sporulation (90%) was unaffected. The role of starch and amylopectin in the formation of heat-resistant spores probably involves the amyloytic production of utilizable short-chain glucose polymers that provide an energy source for the completion of sporulation.     

Effects of Temperature on Activation, Germination, and Outgrowth of Bacillus megaterium Spores

The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.     

The effect of coenzyme leakage and replacement on the growth and metabolism of two fusobacteria.

Suspensions in water of two species of Fusobacterium leaked several coenzymes when incubated at normal growth temperatures. Chromatography of filtrates from these suspensions revealed the presence of NAD, NADP, FMN, tetrahydrofolic acid and, in one of the two, pyridoxal phosphate. Analyses of some enzymic activities in whole organisms demonstrated deficiencies in coenzymes:glutamate dehydrogenase was virtually inactive in the absence of added NAD; tryptophanase activities were diminished by washing but the extent differed between strains; histidase activity was not decreased by washing or suspension in water or saline. Both lag phase and doubling time increased markedly in severely washed organisms inoculated into fresh medium. Addition of appropriate coenzymes shortened the lag phase for both strains and shortened the doubling time in one.     

The effect of sodium chloride and temperature on the rate and extent of growth of Clostridium botulinum type A in pasteurized pork slurry

A selective medium was used to enumerate Clostridium botulinum growing in the presence of natural spoilage organisms in a model cured pork slurry. The growth responses of a mixed spore inoculum of six strains of Cl. botulinum type A were studied at 15 degrees, 20 degrees and 27 degrees C with 1.5, 2.5, 3.5 or 4.5% (w/v) salt added (aw range 0.961-0.990). Gompertz and logistic curves, which have a sigmoid shape, were fitted to the data and lag times, growth rates, generation times and time to maximum growth rates were derived. Variation in germination rates of the spores occasionally gave a falsely extended lag time resulting in an exceptionally high estimate for growth rate. Products containing 4.5% (w/v) NaCl would be capable of supporting growth of proteolytic strains of Cl. botulinum, even at 15 degrees C, although the lag period would be extended. In products where absence of Cl. botulinum cannot be assured additional preservative measures are essential. The information obtained provides a framework to investigate the effects of a wider range of additives or variables on the growth responses of Cl. botulinum.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798.

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

The effect of sodium chloride and temperature on the rate and extent of growth of Clostridium botulinum type A in pasteurized pork slurry

A selective medium was used to enumerate Clostridium botulinum growing in the presence of natural spoilage organisms in a model cured pork slurry. The growth responses of a mixed spore inoculum of six strains of Cl. botulinum type A were studied at 15°, 20° and 27°C with 1˙5, 2˙5, 3˙5 or 4˙5% (w/v) salt added (a_w range 0961–0990). Gompertz and logistic curves, which have a sigmoid shape, were fitted to the data and lag times, growth rates, generation times and time to maximum growth rates were derived. Variation in germination rates of the spores occasionally gave a falsely extended lag time resulting in an exceptionally high estimate for growth rate. Products containing 4˙5% (w/v) NaCl would be capable of supporting growth of proteolytic strains of Cl. botulinum , even at 15°C, although the lag period would be extended. In products where absence of Cl. botulinum cannot be assured additional preservative measures are essential. The information obtained provides a framework to investigate the effects of a wider range of additives or variables on the growth responses of Cl. botulinum .     

Effects of different spices used in production of fermented sausages on growth of and curvacin A production by Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A. The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages. The influence on the growth characteristics and especially on the kinetics of curvacin A production with L. curvatus LTH 1174 was evaluated. Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it. The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production. The maximum attainable biomass concentration (X(max)) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increased X(max). Nutmeg decreased both growth and bacteriocin production considerably. Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant. Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium. Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing.     

Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.

Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.     

Germination properties as marker events characterizing later stages of Bacillus subtilis spore formation

At various stages during spore formation sporangia were shocked by cold treatment or with toluene, and the germination requirements of the prespores were examined. Up to 5 h after induction of sporulation (t5) germination was spontaneous; i.e., it occurred without any added germinants. After t5, during stages V and VI, the capacity for spontaneous germination diminished progressively, and the spores acquired a need for externally added germinants. At t6 this need was satisfied by either L-alanine or a mixture of KCl, glucose, and fructose. By t8, the latter response had disappeared. The spores germinated only with L-alanine, and the response was much slower. Experiments with chloramphenicol showed that the germination properties of the spores appearing between t6 and t8 were the expression of events in protein synthesis that had occurred before t5. Although the germination requirements developed at about the same time as heat resistance, they could be dissociated from heat resistance in wild-type and mutant cells. The germination properties of the developing spores are additional marker events characterizing the later stages of sporulation, as follows: (i) spontaneous germination (up to the end of stage IV); (ii) germination requirements that are satisfied by KCl-glucose-fructose or L-alanine (stage V); and (iii) slow germination response with L-alanine only (stage VI).     

Achievement of complete Bacillus subtilis microcycle sporulation by the addition of S-adenosylmethionine and phospholipids.

In an attempt to find factors that may be responsible for the initiation of sporulation, a system in which the germination and outgrowth phases were separate was applied to Bacillus subtilis. Outgrowth of the germinated spores to only the primary singlet cells was followed in chemically defined medium. Addition of specific metabolites induced the primary singlet cells to sporulate via microcycle sporulation. Experiments are described that led to complete sporulation by the addition of diaminopimelic acid, S-adenosyl-L-methionine and phosphatidylethanolamine.     

Expression of glycosidase activities during germination of Dictyostelium discoideum spores.

Several lysosomal glycosidase activities were examined in vitro during heat-induced germination of Dictyostelium discoideum spores and were found not to be coordinately controlled. The level of beta-glucosidase activity increased significantly during the emergence stage of germination. Both alpha-glucosidase and N-acetyl-beta-glucosaminidase activities remained relatively constant until postemergence, when they increased slightly; alpha-mannosidase activity decreased during all stages of germination. The activity of beta-galactosidase increased slightly during spore swelling, fell below the level initially found in spores at zero time, and increased slightly during postemergence. The expression of all of these enzyme activities, except the increase in beta-galactosidase, appeared to require protein synthesis. Spores in the lag phase of germination which were exposed to severe environmental stress were deactivated and exhibited reduced levels of alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase activities. Prolonged heat activation treatment reduced the levels of lysosomal glycosidase activities in postactivated spores but did not change the subsequent enzyme patterns during the spore-swelling and emergence stages of germination.