Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

Mechanism of chemical manipulation of the heat resistance of Clostridium perfringens spores

The mechanism(s) of chemical manipulation of the heat resistance of Clostridium perfringens type A spores was studied. Spores were converted to various ionic forms by base-exchange technique and these spores were heated at 95°C. Of the four ionic forms, i.e. Ca²⁺, Na⁺, H⁺ and native, only hydrogen spores appeared to have been rapidly inactivated at this temperature, when survivors were enumerated on the ordinary plating medium. However, the recovery of the survivors was improved when the plating medium was supplemented with lysozyme, and more dramatically when the heated spores were pretreated with alkali followed by plating in the medium containing lysozyme. In contrast to crucial damage to germination, in particular to spore lytic enzyme, no appreciable amount of DPA was released from the heat-damaged H-spores. These results suggest that a germination system is involved in the thermal inactivation of the ionic forms of spores, and that exchangeable cation load plays a role in protection from thermal damage of the germination system within the spore. An enhancement of thermal stability of spore lytic enzyme in the presence of a high concentration of NaCl was consistent with the hypothesis.     

AN AUTOMATED METHOD FOR QUANTIFYING THE L-ALANINE TRIGGER OF BACILLUS SUBTILIS SPORE GERMINATION AND COMPETITIVE INHIBITION BY D-ALANINE

We developed an automated method for studying the germination kinetics of Bacillus subtilis spores using a microtiter plate (MP) reader. Phosphate buffer supplemented with L-alanine was used to isolate the germination phase as determined by decrease in optical density (OD₆₃₀). Using a standard 96-well MP, L-alanine triggered germination kinetics were measured by automatic OD measurement every 3 min until the maximum OD₆₃₀ change (OD₆₃₀) was determined. When OD₆₃₀ values were plotted against L-alanine concentration on a double reciprocal plot, a straight line (R²= 0.98) was produced. The addition of D-alanine to the medium demonstrated classical competitive inhibition on double reciprocal plots. A 3-dimensional representation of the untransformed data showed the response surface nature of competitive inhibition. The method automates the tedious task of determining loss of refractility associated with spore germination under defined conditions so that inhibitors to germination can be studied. Since 96 OD₆₃₀ determinations can be done simultaneously in small volumes (200 μL) extensive data can be generated about inhibitors using relatively small spore crops in a single, short (1.4 h) incubation.     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Influence of NaCl, NaNO2 and oxygen on the germination and growth of Bacillus licheniformis, a spoilage organism of chub-packed luncheon meat

The thermal resistance of Bacillus licheniformis spores was increased from a D ₇₀-value of 590 min to one of 900 min by the addition of 4% NaCl to the heating medium [tryptone-yeast extract-glucose (TYG) broth, pH 6.8], but was decreased to 470 min in TYG broth acidified to pH 4.4. Sodium nitrite (0.02%) enhanced spore destruction at 80°C but not at 70°C; addition of 4% NaCl eliminated this effect. Less than half the number of spores surviving heat comparable to commercial cooking were heat-damaged to the extent of being unable to grow aerobically in the presence of 4% NaCl. No growth occurred during anaerobic incubation even when the media contained no added NaCl. Oxygen was not required to trigger spore germination, but trace amounts were needed for the successful outgrowth of germinated spores. Spore germination was accelerated and enhanced by the presence of at least 2% NaCl. Therefore under anaerobic conditions NaCl promotes microbiological stability because the germinated spores cannot develop further and become moribund. It is concluded that the plastic casing of luncheon-meat chubs is not sufficiently oxygen-impermeable to allow the product a long shelf-life other than at chill temperatures unless the chubs are stored in an oxygen-free atmosphere.     

Factors Affecting Germination and the Mode of Germination of Zygospores of Choanephora cucurbitarum

Treatment of zygospores of Choanephora cucurbitarum with KMnO₄, NaClO or H₂O₂ effectively activated the spores and induced their germination. The optimum concentration of KMnO₄ for activation of zygospores was 0.25 to 0.5%. Zygospores were not able to germinate in darkness even after activation by KMnO₄. When zygospores from 40 to 50-day-old cultures were treated with 0.5% KMnO₄ solution for 60 min before incubation on water agar at 24% under light, about 50% germinated in 10 days. KMnO₄ treatment killed more than 99% of residual mycelial fragments, sporangiospores and sporangiola in the zygospore suspension. During germination disappearance of oil droplets in zygospores occurred prior to the cracking on zygospore wall. Both sporangial germination and mycelial germination were found. Moreover, sporangiole germination was observed for the first time.     

The Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores and Changes in the Oxidation-Reduction Potential of Cultures

S ummary . The E_h fall observed during incubation of Clostridium butyricum spores occurred during germination and emergence, not during the log phase; it is attributed to the H₂ tension resulting from metabolism. When the O₂ tension in the medium was increased, the E_h fell only after a few spores outgrew and replicated; germination of remaining spores then followed. It is suggested that the few cells able initially to metabolize can (a) elaborate NADH etc. which reduce the O₂ tension to a level non-inhibitory for the remaining spores, and (b) produce the H₂ tension recorded by the Pt electrode.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Effects of cyanide, salicylhydroxamic acid, and temperature on respiration and germination of spores of the fern Sphaeropteris cooperi

During the first 96 h of culture, germinating spores of the fern Sphaeropteris cooperi (F. v. Muell.) Tryon showed a gradual rise in respiratory activity to a maximum of about 6.5 μl 0₂ h⁻¹ mg⁻¹ dry wt. This was followed by a transitory decline in rate, concluded by a second respiratory rise preceding the emergence of the rhizoid after 192 h of culture. Oxygen uptake during the first 120 h of germination was insensitive to 1 m M potassium cyanide (KCN) but was inhibited by 1 m M salicylhydroxamic acid (SHAM); however, beyond this time cyanide showed increasing inhibitory effectiveness whereas SHAM became less effective. Regardless of time of application, KCN had no effect on germination. Maximum inhibition of germination by SHAM was achieved if applied up to 120 h after culture initiation, after which spores became insensitive to SHAM. Heat treatment (50°C for 90 min) during the cyanide-resistant phase of respiration (0 h–120 h) induced cyanide-sensitive respiration and completely inhibited spore germination. Elevated temperatures had little effect if applied during the cyanide-sensitive phase (beyond 120 h). Temperature inhibited spores regained their ability to germinate if maintained in culture until the cyanide-resistant pathway was restored and then subjected to a second photoinductive light treatment. These results suggest the presence and possible involvement of the cyanide-resistant, alternative respiratory pathway during germination of Sphaeropteris cooperi spores.     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.     

Application of the 'Inoculated Pack Test' method for the determination of the heat resistance of Clostridium sporogenes PA3679 spores in acidified mushrooms

The effect of pH in the range 5.2–6.7 on the thermal destruction of Clostridium sporogenes PA3679 spores suspended in mushrooms in brine acidified with citric acid was examined by the 'inoculated pack test' method. The results indicated that increasing acidity is accompanied by decreasing decimal reduction times at 121.1°C: D _121.1 at pH 6.0 and 5.2 was, respectively, 64% and 17.5% of that at pH 6.7, the pH of natural mushrooms ( D _121.1= 2.22 min). A linear model ( r = 0.988, α= 0.05) was developed where the D _121.1 value was a function of the pH over the range studied. The inoculated pack test seems to be the only method to evaluate the actual microbial heat resistance, whether of spore or of vegetative forms, in order to estimate within reasonably close limits a suitable process time required to eliminate health hazards and to prevent spoilage losses in a given food product.     

On the presence of calmodulin-like protein in mycobacteria

Abstract Endogenous elemental sulfur (S⁰) has been studied in dormant spores and in spores in the early stages of germination, of Phomopsis viticola . S⁰ was measured by high-pressure liquid chromatography (HPLC). The rapid and almost total disappearance of endogenous S⁰ during the early stages of spore germination was directly related to a sharp increase of the respiratory activity and the ATP concentration. This was followed by the synthesis of DNA, RNA, proteins and lipids. Respiratory activity, S⁰ reduction and germination were inhibited in high concentrations of spores. Endogenous S⁰ disappearance, due to its reduction at the level of the mitochondrial respiratory chain with hydrogen sulfide production, may play a key role in the breaking of dormancy and the induction of germination in spores of P. viticola .     

Sporulation of Clostridium putrefaciens and the Resistance of the Spores to Heat, γ-Radiation and Curing Salts

Clostridium putrefaciens grew well in most media used routinely for culturing anaerobes, but produced spores only on lactose-egg yolk agar. The D_80° was 8–14 min, z value was 4°–6° and D _γ, 0.16 Mrad. The inhibitory interactions of pH, NaCl, NaNO₂ and incubation temperature are described.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. III. LABORATORY DETERMINATIONS OF THE HEAT RESISTANCE OF SPORES OF BACILLUS SUBTILIS IN WATER AND IN MILK

SUMMARY: Thermal death curves for spores of Bacillus subtilis 786 have been determined in water and in milk. Generally a non-logarithmic order of death was observed. Numbers of survivors were lower in milk than in water, suggesting that there may be inhibitory factors in UHT sterilized milk which affect the germination and/or subsequent growth of heated spores.
The thermal death curves for spores suspended in milk yielded Q₁₀ values of about 30 in the range 110–120°. This is higher than the figures previously reported in the literature for R. subtilis spores. Spores of a number of strains of B. subtilis were compared with strain 786 and all gave high Q₁₀ values.
The results obtained in this work have been used to predict the destruction of spores at higher temperatures in a UHT plant (Burton et al. 1958). The calculated values agree well with the results obtained in the plant by Franklin et al. (1958).     

The effect of age on the thermal resistance of arthroconidia from Chrysosporium inops

J. L. KINDERLERER. 1996. Food-borne members of the genus Chrysosporium have been isolated relatively infrequently. The heat resistance of arthroconidia of the xerophilic fungus, Chrysosporium inops Carmichael, was determined in 0.1% peptone at 66C. The survival curve was sigmoid in shape. The initial lag period was due to the chains of arthroconidia. Thermal inactivation occurred when one viable conidium was left per chain. The presence of chains of arthroconidia was confirmed with the cryo scanning electron microscope. The decimal reduction times were obtained from the regression line of the linear death phase for the heat-sensitive spores. The decimal reduction time (D₆₆) increased with increasing spore age. It was 1.67 min for 3-week-old spores, 1.95 min for 4-week-old spores and 5.49 min for 6-week-old spores. The older spores could recover from thermal death if they were given sufficient time. There was a significant increase in D₆₆ value for 6-week-old spores from 3.97 min to 5.49 when the counts were obtained after 14 d incubation (compared to counts after incubation for 10 d). This effect was not seen for the 3- and 4-week-old spores. There was a small population of heat-resistant spores. The initial population of arthroconidia was greater than log 7 cfu ml^-1. After heating for 1 h at 66C approximately log 2.2 cfu ml^-1 survived. These survivors represented approximately 0.001% of the original population.     

Production and Pathogenicity to Wheat of Pseudocercosporella herpotrichoides Conidia

Weekly estimates of numbers of Pseudocercosporella herpotrichoides conidia on naturally infected wheat straw, made from February to July 1982, showed there were most conidia (8.1 × 10⁶ per straw) in February and least (1.9 × 10⁴ per straw) at the end of June. The viability of these spores remained high throughout this period, with an average of 85 % germination after 24 h.
After removal of spores produced in the field, straws were incubated at 5, 10, 15, 20 or 25°C and subsequent sporulation assessed after 3 or 5 weeks. The optimum temperature for spore production was 5°C and very few spores were produced at 25°C. There was no difference in viability between spores produced at different temperatures.
Wheat seedlings placed amongst infected straw collected and retained spores on the upper and lower surfaces of all leaf blades and on outer leaf sheaths. Both naturally dispersed spores and spores sprayed on to plants were not removed by subsequent rainfall.
When wheat seedlings were inoculated between the coleoptile and outer leaf sheath with different numbers of P. herpotrichoides spores, lesion development was most rapid in seedlings inoculated with the greatest numbers of spores. However, after incubation for 12 weeks visible lesions were present on all plants inoculated with > c. 10 spores.