Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

Differences between Brachiola (Nosema) algerae isolates of human and insect origin when tested using an in vitro spore germination assay and a cultured cell infection assay

Brachiola (Nosema) algerae is a microsporidian species generally believed to be an intracellular parasite of insects, especially mosquitoes. However, both mosquito and human isolates have been shown to infect mammalian cells. The present study was undertaken to determine if spores of two insect and two human isolates of B. algerae cultured at 30 degrees C and 37 degrees C differed in their ability to germinate and infect cultured green monkey kidney cells at these two temperatures. Spores from all four isolates exhibited an optimum pH of 9.5 for germination. Mercury (Hg2+) inhibited germination of all isolates equally. Germination of spores from all four isolates was significantly greater when the parasite was cultured at 30 degrees C than when cultured at 37 degrees C. However, spores from the insect isolates cultivated at 30 degrees C or 37 degrees C infected significantly fewer mammalian cells at 37 degrees C than did spores from the human isolates under the same conditions. Thus, there is no correlation between the effects of temperature on the germination and the infectivity of an isolate. In addition, while exposure of B. algerae to 37 degrees C has been reported to cause spore dysmorphism, we failed to observe any consistent ultrastructural changes that explained the greater infectivity of the human isolates at 37 degrees C.     

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.     

Sensitivity of chemically treated spores of Clostridium perfringens type A to an initiation protein.

Extraction of Clostridium perfringens type A spores with dithiothreitol (DTT), DTT plus sodium dodecyl sulphate (DTT-SDS), urea-mercaptoethanol (UME), or alkali, solubilized from 18.6 to 46.5 of the total dry weight of spores. The initiation of germination and lysis of such treated spores with lysozyme and an initiation protein (IP) from the culture supernatant fluid of sporulating cells of C. perfringens was studied under various conditions. The ability of lysozyme and the crude IP to induce germination and lysis of extracted spores was concentration dependent up to 0.5 microgram/ml and 5.6 mg/ml respectively. IP showed an optimum of activity between pH 7 and 8 for DTT-SDS and DTT extracted spores, and between pH 6 and 9 for UME extracted spores. The optimum temperature of activity for IP was 55 degrees C. Dissimilarities in the extent to which lysozyme and the IP initiated germination and lysis of spores extracted by various methods may have been a reflection of the differences in amounts of protein solubilized by each treatment.     

Heat activation of Streptomyces viridochromogenes spores.

The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated.     

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores.

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Nutritional conditions for the germination of streptomyces galbus 5ME-13 spores

The nutritional conditions for the germination of spores of Streptomyces galbus 5ME-13 were determined under laboratory conditions. The germination of the spores was intiated by the emergence of 1–2 germ tubes after the second hour of incubation and attained its maximum at the sixth hour. This was accompanied by a steady rise in the optical density of the germinating spore suspension. A malt-extract yeast-extract medium was found to be the best medium for the germination of the spores. Glycerol as the sole source of carbon was the best supporter for spore germination while, as N source, L-alanine was preferred. The optimum pH and temperature for spore germination were 7.2 and 30°C, respectively.     

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Spore germination promoter of Dictyostelium discoideum excreted by Aerobacter aerogenes.

The nutrient medium in which Aerobacter aerogenes was grown, contains a spore germination promoter (SGP) for the cellular slime mould Dictyostelium discoideum. SGP can cuase synchronous spore germination in a short time, and triggers the germination process in just a few minutes. Germination-promoting capacity of SGP decreases as it comes in contact with increasing number of spores. When spores activated by SGP are stored at 4 degrees C, they gradually return to the dormant state. SGP is comparatively heat-stable, but is unstable at pH above 10 or under 3.     

Thermal analysis of the spores of Bacillus cereus with special reference to heat activation.

The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Commitment of bacterial spores to germinate. A measure of the trigger reaction.

The rate of commitment of bacterial spores to germinate after short exposure to L-alanine increases exponentially from the time of addition of L-alanine. This absence of a lag facilitates kinetic analysis and allows the dependence of commitment on temperature and pH to be determined. The pH profile of commitment has been compared with that obtained from measurements of absorbance decreases during germination, and the two profiles exhibit differing pK values. It is suggested that because the decrease in A600 of spore suspensions is a late event in germination, it is an unsuitable parameter for studying germination-triggering reactions. Commitment has been shown to be temperature-dependent, with an optimum at approx. 37 degrees C and an activation energy (mu) of 1.08 X 10(5) J/mol. The data obtained from the present studies have been used to develop a model for the triggering of germination.