Germinability and heat resistance of spores of Clostridium difficile strains

Out of 111 Clostridium difficile strains, 108 produced spores in numbers of more than 10(5)/ml and the remaining three did not produce any spores in brain heart infusion medium. The germination frequency in the medium without lysozyme varied widely from strain to strain, ranging from less than 10(-8) to 10(0), and in 77 of the 108 strains the germination frequency was 10(-5) or less. The spores, when treated with sodium thioglycollate and then inoculated into the medium containing lysozyme, germinated in all of the 108 strains at a frequency of 10(-0.5) or more. The spores of two strains germinated at a frequency of more than 10(-0.5) in all methods. Spores of C. difficile strains were fairly highly heat-resistant; D100C values ranged from 2.5 to 33.5 min.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.     

Development and morphological features of biofilms formed by transgenic and wild type strains of Bacillus subtilis

The study addressed the ability of the transgenic strain (TM) B. subtilis 2335/pBMB105 (Km^rInf⁺) to form biofilms on the surface of liquid media of various compositions, inoculated with vegetative cells and spores. The morphological features of these biofilms do not differ from those of the films formed by the recipient strain (WT) B. subtilis 2335 (Km^s). However, the TM and the natural one differ in the dynamics of biofilm formation and the cellular composition of the films. Biofilms of the TM are formed earlier, develop at a higher rate, but decompose later than the films of the WT. When the medium is inoculated with vegetative cells, sporulation in the biofilms of both strains undergoes glucose repression; no such effect is observed when the medium is inoculated with spores. The TM does not form films when the medium is inoculated with spores and supplemented with glycerin and kanamycin.     

Growth of moulds inoculated into commercial mineral water

The growth of mould spores of Penicillium sp. and Cladosporium sp. inoculated in a commercial mineral water product was studied. The strains had been isolated as fungal foreign bodies in commercial mineral waters. In product A, which was not originally sterilized and was contaminated with psychrophilic bacteria, the inoculated mould spores of the strains did not grow; no increases in viable colony counts or beta-glucans concentration in the samples were observed during storage. In a sterilized product A, inoculated spores of the strains grew into visible foreign bodies. The viable colony counts and the beta-glucans concentration in the samples increased during storage. These results showed that in a sterilized mineral water product, mould spores could grow into visible foreign bodies.     

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.     

Hydrophobicity of Bacillus and Clostridium spores.

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Production of Fumonisins by Fusarium Verticillioides Strains on Solid and in a Defined Liquid Medium — Effects of L-Methionine and Inoculum

In order to evaluate the toxicological and carcinogenic effects of fumonisins, large amounts of fumonisins need to be purified, which requires optimal conditions for production in culture. Five strains of F. verticillioides were compared for their ability to produce fumonisins in solid and liquid media with and without the addition of methionine, a fumonisin precursor. Inoculations were made either with lyophilized cultures or a concentrated inoculum. Growth in liquid medium, measured by biomass, was directly correlated to total fumonisin production when a lyophilized inoculum was used. Fumonisin production was stimulated by the addition of 0.2% L-methionine to solid media for most strains. Levels ranged from 1500-3900 mg/kg in rice, and 2900-12500 mg/kg in maize cultures inoculated with lyophilized cultures; 200-4800 mg/kg in rice, and 1500-4200 mg/kg in maize cultures inoculated with concentrated inoculum. Strains that produced relatively high levels of fumonisins in solid media did not necessarily do so in liquid medium and vice versa. Total fumonisin levels in liquid medium ranged from 40-590 mg/l inoculated with lyophilized cultures and < 1-110 mg/l inoculated with concentrated inoculum, without adding the precursor. F. verticillioides strains therefore varied in their ability to produce fumonisins, and conditions for production need to be optimized individually for each strain.     

Spore germination of Frankia strains isolated from Colletia hystrix and Retanilla ephedra (Rhamnaceae)

Two Frankia strains, ChI1 from Colletia hystrix and ReI6 from Retanilla ephedra, were assayed to determine the germinability of their spores and the stages of the life cycle in different media. In BAP medium, both strains showed approximately 18% spore germination. However, in BAP medium lacking micronutrients and FeEDTA, strain ChI1 spores exhibited approximately 53% germination and strain ReI6 spores about 42%. The survey also showed that the spores were not heat-resistant, the ReI6 spores being more sensitive than ChI1 spores. Both strains exhibited a small increase in their percentage of germination with a 15-pulse ultrasonic treatment.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

Conservation in probiotic preparations of Lactobacillus with inhibitory capacity on other species

Strains of Lactobacillus isolated from dairy products and genital tract competed with Candida albicans through a membrane of 12000 dalton cut-off. This inhibition was due to hydrogen peroxide and was trypsin-stable, heat-sensitive and antagonized by catalase. Lactobacillus coming from "starters" showed antimicrobial activity against fungus isolated in a yogurt factory. Penicillium, Alternaria, Phialophora, Microsporum and Candida spp. were inhibited when 10(2) spores were inoculated in the assay. No inhibition was observed with 10(5) spores. Besides, one of 21 Lactobacillus strains isolated from the vaginas of healthy women inhibited pathogenic bacteria by means a bacteriocin trypsin-sensitive, heat-stable and retained by dialysis membrane. Tablets for future probiotic use were prepared and the viability of bacteria was assayed using media with different compositions. Pharmaceutical preparations with polyethyleneglycol was the best formulation for the Lactobacillus viability, the counts remained between 10(7) and 10(6) cfu/tablet for up to 1 year.     

Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat

A cocktail of washed spores from six psychrotrophic Clostridium strains isolated from blown vacuum-packed meats was inoculated onto lamb chumps. A second washed spore cocktail of four toxigenic reference Cl. botulinum strains, types A, B (two strains) and E, and a Cl. butyricum type E strain, was similarly inoculated onto lamb chumps. All inoculated lamb chumps were individually vacuum-packed and placed into storage at various temperatures typical of good to grossly abusive chilled storage (-1 degree C to 15 degrees C). All packs were observed for gas production (pack-'blowing') over a 12 week storage period. On gas production, or after 12 weeks of storage, packs were examined by mouse bioassay for botulinum toxin production. The packs inoculated with the meat isolate cocktail showed evidence of gas production earlier than packs inoculated with reference strains. No botulinum toxin was recovered from the meat isolate inoculated packs, while botulinal toxin was detected in reference strain inoculated packs down to a nominal storage temperature of 2 degrees C.     

Growth and toxin production by non-proteolytic and proteolytic Clostridium botulinum in cooked vegetables

Growth and toxin production by proteolytic and non-proteolytic strains of Clostridium botulinum have been followed in 28 cooked puréed vegetables prepared under strict anaerobic conditions and incubated at 30°C for up to 60 d. Toxin production was confirmed in 25 of the cooked vegetables inoculated with a suspension of spores of proteolytic strains of types A and B, and in 13 inoculated with a suspension of spores of non-proteolytic strains of types B, E and F. For both proteolytic and non-proteolytic strains, a trend was identified correlating growth and toxin production with the pH of the cooked puréed vegetables.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27.5 degrees C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10(4) spores/g whereas with 800 g loaves survival occurred with doughs containing 5.0 X 10(3) spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0.2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

The effect of sugar concentration and temperature on growth and volatile phenol production by Dekkera bruxellensis in wine

The wine spoilage yeast Dekkera bruxellensis was evaluated for the production of 4-ethylphenol under low concentrations (0.02-20 g L(-1)) of glucose and fructose in synthetic media. Measurable amounts of 4-ethylphenol were produced over 0.2 g L(-1) of each sugar. The yeast growth rate and amount of biomass formed increased from 0.2 to 20 g L(-1) of glucose or fructose, being accompanied by increasing production of 4-ethylphenol. In red wines, the production of 4-ethylphenol was only observed in the presence of growing populations of indigenous or inoculated strains of D. bruxellensis. The production rate of 4-ethylphenol varied between 22 and 93 mug day(-1) either with inoculated strains or wild populations in bottled wines. The production rate of 4-ethylphenol as a function of the increase in the number of cells varied from 349 to 1882 mug L(-1) per one log CFU mL(-1). The effect of temperature on cellular viability and 4-ethylphenol production was tested in red wines with indigenous or inoculated strains of D. bruxellensis. Incubation temperatures of 15, 20 and 25 degrees C allowed cellular growth and volatile phenol production. Increasing incubation temperatures to 36 degrees C induced full viability loss of 10 strains of D. bruxellensis within <12 h.     

Reversal of the inhibitory effect of surfactants upon germination and growth of a consortium of two strains of Bacillus

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth. Received: 9 July 1998 / Received revision: 26 October 1998 / Accepted: 30 October 1998     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

航天诱变对昆虫病原真菌的生物学效应

8个昆虫病原真菌菌株经过航天搭载后,有5个菌株的孢子在航天搭载后全部死亡,菌株M2029和MR8的存活率仅约10-7,菌株M189的存活率可达到20%以上。分离得到航天诱变的单菌落菌株,检测表明航天诱变使昆虫病原真菌的形态、生长速度、产孢量、致病力等多项生物学特性发生了不同程度的变异,变异率高,变异趋向有正负双向性。测定结果表明航天诱变菌株HM189-68s、HM189-32c、HM189-127和HM189-17的生长特性和致病力优于原始菌株。航天诱变为选育生物防治优良菌株提供了新途径。     

Evaluation of Media, Time and Temperature of Incubation, and Method of Enumeration of Several Strains of Clostridium perfringens Spores

Two basal media, containing the ingredients found in common in both SPS (BBL) and TSN (BBL) media and in the previously described media of Schaedler et al. (1965) and Starr et al (1971), but minus antibiotics, were selected as the most suitable for the enumeration of Clostridium perfringens spores in a model system. These media were also used to study the influence of the presence of glucose, xylose, or ribose in various concentrations (0, 0.01, 0.1, and 1.0%) on colony morphology and spore recovery. As the sugar concentration in the basal agar medium increased, the colonies of all the test organisms also increased in size, and more of the black colonies became white in color. At the 1.0% sugar level, glucose permitted only white colony development, whereas the pentoses were completely inhibitory. Both pour plates and most-probable-number tubes were inoculated with the spores of several strains of C. perfringens and incubated at 20, 30, 37, and 45 C for 24, 48, and 72 h. Statistical analyses of the enumeration data indicated, at the 99% confidence level, that a Trypticase(BBL)-yeast extract-glucose-sulfite-iron agar gave maximal population estimates at 37 C in 72 h.