Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1°0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1.0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Possible Involvement of β-Lactamase in Sporulation in Bacillus cereus

Nonreverting beta-lactamase-negative strains were isolated from the beta-lactamase-constitutive strain, Bacillus cereus 569 H. These strains differed from both beta-lactamase-inducible and -constitutive strains not only in failure to produce beta-lactamase but also in failure to autolyze on aging, delayed sporulation, and failure to release free spores from sporangia when produced. The addition of B. cereus beta-lactamase of 15% purity to a final concentration of 10 IU/ml stimulates sporulation and particularly the release of free spores in culture from sporangia of strain 569 (inducible wild-type), 569/H (constitutive mutant of 569), and HPen(-), a nonreverting beta-lactamase strain isolated from 569/H in this laboratory. Cultures of HPen(-) did not release free spores without this treatment. Similar stimulation of sporulation and spore release by beta-lactamase from B. cereus were observed in another beta-lactamase-negative strain derived from 569/H as well as in certain sporogeny mutants of B. subtilis. The beta-lactamase preparation used in these experiments was free of peptidases, proteases, and autolysins capable of solubilizing wall from vegetative cells. These results, taken with our previous finding that a soluble peptidoglycan inducer becomes available in cultures of B. cereus only at sporulation and that normal derepression of beta-lactamase accompanies normal sporulation, suggest that beta-lactamase in B. cereus may be involved in peptidoglycan metabolism during sporulation and possibly the breakdown of sporangial wall with the concomitant release of mature spores.     

Direct Transition of Outgrowing Bacterial Spores to New Sporangia Without Intermediate Cell Division.

Vinter, Vladimir (Syracuse University, Syracuse, N.Y.), and Ralph A. Slepecky. Direct transition of outgrowing bacterial spores to new sporangia without intermediate cell division. J. Bacteriol. 90:803-807. 1965.-A direct transition was observed of the primary cell developed after germination of Bacillus cereus spores into new sporangia without intermediate division stages. Two simple methods were used for replacement of outgrowing spores into diluted medium or saline. Elongated primary cells prevented from division by limitation of nutrients in the suspending medium were able to form new forespores in 8 hr and sporangia in 12 hr. These new sporangia were still marked by attached envelopes of the original spore. Under the same conditions, cells replaced during the first divisions quickly lysed. Spores formed in the elongated primary cell during "microcycle sporogenesis" possessed normal heat resistance and refractility and were later released from sporangia.     

Growth of Bacillus cereus during rice tapé fermentation

Growth of Bacillus cereus NCIB 8579 was studied on four varieties of rice with and without tapé fermentation. Fermented and unfermented rice supported growth of B. cereus to 10⁷–10⁹cfu/g. With fermentation the pH fell and numbers of B. cereus remained high ( ca 10⁸cfu/g) except on black glutinous rice where numbers declined. Cells added at different fermentation times survived less well as fermentation progressed and the pH fell. Once growth on rice is established, B. cereus is able to survive fermentation, probably as spores.     

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.     

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Spores of microorganisms

The addition of penicillin (300–1,000 units/ml.) to a culture ofBacillus cereus during formation of the refractive prespores leads to lysis of the sporangia and to the release of spore components (calcium and dipicolinic acid) from the cells. Penicillin mildly raises the incorporation of amino acids, including diaminopimelic acid, into hot-TCA precipitate of cells, while chloramphenicol lowers it. In the later phases of penicillin inhibition, DAP-containing structures are also destroyed, including the fraction firmly bound to the envelope structures of the spore (in the control culture this fraction is not released until later, during digestion by enzymes localized in the envelope structures themselves). Penicillin inhibition of sporogenesis can be reversed by adapting the culture to penicillin or by simultaneously adding chloramphenicol. After the presporulation phase, sporogenesis is relatively resistant to chloramphenicol, but the whole process is considerably slowed down. Chloramphenicol also affects the morphology of the spores during their formation and inhibits their release from the sporangia until the late phase of sporulation.     

The use of Bacillus diarrhoeal enterotoxin (BDE) detection using an ELISA technique in the confirmation of the aetiology of Bacillus-mediated diarrhoea

A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces. The ability of isolates of Bacillus spp., including Bacillus cereus , to produce BDE in Brain Heart Infusion broth containing 0·1% glucose was also checked by use of the kit. Results show that 29 out of 31 B. cereus isolates were enterotoxigenic. Foods positive for preformed BDE were always contaminated with >10⁵ B. cereus cfu g⁻¹, but not all foods contaminated with large numbers of B. cereus were positive for BDE. Bacillus spp., other than one isolate which closely resembled B. subtilis , were negative for BDE production. Criteria for the confirmation of Bacillus -mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.     

An Indirect Radiolabelled Antibody Staining Technique for the Rapid Detection and Identification of Bacteria

Small numbers of Bacillus subtilis var. niger spores, Serratia marcescens and Francisella tularensis can be detected and identified in an hour using an indirect radiolabelled antibody staining technique with homologous rabbit antibacterial antibody and radiolabelled goat anti-rabbit globulin. Tritium is better than ¹²⁵I, ¹⁴C and ³⁵S for labelling the globulin. The reagent obtained is sensitive and adequately stable.     

Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms

Aims:  The hypothesis that surrogate planktonic pathogens ( Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested.
Methods and Results:  Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0·15 and 1·5 N m⁻². The amount of surrogate pathogens introduced ranged from approximately 10⁵ CFU ml⁻¹ to 10¹⁰ spheres ml⁻¹. The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0·4–3·0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated.
Conclusions:  Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination.
Significance and Impact of the Study:  This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.     

Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto

Aims:  To isolate and identify a benefic bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp feed to promote shrimp growth performance.
Methods and Results:  A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50°C), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40°C, a pH of 6–8 and 0% NaCl. No shrimp died after being injected with B. subtilis E20 [up to 10⁹ colony-forming units (CFU) per shrimp]. Bacillus subtilis E20 was incorporated in diets at the levels of 0 (control), 10⁶, 10⁷, and 10⁸ CFU kg⁻¹ for shrimp grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (10⁸ CFU kg⁻¹ of diet), shrimp had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20.
Conclusions:  Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp growth performance through increasing the digestibility of food.
Significance and Impact of the Study:  Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp growth performance, and consequently reduce feed costs.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10⁸ cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10⁶–10⁷ cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus . In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5·5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

RAPID DETECTION OF GERMINATING BACILLUS CEREUS CELLS USING FLUORESCENT IN SITU HYBRIDIZATION

Methods for the specific detection of Bacillus spores are needed in many situations such as the recognition of food poisoning. This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples.
B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus -specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra heat-treated milk samples. FISH-labeled cells were detected by using flow cytometry, and the results were confirmed by fluorescence microscopy. The optimal condition allows the detection of B. cereus spores in less than 2 h. Overall, a ninefold reduction in total time for detection was achieved when comparing with previous works. Therefore, the permeabilization and hybridization optimizations mentioned in this study are major improvements for the detection time of B. cereus spores.

PRACTICAL APPLICATIONS

By using the optimized conditions of germination/outgrowth, permeabilization and hybridization, the detection of 10³ cfu/mL of Bacillus cereus spores using fluorescent in situ hybridization is possible within 2 h in milk sample.     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.     

Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus

J assim , H.K., F oster , H.A. & F airhurst , C.P. 1990. Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus. Journal of Applied Bacteriology 69 , 563–568.
The effects of exposing fifth instar larvae of Scolytus scolytus and S. multistriatus to spore suspensions of Bacillus spp. were investigated. Bacillus thuringiensis ser 3a, 3b increased the mortality of larvae cultured on an artificial medium from approximately 20% in control cultures to over 80% in cultures exposed to the bacteria. The mortality was dose-dependent for S. multistriatus and the approximate LC₅₀ value was 2.2 times 10³ spores/ml. Different serotypes of B. thuringiensis caused different levels of mortality: H6 produced the highest mortality and H1 the lowest. Bacillus alvei and B. cereus were also pathogenic but B. megaterium was not. The results are discussed in relation to the mechanism of pathogenicity and the potential for the use of B. thuringiensis for the control of the vectors of Dutch elm disease.     

Influence of sporulation medium and divalent ions on the heat resistance of Alicyclobacillus acidoterrestris spores

The influence of divalent cations on the heat resistance of spores of the thermoacidophilic spoilage bacterium Alicyclobacillus acidoterrestris was studied. The heat resistance of A. acidoterrestris spores was not affected by the presence of the different divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, Mn²⁺ and Sr²⁺) in the sporulation medium, and by the demineralization or remineralization. And the Ca and Mn contents in A. acidoterrestris spores were scarcely changed by these treatments. However, the heat resistance of Bacillus subtilis spores was affected with the changes of Ca content in the spores. The Ca contents in A. acidoterrestris spores of the different forms were greater than the DPA content. In contrast, the DPA content in the B. subtilis spores was greater than the Ca content. These findings suggest that the presence of constant amount of Ca-DPA and a stronger binding characteristic of divalent ions, especially Ca and Mn, is reflected in the specific heat resistance of A. acidoterrestris spores.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Effect of polymyxins on Bacillus polymyxa sporogenesis

Bacillus polymyxa var. Ross. producing polymyxin M and Bacillus polymyxa 153 producing polymyxin B form spores during submerged cultivation when the rate of biosynthesis of antibiotic peptides is low and when the production of antibiotics is over. However, sporogenesis is stimulated if polymyxins are added at the early stage of cultural growth. Inhibition of the synthesis of antibiotics suppresses the formation of spores. Substances other than polymyxins do not exhibit such a specific effect on sporogenesis. The fact that the culture requires endogenous polymyxins which are most effective in the period prior to the appearance of spores in the culture suggests the regulatory action of these peptides at the stage between vegetative growth and spore formation in Bacillus polymyxa.     

Hypochlorite effects on spores and spore forms of Bacillus subtilis and on a spore lytic enzyme

G orman , S.P., S cott , E.M. & H utchinson , E.P. 1984. Hypochlorite effects on spores and spore forms of- Bacillus subtilis and on a spore lytic enzyme. Journal of Applied Bacteriology 56 , 295–303.
Spores of Bacillus subtilis NCTC 10073 were converted to ion-exchange (Ca, H) forms and coat-defective (urea-mercaptoethanol, urea-dithiothreitol-sodium lauryl sulphate) forms. The resistance of these to sodium hypochlorite (1000 parts/10⁶ free chlorine) was compared and related to uptake from which the assumed monolayer capacities were calculated. Hypochlorite effects on spore protoplasts and cortical fragments were also examined in relation to DPA and hexosamine release. A spore lytic enzyme was extracted and examined in respect of hypochlorite activity. The results are discussed in terms of the mechanism and site of action of hypochlorite on the bacterial spore.