Wet and dry density of Bacillus anthracis and other Bacillus species

Aims: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. Methods and Results: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1·162 and 1·165 g ml^?1 and the other group included strains whose spores showed higher density values between 1·174 and 1·186 g ml^?1. Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R² = 0·95) between density and volume for the spores of all strains and species studied. Conclusions: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.     

Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets

We have previously shown that galacto-rhamnogalacturonan fibers can be enzymatically extracted from potato pulp and that these fibers have potential for exerting a prebiotic effect in piglets. The spore-forming Bacillus species are widely used as probiotics in feed supplements for pigs. In this study, we evaluated the option for further functionalizing Bacillus feed supplements by selecting strains possessing the enzymes required for extraction of the potentially prebiotic fibers. We established that it would require production and secretion of pectin lyase and/or polygalacturonase but no or limited secretion of galactanase and β-galactosidase. By screening a library of 158 Bacillus species isolated from feces and soil, we demonstrated that especially strains of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus mojavensis have the necessary enzyme profile and thus the capability to degrade polygalacturonan. Using an in vitro porcine gastrointestinal model system, we revealed that specifically strains of B. mojavensis were able to efficiently release galacto-rhamnogalacturonan from potato pulp under simulated gastrointestinal conditions. The work thus demonstrated the feasibility of producing prebiotic fibers via a feed containing Bacillus spores and potato pulp and identified candidates for future in vivo evaluation in piglets.

     

Phylogenetic diversity of thermophilic carbohydrate degrading bacilli from Bulgarian hot springs

Phylogenetic diversity of culturable bacteria from genus Bacillus and related genera, isolated from 18 Bulgarian hot springs was investigated in association with their functional diversity. Sixty-seven thermophilic and facultative thermophilic strains were isolated under aerobic conditions at 60°C. Sixty-six of them belonged to eight species in four genera from Bacillus group: Anoxybacillus, Geobacillus, Brevibacillus and Bacillus. Representatives of the genus Anoxybacillus predominated. Based on phylogenetic analysis (<97% sequence similarity) four strains belonged to groups representing potentially novel species. Producers of carbohydrases, degrading 12 from the tested 13 substrates were isolated. About half of the isolates degraded amylose by exo- or endo-mechanism of action of their enzymes. The isolates degrading hemicellulose carbohydrates like arabinan, arabinoxylan, β-glucan, galactan, galactomannan and xyloglucan were reached to. Some of the microorganisms were able to uptake microbial polysaccharides like curdlan and gellan and their enzymes were between first reported thermostable enzymes in their groups, like gellan lyase and curdlan lyase A relation between species affiliation and their functional activity was observed—all A. gonensis strains were producer of amylolytic enzymes, most of Brevibacillus ruber strains were able to grow in a minimal medium with xanthan.     

Gas discharge plasmas are effective in inactivating <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Clostridium</Emphasis> spores

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Improvement in symbiotic efficiency of chickpea (Cicer arietinum) by coinoculation of Bacillus strains with Mesorhizobium sp. Cicer

Rhizobacteria belonging to Bacillus sp. were isolated from the rhizosphere of chickpea (Cicer arietinum). Ten Bacillus strains were studied for their antifungal activity, effect on seedling emergence and plant growth promotion. Two Bacillus strains CBS127 and CBS155 inhibited the growth of all the four pathogenic fungi tested on nutrient agar medium plates in vitro. Seed inoculation with different Bacillus strains showed stimulatory effect on root and shoot growth at 10 d of observation in comparison to control whereas four Bacillus strains CBS24, CBS127, CBS129 and CBS155 caused retardation of shoot growth at 10 d. Maximum nodule-promoting effect was observed with Bacillus strains CBS106, CBS127 and CBS155. The symbiotic effectiveness of Mesorhizobium sp. Cicer strain Ca181 was further improved on coinoculation with six Bacillus strains i.e. CBS9, CBS17, CBS20, CBS106, CBS127 and CBS155 at 80 d of plant growth under sterile conditions and shoot dry weight ratios increased 1.62 to 1.74 times those of Mesorhizobium-inoculated treatments, suggesting the usefulness of introduced rhizobacteria in improving crop productivity.     

Bacillus probiotics: an alternative to antibiotics for livestock production

The use of probiotics as feed supplements in animal production has increased considerably over the last decade, particularly since the ban on antibiotic growth promoters in the livestock sector. Several Bacillus sp. are attractive for use as probiotic supplements in animal feed due to their ability to produce spores. Their heat stability and ability to survive the low pH of the gastric barrier represent an advantage over other probiotic micro‐organisms. This review discusses important characteristics required for selection of Bacillus probiotic strains and summarizes the beneficial effect of Bacillus‐based feed additives on animal production. Although the mechanism of action of Bacillus probiotics has not been fully elucidated, they are effective in improving the growth, survival and health status of terrestrial and aquatic livestock. Bacillus strains also have utility in bioremediation and can reduce nitrogenous waste, thereby improving environmental conditions and water quality. Finally, recent innovative approaches for using Bacillus spores in various applications are discussed.     

CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis

Bacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation-specific and heat-labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo-regulator CotH contributes to the switch between the coat of 25°C and that of 42°C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components.     

Analysis of Metabolism in Dormant Spores of Bacillus Species by 31P Nuclear Magnetic Resonance Analysis of Low-Molecular-Weight Compounds

This work was undertaken to obtain information on levels of metabolism in dormant spores of Bacillus species incubated for weeks at physiological temperatures. Spores of Bacillus megaterium and Bacillus subtilis strains were harvested shortly after release from sporangia and incubated under various conditions, and dormant spore metabolism was monitored by ³¹P nuclear magnetic resonance (NMR) analysis of molecules including 3-phosphoglyceric acid (3PGA) and ribonucleotides. Incubation for up to 30 days at 4, 37, or 50°C in water, at 37 or 50°C in buffer to raise the spore core pH from ∼ 6.3 to 7.8, or at 4°C in spent sporulation medium caused no significant changes in ribonucleotide or 3PGA levels. Stage I germinated spores of Bacillus megaterium that had slightly increased core water content and a core pH of 7.8 also did not degrade 3PGA and accumulated no ribonucleotides, including ATP, during incubation for 8 days at 37°C in buffered saline. In contrast, spores incubated for up to 30 days at 37 or 50°C in spent sporulation medium degraded significant amounts of 3PGA and accumulated ribonucleotides, indicative of RNA degradation, and these processes were increased in B. megaterium spores with a core pH of ∼7.8. However, no ATP was accumulated in these spores. These data indicate that spores of Bacillus species stored in water or buffer at low or high temperatures exhibited minimal, if any, metabolism of endogenous compounds, even when the spore core pH was 7.8 and core water content was increased somewhat. However, there was some metabolism in spores stored in spent sporulation medium.     

A genetic algorithm-Bayesian network approach for the analysis of metabolomics and spectroscopic data: application to the rapid identification of Bacillus spores and classification of Bacillus species

Background  

The rapid identification of Bacillus spores and bacterial identification are paramount because of their implications in food poisoning, pathogenesis and their use as potential biowarfare agents. Many automated analytical techniques such as Curie-point pyrolysis mass spectrometry (Py-MS) have been used to identify bacterial spores giving use to large amounts of analytical data. This high number of features makes interpretation of the data extremely difficult We analysed Py-MS data from 36 different strains of aerobic endospore-forming bacteria encompassing seven different species. These bacteria were grown axenically on nutrient agar and vegetative biomass and spores were analyzed by Curie-point Py-MS.     

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to 98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.     

基于可培养方法分析云南腾冲小空山火山谷芽胞杆菌分布特征

【目的】为了解云南腾冲小空山火山谷土壤中可培养芽胞杆菌种类分布特征。【方法】采用可培养手段对小空山火山谷阳坡、谷底和阴坡土壤中的芽胞杆菌进行分离培养,根据16S rRNA基因序列同源性对分离菌株进行鉴定,并分析系统发育地位。利用Canoco5软件分析采样点芽胞杆菌种类分布特征与土壤样品理化性质的相关性。【结果】从火山谷土壤样品中共分离获得180株芽胞杆菌,16S rRNA基因测序鉴定结果表明分离菌株隶属于芽胞杆菌纲2个科(芽胞杆菌科和类芽胞杆菌科)、6个属、34个种,其中芽胞杆菌属(Bacillus) 11个种,类芽胞杆菌属(Paenibacillus) 14个种,短芽胞杆菌属(Brevibacillus)3个种,赖氨酸芽胞杆菌属(Lysinibacillus)4个种,嗜冷芽胞杆菌属(Psychrobacillus)1个种和绿芽胞杆菌属(Viridibacillus)2个种,其中7个菌株与其最近模式菌株16SrRNA相似性低于种的界定阈值(98.65%),为芽胞杆菌潜在新物种。优势属为芽胞杆菌属和类芽胞杆菌属,优势种为蕈状芽胞杆菌(Bacillusmycoides),图瓦永芽胞杆菌(Bacillustoyonensis),蜡状芽胞杆菌(Bacilluscereus),解木糖赖氨酸芽胞杆菌(Lysinibacillusxylanilyticus),蜂房类芽胞杆菌(Paenibacillusalvei)和沙地绿芽胞杆菌(Viridibacillus arenosi)。其中16个种分离自阳坡,29个种分离自阴坡,9个种分离自谷底,三者共同种类为6种。阳坡、谷底和阴坡的芽胞杆菌种群分布Bray-Curtis相似性为62.4%,多样性分析结果表明,Shannon指数(H′)大小次序为阴坡阳坡谷底。环境因子分析发现,芽胞杆菌种群分布多样性特征与其土壤的海拔高度、碳氮比和硫含量呈负相关,而和碳源和氮源含量呈正相关。【结论】从以上结果得出,云南腾冲火山谷有着较为丰富的芽胞杆菌资源,且还存在可分离培养的芽胞杆菌的潜在新物种,为利用火山微生物资源提供了保障。     

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

Aims: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results: Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T_lag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT_release) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average T_lag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T_lag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average T_lag and ΔT_release values in dodecylamine germination that does not utilize GRs. Conclusions: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.     

A gene encoding alanine racemase is involved in spore germination in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted l-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of l-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, d-alanine was an effective and competitive inhibitor of l-alanine-induced germination of B. thuringiensis spores. d-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing l-alanine-induced germination by inhibiting alanine racemase. d-Alanine also increased the rate of inosine-induced germination of wild-type spores. However, d-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted d-alanine to l-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.     

Inability of detect cyclic AMP in vegetative or sporulating cells or dormant spores of Bacillus megaterium

Cyclic AMP was not found in vegetative cells or sporulating cells or dormant spores of $\text{Bacillus}\text{megaterium}$ using an assay which would have detected an $\text{in}\text{vivo}$ concentration of 1 – 2 × 10^?9 M. Adenyl cyclase and cyclic AMP phosphodiesterase were also not detected in sonicates of vegetative or sporulating $\text{B.}\text{megaterium}$ cells.     

Influence of the surface temperature of packaging specimens on the inactivation of Bacillus spores by means of gaseous H(2) O(2)

Aims: To determine the influence of condensation as a function of the surface temperature of aseptic packaging, on the inactivation of Bacillus spores [Bacillus subtilis (DSM 347), B. subtilis SA22, Bacillus atrophaeus] having different surface properties by means of vaporized H₂O₂. Methods and Results: The packaging specimens inoculated with Bacillus spores were tempered and subsequently exposed to H₂O₂‐vapour. During the exposure, surface temperature curves were measured and the spore survival was determined. Results showed that decreasing the initial surface temperature of the packaging specimens had a positive effect on the sporicidal activity of H₂O₂‐vapour, where the effect was less pronounced for less hydrophilic spores. The surfaces of spores were characterized by means of the water contact angle. Conclusions: For starting surface temperatures below the dew point temperature of the sterilant gas, the condensation of highly concentrated liquid H₂O₂ on the packaging surface accelerates the killing of the spores, while the inferior wettability of more hydrophobic spores compared to more hydrophilic ones diminishes the effect. Significance and Impact of the Study: Regarding industrial packaging sterilization, a mixed microflora has to be inactivated. Promoting the condensation of H₂O₂ improves in general the killing of different species of spores, however, at various degrees depending on the wettability of spores.     

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     

Species-level identification of <Emphasis Type="Italic">Bacillus</Emphasis> strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis

The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.     

Effects of the Use of a Combination of Two Bacillus Species on Performance,Egg Quality,Small Intestinal Mucosal Morphology,and Cecal Microbiota Profile in Aging Laying Hens

Sixty-week-old Hy-Line brown laying hens were randomly divided into five groups and fed different diets over a period of 84 days. Experimental treatments included a basal diet (control); the basal diet supplemented with 1.0 × 10⁶B. licheniformis yb-214245; the basal diet supplemented with 1.0 × 10⁶B. subtilis yb-114246; a combination of both strains in a 2:1 ratio (6.6 × 10⁵:3.3 × 10⁵B. licheniformis yb-214245:B. subtilis yb-114246); and the latter, added with 5 mg/kg flavomycin. Basal diet supplementation with the combined Bacillus species improved egg-laying performance in aging hens significantly (P < 0.05). Eggshell strength improved significantly with this treatment, compared to the control or the antibiotic-supplemented groups (P < 0.05). The levels of total cholesterol, triglycerides, and very low-density lipoprotein cholesterol in egg yolk declined significantly more in the Bacillus-treated group than in the control or the antibiotic-supplemented groups (P < 0.01). Small intestinal morphology was better in the hens treated with the Bacillus combination than in the hens in the control group (P < 0.05). The total number of aerobic bacteria (Bacillus, Lactobacillus, and Bifidobacterium) in the cecum was significantly higher in all the Bacillus-supplemented hens than either in the control or the antibiotic-supplemented hens (P < 0.01); additionally, the number of E. coli and Salmonella was significantly lower than in the control group (P < 0.01). In conclusion, diet supplementation with the combination of Bacillus species used here for aging laying hens improved their growth performance, cecal bacterial composition, egg quality, and small intestine morphology.

     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.