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Extended shelf-life (ESL) refrigerated ready-to-eat foods are thermally pasteurized to ensure food safety and stability. However, surviving psychrotrophic Bacillus cereus spores can still pose a challenge. Studies predicting their behavior often overlook sporulation conditions. This study investigated the effect of sporulation temperature on germination of three Bacillus weihenstephanensis strains in specific nutrients (inosine and/or amino acids) with or without prior heat activation (80 °C, 10 min). Sporulation temperature variably affected germination, with stronger effects in moderately responsive strains and nutrients. Heat activation strongly stimulated germination, particularly in nutrients with poorer responses, mitigating differences induced by sporulation temperature. The influence of sporulation temperature on germination and growth in an ESL matrix at refrigeration temperatures (4 °C or 8 °C) in vacuum packaging after heat activation or commercial pasteurization (90 °C, 10 min) was also studied. The latter treatment increased germination rates of surviving spores; however, some strains suffered damage and lost viability upon germination at 4 °C but recovered and grew at 8 °C. These findings highlight the need to account for variability in spore recovery and outgrowth during quantitative risk assessments for psychrotrophic B. cereus in ESL foods. [ABSTRACT FROM AUTHOR]

Members of the B. cereus group are spore-forming organisms commonly associated with spoilage of milk and dairy products. We have determined the genetic identity and growth characteristics of 57 B. cereus isolates collected from a Norwegian ice cream production plant. Our findings revealed persistence of B. cereus spp. strains for up to 19 months, suggesting the plant's susceptibility to long-term colonization. One of the mesophilic isolates, NVH-YM303, carried a complete cereulide synthetase operon. To assess the potential food poisoning risk associated with the presence of cereulide-producing strains in the production line, we examined the production of cereulide in ice cream and milk at different temperatures by NVH-YM303 and by the emetic psychrotrophic B. weihenstephanensis strain BtB2-4. Our findings revealed that NVH-YM303 produced higher levels of cereulide in ice cream as compared to milk. Furthermore, it was observed that NVH-YM303 produced more cereulide in ice cream at 25 °C compared to 15 °C. Conversely, BtB2-4 produced more cereulide in ice cream at 15 °C than at 25 °C. The results obtained in this study contribute to knowledge important for risk assessment of the potential hazards posed by the presence of B. cereus within ice cream production facilities. [ABSTRACT FROM AUTHOR]

Extended shelf-life (ESL) refrigerated ready-to-eat foods are thermally pasteurized to ensure food safety and stability. However, surviving psychrotrophic Bacillus cereus spores can still pose a challenge. Studies predicting their behavior often overlook sporulation conditions. This study investigated the effect of sporulation temperature on germination of three Bacillus weihenstephanensis strains in specific nutrients (inosine and/or amino acids) with or without prior heat activation (80 °C, 10 min). Sporulation temperature variably affected germination, with stronger effects in moderately responsive strains and nutrients. Heat activation strongly stimulated germination, particularly in nutrients with poorer responses, mitigating differences induced by sporulation temperature. The influence of sporulation temperature on germination and growth in an ESL matrix at refrigeration temperatures (4 °C or 8 °C) in vacuum packaging after heat activation or commercial pasteurization (90 °C, 10 min) was also studied. The latter treatment increased germination rates of surviving spores; however, some strains suffered damage and lost viability upon germination at 4 °C but recovered and grew at 8 °C. These findings highlight the need to account for variability in spore recovery and outgrowth during quantitative risk assessments for psychrotrophic B. cereus in ESL foods.

Members of the B. cereus group are spore-forming organisms commonly associated with spoilage of milk and dairy products. We have determined the genetic identity and growth characteristics of 57 B. cereus isolates collected from a Norwegian ice cream production plant. Our findings revealed persistence of B. cereus spp. strains for up to 19 months, suggesting the plant’s susceptibility to long-term colonization. One of the mesophilic isolates, NVH-YM303, carried a complete cereulide synthetase operon. To assess the potential food poisoning risk associated with the presence of cereulide-producing strains in the production line, we examined the production of cereulide in ice cream and milk at different temperatures by NVH-YM303 and by the emetic psychrotrophic B. weihenstephanensis strain BtB2-4. Our findings revealed that NVH-YM303 produced higher levels of cereulide in ice cream as compared to milk. Furthermore, it was observed that NVH-YM303 produced more cereulide in ice cream at 25 °C compared to 15 °C. Conversely, BtB2-4 produced more cereulide in ice cream at 15 °C than at 25 °C. The results obtained in this study contribute to knowledge important for risk assessment of the potential hazards posed by the presence of B. cereus within ice cream production facilities.

Cereulide-producing Bacillus cereus, which causes foodborne illnesses with vomiting, and psychrotolerant B. cereus group strains such as Bacillus mycoides, which can grow at ≥7°C and cause spoilage of refrigerated foods, are significant concerns for the food industry. Rapid and simple methods to discriminate the cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains are needed. We developed a novel, rapid, and simple method with matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) analysis for simultaneous discrimination of these two groups from other B. cereus group strains. A potassium adduct of cereulide was used to detect cereulide-producing B. cereus, and three ribosomal subunit proteins (L30, S16, and S20) were used to detect psychrotolerant B. cereus group. A total of 51 B. cereus group strains were analyzed by MALDI-TOF MS. The biomarkers allowed successful discrimination of 16 cereulide-producing B. cereus and 15 psychrotolerant B. cereus group strains from other B. cereus group strains. The results showed that this MALDI-TOF MS analysis allows simultaneous discrimination of cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains. This efficient method has the potential to be a valuable tool for ensuring food safety. [ABSTRACT FROM AUTHOR]

Bacillus weihenstephanensis can grow at refrigeration temperature and cause food poisoning. It has been isolated from liquid whole egg products. The moderate heat used for pasteurization of liquid egg products is ineffective for killing spore-forming bacteria, including Bacillus. Available predictive models and a pretrial study in broth suggested the potential for growth of Bacillus spp. under the tested conditions. Hence, hurdles such as storage of product below 4°C or use of preservatives would be needed to ensure the food safety of pasteurized egg products. This study evaluated the growth inhibition of B. weihenstephanensis in pasteurized liquid whole egg product formulated with 6.25 ppm of nisin during storage at refrigerated and refrigerated temperatures at abuse levels for a total 13 weeks in three replicate trials. At day 0, the product had a pH of 7.52 ± 0.29, while background microflora, such as aerobic plate counts (APC), presumptive Bacillus cereus and yeast and molds were <10 CFU/g. Product inoculated with target 2.5 log CFU/g of B. weihenstephanensis, stored at 4°C for 4 weeks and subsequently at 7 or 10°C for 9 weeks, exhibited no growth in all three replicate trials. Average counts reduced (P < 0.05) by at least 1 log in 6 weeks in all samples stored at either 7 or 10°C. Similarly, growth of total plate counts, presumptive Bacillus spp., and yeast and mold counts was not observed in uninoculated controls stored at 4°C for 4 weeks and subsequently at 7 or 10°C for 9 weeks. Visual and odor evaluation performed at each sampling time point showed no abnormalities. This study assessed the efficacy of the maximum level of nisin allowed for use in pasteurized liquid whole eggs and validated the inhibition of B. weihenstephanensis in the product for an extended shelf life of up to 13 weeks. [ABSTRACT FROM AUTHOR]

Some strains of the Bacillus cereus group can be implicated in two types of foodborne intoxication syndromes, namely diarrheal and emetic. The emetic syndrome results from the ingestion of the cereulide toxin produced by particular strains of B. cereus and Bacillus weihenstephanensis. It represents a major concern since it can lead, in severe cases, to the death of the intoxicated patients. This study aimed to characterize the virulent phage Deep-Blue and investigate its biocontrol potential against psychrotolerant B. weihenstephanensis. This myovirus was isolated from an agricultural soil and is stable for at least 6 months at 4 °C. It is specific to members of the B. cereus group and shows a lytic activity against five of twelve B. weihenstephanensis. Its lytic cycle lasts less than 1 h with a final phage progeny of ca. 300 virions. Regarding its bactericidal potential, Deep-Blue was able to reduce B. weihenstephanensis contamination under laboratory conditions (LB broth) and in milk, and to control bacterial growth in food matrices such as milk rice. Finally, the application of Deep-Blue as preventive treatment was shown to efficiently control the development of B. weihenstephanensis biofilm. • Phage Deep-Blue shows lytic activity against psychrotolerant B. weihenstephanensis. • Deep-Blue can reduce contamination under laboratory conditions (LB and in milk). • Deep-Blue can control bacterial growth in food matrices such as milk rice. • Deep-Blue prophylactic application efficiently controls B. weihenstephanensis biofilm development. [ABSTRACT FROM AUTHOR]

pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis is able to efficient self-transfer, to mobilize and retro-mobilize non-conjugative plasmids, including "non-mobilizable" plasmids, and to transfer chromosomal loci. It also displays a remarkable aggregation phenotype associated with conjugation under liquid conditions. However, it was recently shown that aggregation boosts pXO16 transfer but is not mandatory. In this paper, we have further explored pXO16 transfers under various mating conditions and with different members of the Bacillus cereus group. The results indicated that colony or filter mating largely compensate the transfer deficit observed when using a pXO16 aggregation-minus mutant. Using filter mating, pXO16 transfer efficiency and host range were both improved. For instance, pXO16 was shown to transfer itself, and to mobilize the small pUB110 plasmid, from B. thuringiensis serovar israelensis to the thermotolerant Bacillus cytotoxicus at frequencies of 3.3 × 10−3 and 5.2 × 10−4 transconjugants per donor (T/D), respectively. All together, these results indicate that pXO16 can potentially "circulate" among members of the Bacillus cereus group. Yet, this is contrasting with pXO16's known natural distribution, which is apparently limited to the israelensis serovar of B. thuringiensis. • Filter and colony matings can substitute for the aggregation of the conjugative plasmid pXO16. • pXO16 can efficiently conjugate to various members of B. cereus s.l. , including its most distantly member B. cytotoxicus. • pXO16 mobilization of small, non-conjugative plasmids, is also extended to a broader host range. [ABSTRACT FROM AUTHOR]

Abstract Predicting the microbial safety of food products stored in modified atmosphere packaging implies taking into account the effect of oxygen reduction on microbial growth. According to their respiratory-type, the micro-organisms are not impacted similarly by the oxygen concentration. The aim of this article was to quantify and model the oxygen effect on the growth rates of 5 bacterial species: Listeria monocytogenes and Bacillus weihenstephanensis (facultative anaerobic), Pseudomonas fluorescens (strict aerobic), Clostridium perfringens and Clostridium sporogenes (strict anaerobic). The results showed the oxygen concentration doesn't modify the behavior of both facultative anaerobic strains. The growth rate of P. fluorescens decreased with the oxygen concentration, but the effect is only noticeable when the oxygen concentration fell below 3% in the gaseous phase. Conversely, the oxygen acted as a growth inhibitor for both Clostridium species. But total inhibition is reached only for 3.26% and 6.61% respectively for C. sporogenes and C. perfringens. Two models have been fitted for both respiratory-types, the first is the Monod model considering oxygen as a substrate for growth, and the second is the classic inhibitory model based on minimal inhibitory concentration. Highlights • C. perfringens and C. sporogenes tolerate low oxygen presence for growth. • P. fluorescens can grow with oxygen as low as 0.1%. • L. monocytogenes and B. weihenstephanensis growth is unaffected by the low oxygen level. • The inhibitory model describes the behavior of both Clostridii in the presence of oxygen. • Oxygen is a limiting nutrient for P. fluorescens ; this is described by the Monod model. [ABSTRACT FROM AUTHOR]

Nitrate reductase catalyses the oxidation of NAD(P)H and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(P)H. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D). This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min−1 for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48-75.9%), and rhamnolipids (52.7-63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance. [ABSTRACT FROM AUTHOR]

Abstract In this comparative genomics study our aim was to unravel genes under positive selection in the core genome of the Bacillus cereus group. Indeed, the members of this group share close genetic relationships but display a rather large phenotypic and ecological diversity, providing a unique opportunity for studying how genomic changes reflect ecological adaptation during the divergence of a bacterial group. For this purpose, we screened ten completely sequenced genomes of four pathogenic Bacillus species, finding that 254 out of 3093 genes have codon sites with d N / d S (ω) values above one. These results remained unchanged after having disentangled the confounding effects of recombination and selection signature in a Bayesian framework. The presumably adaptive nucleotide polymorphisms are distributed over a wide range of biological functions, such as antibiotic resistance, DNA repair, nutrient uptake, metabolism, cell wall assembly and spore structure. Our results indicate that adaptation to animal hosts, whether as pathogens, saprophytes or symbionts, is the major driving force in the evolution of the Bacillus cereus group. Future work should seek to understand the evolutionary dynamics of both core and accessory genes in an integrative framework to ultimately unravel the key networks involved in host adaptation. Highlights • Adaptation to animal hosts in the Bacillus cereus group is driven by a significant amount of genes under positive selection • Recombination signatures do not erase the selection signal • Adaptive SNPs affect genes involved in antibiotic resistance, DNA repair, nutrient uptake and spore genesis • Pangenome alone does not explain the full adaptive landscape of the B. cereus group. [ABSTRACT FROM AUTHOR]

Polyethylene has achieved an inseparable place in our life due to its recalcitrant nature, durability and cost, especially as a packaging material. Attributable to its inactive nature and degradation resistant property, its collection in nature has turned out to be tremendous. In the present study, polyethylene degrading bacterium was isolated from the garbage soil from Kolhapur and screened under in vitro condition. Through the 16S ribosomal RNA gene partial sequence, the isolated bacterium was identified as Bacillus weihenstephanensis. Polyethylene sheets, only source of carbon, along with synthetic media were incubated on a rotary shaker at 30°C and 110 rpm for 6 months. The biodegraded samples of LDPE and HDPE exhibited weight loss (7.02% and 7.08%, respectively). The biodegradation of LDPE and HDPE sheets was further investigated through FTIR spectroscopy which has confirmed the weakening and breaking of existing bonds and also the formation of new functional carbonyl group (C-O) at 1262 cm-1, 1745 cm-1 and 799 cm-1 which is a result of microbial activity. [ABSTRACT FROM AUTHOR]

Omics databases have exploded, opening the avenue to take strain diversity or physiological variability into consideration in microbiological risk assessment (MRA). However, one obstacle to the integration of omics data in MRA is the production of quantitative data that may be used to build mathematical models. Gene expression is recognized as relevant biomarker to describe bacterial behavior and reverse transcription quantitative PCR (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. However, numerous critical points may arise throughout the entire workflow of RT-qPCR data acquisition influencing accuracy of the results and reliability of the conclusions. Although recommendations about the minimum information that should be found in publications about quantitative real-time PCR experiments, heterogeneity in the reporting of RT-qPCR quality controls in publications remains. Herein, the step-by step RT-qPCR quality controls established for the selection of Bacillus weihenstephanensis resistance biomarkers were described. Throughout this example, appropriate quality procedures and quality controls that shall be set up and carefully assessed to ensure reliable interpretations in RT-qPCR were depicted. [ABSTRACT FROM AUTHOR]

EPR spectroscopy was used to examine the properties of and free radical concentrations in atypical water-soluble melanin-like pigments from Bacillus weihenstephanensis strains. The same EPR spectral shape was observed in bacterial melanins as in eumelanin. The EPR lines were homogeneously broadened. Continuous microwave saturation of the EPR lines indicated slow spin-lattice relaxation processes in the samples. Strong dipolar interactions characterized the tested melanin samples. Higher free radical concentrations were found in bacterial melanin than in synthetic melanin. The free radical concentrations in melanin from B. weihenstephanensis increased in the following order: strain JAS 81/4 < JAS 83/3 < JAS 86/1 < JAS 39/1. [ABSTRACT FROM AUTHOR]

Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities. This indicates that chitinases contribute to antifungal activities. The present study identified B. thuringiensis isolates from tomato roots which exhibited in vitro antifungal activity against Verticillium species. [ABSTRACT FROM AUTHOR]

Nitrate reductase catalyses the oxidation of NAD(P)H and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(P)H. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D). This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking. [ABSTRACT FROM AUTHOR]

Bacillus weihenstephanensis RI12, isolated from hydrocarbon contaminated soil, was assessed for Congo Red bio-treatment potency. Results suggested the potential of this bacterium for use in effective treatment of Congo Red contaminated wastewaters under shaking conditions at acidic and neutral pH value. The strain could tolerate higher doses of dyes as it could decolorize up to 1,000 mg/l of Congo Red. When used as microbial surfactant to enhance Congo Red biodecolorization, Bacillus subtilis SPB1-derived lipopeptide accelerated the decolorization rate and maximized the decolorization efficiency at an optimal concentration of biosurfactant of about 0.075%. Studies ensured that Congo Red removal by this strain could be due to an adsorption phenomena. Germination potencies of tomato seeds using the treated dyes under different conditions showed the efficient biotreatment of the azo dye Congo Red especially with the addition of SPB1 biosurfactant. To conclude, the addition of SPB1 bioemulsifier reduced energy costs by reducing the effective decolorization period; the biosurfactant stimulated bacterial decolorization method may provide a highly efficient, inexpensive and time-saving procedure in the treatment of textile effluents. [ABSTRACT FROM AUTHOR]

The article discusses a study in India that tested the effectiveness of bacterial agents Bacillus cereus CWBI-B1082, Bacillus weihenstephanensis WSBC and Bacillus weihenstephanensis KBAB4 against the larvae of Culicoides spp. It cites the isolation of the bio-control agents (BCA) from the flies' natural breeding sites and the results of their in-vivo and in-vitro trials. It also notes the BCAs' characteristics and their efficacy in reducing the larval count from drainage channel.

Background Cereulide is a cyclic dodecadepsipeptide ionophore, produced via non-ribosomal peptide synthetases (NRPS), which in rare cases can lead to human death. Early studies had shown that emetic toxin formation belongs to a homogeneous group of Bacillus cereus sensu stricto and the genetic determinants of cereulide (a 24-kb gene cluster of cesHPTABCD) are located on a 270-kb plasmid related to the Bacillus anthracis virulence plasmid pXO1. Results The whole genome sequences from seven emetic isolates, including two B. cereus sensu stricto and five Bacillus weihenstephanensis strains, were compared, and their inside and adjacent DNA sequences of the cereulide biosynthesis gene clusters were analyzed. The sequence diversity was observed, which classified the seven emetic isolates into three clades. Different genomic locations of the cereulide biosynthesis gene clusters, plasmid-borne and chromosome-borne, were also found. Potential mobile genetic elements (MGEs) were identified in the flanking sequences of the ces gene cluster in all three types. The most striking observation was the identification of a putative composite transposon, Tnces, consisting of two copies of ISces element (belonging to IS6 family) in opposite orientations flanking the ces gene cluster in emetic B. weihenstephanensis. The mobility of this element was tested by replacing the ces gene cluster by a KmR gene marker and performing matingout transposition assays in Escherichia coli. The results showed that Tnces::km transposes efficiently (1.04 × 10-3 T/R) and produces 8-bp direct repeat (DR) at the insertion sites. Conclusions Cereulide biosynthesis gene clusters display sequence diversity, different genomic locations and association with MGEs, in which the transposition capacity of a resistant derivative of the composite transposon Tnces in E. coli was demonstrated. Further study is needed to look for appropriate genetic tools to analysis the transposition of Tnces in Bacillus spp. and the dynamics of other MGEs flanking the ces gene clusters. [ABSTRACT FROM AUTHOR]

Abstract: This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (zpH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods. [Copyright &y& Elsevier]

Abstract: Superdormant spores are those which germinate extremely slowly compared to the majority of the spore population. The development of a method to isolate large quantities of superdormant spores in a laboratory setting has allowed for further study of these hardy organisms. In this study, the ability of nonthermal processing technologies, including a combined hurdle technology of aqueous ozone and high pressure processing (HPP), to inactivate superdormant spore populations of mesophilic and psychrotolerant isolates of Bacillus cereus was investigated. Superdormant spores were approximately 20% more resistant to ozone treatment than heterogeneous spore populations (p=0.02) and psychrotolerant species were approximately 31.9% more resistant than mesophilic species (p=0.004). The combined ozone-HPP hurdle technology achieved a maximum 2.67-logCFU/mL reduction of superdormant spores. Industrial relevance: Nonthermal processing technologies including high pressure processing (HPP) and ozone have high implications for reducing the number of spores in foods; however, these technologies are not widely utilized in the industry due to their inability to completely inactivate all the spores within a product. The spores that do not germinate as easily as the majority of a spore population are known as superdormant spores. This study demonstrates that an ozone-HPP combined hurdle technology may be able to effectively reduce the number of superdormant spores, specifically psychrotolerant sporeforming toxin-producing species such as B. cereus and Bacillus weihenstephanensis, in refrigerated food products such as RPFEDs and reduce the risk of foodborne illness. [Copyright &y& Elsevier]

The Bacillus cereus group comprises seven bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, and Bacillus weihenstephanensis. Bacillus weihenstephanensis is distinguished based on its capability to grow at 7 °C but not at 43 °C, and the presence of specific signature sequences in the 16S rRNA and cspA genes and in several housekeeping genes: glpF, gmK, purH, and tpi. Bacillus weihenstephanensis-specific signature sequences were found in some B. cereus and B. mycoides strains suggesting psychrotolerance. This was confirmed by growth at 7 °C but not at 43 °C. The other B. cereus and B. mycoides strains and all B. anthracis, B. thuringiensis, and B. pseudomycoides harbored the mesophilic signature sequences. The strains tested grew at 43 °C but did not grow at 7 °C. A maximum-likelihood phylogenetic tree was inferred from comparisons of the concatenated nucleotide sequences. Three groups and one branch were revealed. Group I, II, and III comprised the mesophilic B. cereus, some mesophilic B. mycoides, and all B. anthracis and B. thuringiensis strains; the psychrotolerant B. cereus and B. mycoides, and all B. weihenstephanensis strains; and some mesophilic B. mycoides and all B. pseudomycoides strains, respectively. The branch corresponds to the single B. cytotoxicus strain. Based on psychrotolerance and multilocus sequence analysis, further confirmed by comparisons of amino acid sequences, we show that some B. cereus and B. mycoides strains should be reclassified as B. weihenstephanensis. [ABSTRACT FROM AUTHOR]

The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes ( glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified. [ABSTRACT FROM AUTHOR]

Abstract: Cereulide-producing Bacillus cereus, which causes foodborne illnesses with vomiting, and psychrotolerant B. cereus group strains such as Bacillus mycoides, which can grow at ≥7°C and cause spoilage of refrigerated foods, are significant concerns for the food industry. Rapid and simple methods to discriminate the cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains are needed. We developed a novel, rapid, and simple method with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis for simultaneous discrimination of these two groups from other B. cereus group strains. A potassium adduct of cereulide was used to detect cereulide-producing B. cereus, and three ribosomal subunit proteins (L30, S16, and S20) were used to detect psychrotolerant B. cereus group. A total of 51 B. cereus group strains were analyzed by MALDI-TOF MS. The biomarkers allowed successful discrimination of 16 cereulide-producing B. cereus and 15 psychrotolerant B. cereus group strains from other B. cereus group strains. The results showed that this MALDI-TOF MS analysis allows simultaneous discrimination of cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains. This efficient method has the potential to be a valuable tool for ensuring food safety., (Copyright ©, International Association for Food Protection.)

Abstract: The aim of this study was to define an integrative approach to identify resistance biomarkers using gene expression quantification and mathematical modelling. Mid-exponentially growing cells were transferred into acid conditions (BHI, pH 4.6) to obtain inactivation kinetics, performed in triplicate. The inactivation curve was fitted with a mixed Weibull model. This model allowed to differentiate two subpopulations with various acid resistances among the initial population. In parallel, differential gene expression was quantified by RT-qPCR. While narL was down-regulated throughout acid inactivation, sigB and katA were up-regulated. sigB expression up-regulation peak was correlated to the less resistant subpopulation when katA up-regulation, was correlated to the more resistant subpopulation. Moreover, differences in population structure were highlighted between each replicate. The higher proportion of the more resistant subpopulation was linked to a higher katA gene expression. These results suggest that sigB and katA might be used as different types of biomarkers, for instance to track moderate and high acid-resistance, respectively. The use of this approach combining RT-qPCR and predictive modelling to track cellular biomarker variations appears as an interesting tool to take into account physiological cell responses into mathematical modelling, allowing an accurate prediction of microbial behaviour. [Copyright &y& Elsevier]

Abstract: Sporulation niches in the food chain are considered as a source of hazard and are not clearly identified. Determining the sporulation environmental boundaries could contribute to identify potential sporulation niches. Spore formation was determined in a Sporulation Mineral Buffer. The effect of incubation temperature, pH and water activity on time to one spore per mL, maximum sporulation rate and final spore concentration was investigated for a Bacillus weihenstephanensis and a Bacillus licheniformis strain. Sporulation boundaries of B. weihenstephanensis and of B. licheniformis were similar to, or included within, the range of temperatures, pH and water activities supporting growth. For instance, sporulation boundaries of B. weihenstephanensis were evaluated at 5°C, 35°C, pH 5.2 and a w 0.960 while growth boundaries were observed at 5°C, 37°C, pH 4.9 and a w 0.950. Optimum spore formation was determined at 30°C pH 7.2 for B. weihenstephanensis and at 45°C pH 7.2 for B. licheniformis. Lower temperatures and pH delayed the sporulation process. For instance, the time to one spore per mL was tenfold longer when sporulation occurred at 10°C and 20°C, for each strain respectively, than at optimum sporulation temperature. The relative effect of temperature and pH on sporulation rates and on growth rates is similar. This work suggests that the influence of environmental factors on the quantitative changes in sporulation boundaries and rates was similar to their influence on changes in growth rate. [Copyright &y& Elsevier]

Abstract: Bacillus weihenstephanensis is a psychrotolerant bacterium belonging to the Bacillus cereus group. Some strains may be cytotoxic although they have not been described as food-poisoning agents so far. The objective of this work is to model the effects of temperature and a w downshifts on the lag time of B. weihenstephanensis and the dependence of μ max on the growth conditions (temperature and a w). Effects of temperature downshifts were studied on 30 experimental conditions (shifts magnitude ranging from 2 to 20°C, temperature after shift from 10 to 20°C and a w ranging from 0.977 to 0.997). Osmotic shifts were studied for 13 conditions (shift magnitude ranging from 0.008 to 0.020 units of a w, temperature from 10 to 30°C, a w after shift from 0.977 to 0.997). Experimental results show that temperature downshifts were able to induce considerable lag times (up to 20days) when occurring near the growth limits. At lower temperatures, osmotic shifts had also a significant effect. Validation experiments in food subjected to changing conditions of temperature showed that the model provided valid predictions in diluted creamed pasta but overestimated bacterial growth in carrot soup (fail safe predictions). [Copyright &y& Elsevier]

Abstract: Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T min(R), T opt(R), T max(R), pHmin(R) and pHopt(R)) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry. [Copyright &y& Elsevier]

Abstract: The major objective of this study was to determine the influence of the initial headspace and dissolved O2 level and vacuum packaging on growth and diarrhoeal enterotoxin production by Bacillus weihenstephanensis on potato based ready-to-eat food products. In general, the lower the initial headspace or dissolved O2 level the slower the maximum growth rate (μ max, log10 CFU g−1 d−1), the longer the lag phase duration (λ, d) and the smaller the maximum population density (N max, log10 CFU g−1) became. The slowest μ max, the longest λ and the smallest N max were generally found for growth under vacuum packaging. This implies shorter shelf-lives will occur at higher initial headspace or dissolved O2 levels as the growth of B. weihenstephanensis to the infective dose of 105 CFU g−1 in such atmospheres takes a shorter time. Significant consumption of dissolved O2 only occurred when growth shifted from the lag to the exponential phase and growth generally transitioned from the exponential to the stationary phase when the dissolved O2 levels fell below ca. 75 ppb. Diarrhoeal enterotoxin production (determined via detection of the L2 component of haemolytic BL) was similar for growth under initial headspace O2 levels of 1–20.9%, and was only reduced when growth took place under vacuum packaging. The reduction in L2 production when growth took place under vacuum was most probably related to the low final cell densities observed under this condition. Both growth and L2 production were inhibited over a 32-day incubation period at 7 °C by 40% CO2 irrespective of the headspace or dissolved O2 levels. The results illustrate the importance of residual O2 and CO2 on the shelf-stability and safety of modified atmosphere packaged potato based ready-to-eat food products with regards to B. weihenstephanensis. [Copyright &y& Elsevier]

Eighty soil-borne Bacillus cereus group isolates were collected from two neighbouring geographical sites in Belgium. Their genetic relationships and population structure were assessed using Multilocus sequence typing analysis of five chromosomal genes, while the contribution of extrachromosomal elements to the population dynamics was gauged by the presence, diversity and transfer capacity of pXO1- and pXO2-like plasmids. Globally, the bacterial population displayed a broad diversity, including an important subpopulation of psychrotolerant isolates related to Bacillus weihenstephanensis. pXO1- and pXO2-like replicons were present in 12% and 21% of the isolates, but no Bacillus anthracis-related toxin genes were found. Furthermore, only one of the isolates containing a pXO2-related plasmid was shown to be able to mobilize small non-self-conjugative plasmids. Interestingly, several B. cereus sensu lato isolates displaying the same sequence type were observed to have different plasmid contents, suggesting the occurrence of horizontal gene exchange. Similarly, a number of pXO2-like replicons with identical sequences were found in distinct bacterial isolates, therefore strongly arguing for lateral transfers among sympatric bacteria. [ABSTRACT FROM AUTHOR]

Abstract: Emetic toxin (cereulide) formation was recently identified in a psychrotolerant species, Bacillus weihenstephanensis [Thorsen, L., Hansen, B.M., Nielsen, K.F., Hendriksen, N.B., Phipps, R.K., Budde, B.B., 2006. Characterization of emetic Bacillus weihenstephanensisis, a new cereulide-producing bacterium. Applied and Environmental Microbiology, 72, 5118–5121.]. Although recent findings indicated B. weihenstephanensis as a cereulide producer only limited information is available regarding environmental conditions affecting cereulide production. In the present study a model agar system was used to compare cereulide production during surface growth of B. weihenstephanensis MC67, and two well known mesophilic cereulide producing Bacillus cereus strains, NC7401 and NS117. Cereulide production was quantified by use of Liquid-Chromatography Mass Spectrometry/Mass Spectrometry. Cereulide production of B. weihenstephanensis MC67 occurred in stationary growth phase, as previously observed for B. cereus, and biomass formation and cereulide formation showed a linear correlation. During incubation at 5 °C for 1, 2 and 3 weeks growth was inhibited and as a consequence no detectable cereulide production occurred for any of the three strains. Similar results were obtained for the mesophilic B. cereus strains when incubated at 8 °C, whereas B. weihenstephanensis MC67 grew to stationary phase and produced 0.002 μg cereulide/cm2 agar surface in 1 week. Raising the temperature from 5 °C to 25 °C for 24 h after 1 week of incubation resulted in growth to stationary phase and production of variable levels of cereulide. B. weihenstephanensis MC67 produced 6.18 μg cereulide/cm2, B. cereus NS117 0.91 μg cereulide/cm2 and B. cereus NC7401 0.09 μg cereulide/cm2. Similar levels of cereulide was produced by the mesophilic strains when raising the temperature from 8 °C (instead of from 5 °C) to 25 °C for 24 h, while a considerably lower level was produced by B. weihenstephanensis MC67 (0.10 μg cereulide /cm2). If the temperature was raised from 5 °C and 8 °C to 25 °C for 24 h after an increased incubation time for 2 and 3 weeks, all three strains produced considerably less cereulide. B. weihenstephanensis MC67 produced 100–6000 times less and the mesophilic B. cereus strains produced 9–40 times less cereulide. These results can partly be explained by differences in the growth at the temperature abuse. Effect of chill storage on cereulide production at temperature abuse has not been investigated previously. Results of the present study indicate that storage at 5 and 8 °C will not lead to emetic intoxications, however the time at, and choice of chill temperature will determine the amount of cereulide produced in a temperature abuse situation. These results are of relevance for the safety of chilled foods of extended durability. [Copyright &y& Elsevier]

Aim: To determine the potential of Bacillus thuringiensis, known primarily for its entomopathogenicity, to be a psychrotolerant contaminant of stored products. Methods and Results: We determined the genetic properties and diversity of cold-adapted isolates of B. thuringiensis based on (i) the presence of cspA, a genetic determinant that confers psychrotolerance in Bacillus weihenstephanensis, (ii) 16S rRNA genes, and (iii) pulse-field gel electrophoretic (PFGE) genome profiles. We assessed the pathogenic potential of these isolates based on whether they harboured various combinations of known toxigenic-associated determinants ( nheA, hblA, cytK). Of 36 nonclonal B. thuringiensis cultured from soil and milk, 21 harboured cspA, and of these, 16 (76%) were psychrotolerant and possessed genetic signatures typical of psychrotrophic Bacillus species. The majority of psychrotolerant isolates contained various combinations of nheA, hblA, and cytK. Conclusion: Our results show that natural isolates of psychrotolerant B. thuringiensis occur in soil and milk, and suggest that psychrotolerance is determined by cspA. Significance and Impact of the study: The presence of cspA in combination with nheA, hblA, and cytK could be of concern if commercial products are contaminated with strains that harbour these determinants. [ABSTRACT FROM AUTHOR]

Abstract: The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO2. The sensitivity to 20% CO2 was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO2 was combined with 2% O2 and in 3 weeks when combined with “0”% O2 (the remaining atmosphere was made up from N2). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m2/24 h and the atmospheres were 2% O2/20% CO2 and “0”% O2/20% CO2. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m2/24 h and “0”% O2/20 % CO2) inhibited growth of the three strains during 4 weeks storage at 8 °C. Cereulide production was undetectable during storage at 8 °C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 °C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week in MAP with OTR of 1.3 or 40 ml/m2/24 h and 2% O2 resulted in cereulide concentrations of 0.816–1.353 µg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m2/24 h, “0”% O2/20 % CO2) resulted in minimal cereulide production (0.004 µg/g meat sausage) at abuse condition. Extension of MAP storage at 8 °C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production. Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability. [Copyright &y& Elsevier]

The article cited a study on a metal-resistant bacterial strain SM3 characterized as Bacillus weihenstephanensis isolated from Portugal's serpentine soil. The study was based on the biochemical characteristics and on the comparative analysis of the partial 16S ribosomal DNA sequence. In the analysis, bacillus weihenstephanensis SM3 showed a high degree of resistance to nickel, copper, zinc, and also to antibiotics. The study also elucidates the multifarious role of strain SM3 in plant growth promotion and its metal mobilizing potential.

Abstract: Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In the present study, the bacteriocin was tested alone and in combination with other antimicrobials for decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by 1.0–1.5 and by 1.5–2.38log units right after application of treatment, respectively. In both cases, the bacteriocin was effective in reducing the remaining viable population below detection levels during further storage of the samples at 6°C, but failed to prevent regrowth in samples stored at 15 or 22°C. Application of washing treatments containing enterocin AS-48 in combination with several other antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride provided the best results. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not detected or remained at very low concentrations in the treated samples during a 1-week storage period at 15°C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48 producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and produce bacteriocin on sprouts both at 15 and 22°C. At 15°C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22°C. The results obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperature-abuse conditions. [Copyright &y& Elsevier]

Abstract: Characteristics concerning diarrhoeal potential were investigated in B. cereus dairy strains. The thirty-nine strains, isolated from whipping cream, were tested for cytotoxicity after culturing at human body temperature as well as 25 °C and 32 °C. At 37 °C, none of the strains were highly cytotoxic. This observation suggests that those strains should be considered to pose a minor risk with regard to diarrhoeal food poisoning. However, some strains were moderately or highly cytotoxic when grown at 25 °C and 32 °C. While the majority of the strains were able to grow at refrigeration temperatures, only four B. weihenstephanensis strains were identified among them when subjected to discriminative PCR assays and growth temperatures which delimit this species. [Copyright &y& Elsevier]