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Bacillus cereus is rarely implicated when diarrheal cases in children are diagnosed in developing countries due to the lack of molecular methods to identify its enterotoxigenic genes. We report that out of 62 enterobacteria isolated from 70 stool samples collected from children hospitalized at the Mile 4 Hospital, Ebonyi State, Nigeria, 24 isolates were identified as B. cereus based on 16SrRNA gene sequence. The enterotoxins genes nheA and cytK2 were detected in 23 out of the 24 isolates, while hblC was detected in 19 isolates. B. cereus may be responsible for greater number of yearly incidences of acute childhood gastroenteritis in Nigeria., Competing Interests: Declaration of competing interest Authors Declare No Competing Interest., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Bacillus paranthracis , a Gram-positive conditional pathogen of Bacillus cereus group species, is capable of causing foodborne and waterborne illnesses, leading to intestinal diseases in humans characterized by diarrhoea and vomiting. However, documented cases of B. paranthracis infection outbreaks are rare in the world, and the genomic background of outbreak strains is seldom characterized. This study retrospectively analyzed strains obtained from an outbreak in schools, as well as from water systems in peri-urban areas, China, in 2020. In total, 28 B. cereus group isolates were retrieved, comprising 6 from stool samples and 22 from water samples. Epidemiological and phylogenetic investigations indicated that the B. paranthracis isolate from drinking water as the causative agent of the outbreak. The genomic comparison revealed a high degree of consistency among 8 outbreak-related strains in terms of antimicrobial resistance gene profiles, virulence gene profiles, genomic content, and multilocus sequence typing (MLST). The strains related to the outbreak show highly similar genomic ring diagrams and close phylogenetic relationships. Additionally, this study shed light on the pathogenic potential and complexity of B. cereus group through its diversity in virulence genes and mice infection model. The findings highlight the usefulness of B. paranthracis genomes in understanding genetic diversity within specific environments and in tracing the source of pathogens during outbreak situations, thereby enabling targeted infection control interventions.

Bacillus cereus sensu stricto (s.s.) is a well-known foodborne pathogen that produces a range of enterotoxins and is able to cause two different types of foodborne illnesses-the emetic and the diarrheal syndromes. In this study, 54 B. cereus s.s. strains isolated from foodstuff and foods involved in food poisoning outbreaks were characterized according to the presence of toxin-encoding genes, virulence-encoding genes, and panC typing. Most isolates were assigned to panC groups IV (61.1%) and III (25.9%), but members of groups II and V could also be found. Investigation of specific alleles revealed high numbers of isolates carrying toxin and other virulence genes including nheA (100%), nheB (100%), hblA (79.6%), hblC (79.6%), hblD (74.1%), cytK-2 (61.1%), clo (100%), pc-plc (75.9%), sph (68.5%), pi-plc (66.6%), hlyIII (62.9%), and hlyII (24.1%). All isolates were negative for ces and cytK-1. In summary, we detected various enterotoxin and other virulence factor genes associated with diarrheal syndrome in strains analyzed, implicated or not with food poisoning. Furthermore, the most isolates analyzed belong to high-risk phylogenetic groups' panC types III and IV. Our study provides a convenient molecular scheme for characterization of B. cereus s.s. strains responsible for food poisoning outbreaks in order to improve the monitoring and investigation and assess emerging clusters and diversity of strains., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Microorganisms that cause foodborne illnesses challenge the food industry; however, environmental studies of these microorganisms on raw grain, prior to food processing, are uncommon. Bacillus cereus sensu lato is a diverse group of bacteria that is common in our everyday environment and occupy a wide array of niches. While some of these bacteria are beneficial to agriculture due to their entomopathogenic properties, others can cause foodborne illness; therefore, characterization of these bacteria is important from both agricultural and food safety standpoints. We performed a survey of wheat and flax grain samples in 2018 (n = 508) and 2017 (n = 636) and discovered that B. cereus was present in the majority of grain samples, as 56.3% and 85.2%, in two years respectively. Whole genome sequencing and comparative genomics of 109 presumptive B. cereus isolates indicates that most of the isolates were closely related and formed two genetically distinct groups. Comparisons to the available genomes of reference strains suggested that the members of these two groups are not closely related to strains previously reported to cause foodborne illness. From the same data set, another, genetically more diverse group of B. cereus was inferred, which had varying levels of similarity to previously reported strains that caused disease. Genomic analysis and PCR amplification of genes linked to toxin production indicated that most of the isolates carry the genes nheA and hbID, while other toxin genes and gene clusters, such as ces, were infrequent. This report of B. cereus on grain from Canada is the first of its kind and demonstrates the value of surveillance of bacteria naturally associated with raw agricultural commodities such as cereal grain and oilseeds., Competing Interests: The authors have declared that no competing interests exist.

B. cereus is a human pathogen associated with food poisoning leading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum of B. cereus ranges from strains used as probiotics in humans to lethal highly toxic strains. In this study, we gathered a collection of 100 strains representative of the pathological diversity of B. cereus in humans, and characterized these strains for their cytotoxic potential towards human cells. We analyzed the correlation between cytotoxicity to epithelial and macrophage cells and the combination of 10 genes suspected to play a role during B. cereus virulence. We highlight genetic differences among isolates and studied correlations between genetic signature, cytotoxicity and strain pathological status. We hope that our findings will improve our understanding of the pathogenicity of B. cereus, thereby making it possible to improve both clinical diagnosis and food safety., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Three Gram-positive bacterial strains, BML-BC004, BML-BC017 and BML-BC059, isolated from blood samples from three inpatients in Japan, were identified as members of Bacillus cereus using matrix-assisted laser desorption ionization time-of-flight MS. The 16S rRNA gene sequences of these three strains were more than 97.1 % similar to 18 type strains belonging to the B. cereus group. Whole-genome comparisons, using average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), confirmed that the three strains represented three individual distinct species belonging to the B. cereus group. A phylogenetic tree showed that BML-BC004, BML-BC017 and BML-BC059 were located close to B. luti , B. mobilis and B. paramycoides , respectively. Based on these phylogenetic and phenotypic data, including values below the threshold for ANI and dDDH, the three strains should be classified as representing three different novel species of the B. cereus group: Bacillus sanguinis sp. nov., with type strain BML-BC004 T (=DSM 111102 T =JCM 34122 T ), Bacillus paramobilis sp. nov., with type strain BML-BC017 T (=DSM 111100 T =JCM 34124 T ) and Bacillus hominis sp. nov., with type strain BML-BC059 T (=DSM 111101 T =JCM 34125 T ).

A potential pesticide degrading bacterial isolate (2D), showing maximum tolerance (450 mg∙L-1) for cypermethrin, fipronil, imidacloprid and sulfosulfuron was recovered from a pesticide contaminated agricultural field. The isolate degraded cypermethrin, imidacloprid, fipronil and sulfosulfuron in minimal salt medium with 94, 91, 89 and 86% respectively as revealed by high performance liquid chromatography (HPLC) and gas chromatography (GC) analysis after 15 days of incubation. Presence of cyclobutane, pyrrolidine, chloroacetic acid, formic acid and decyl ester as major intermediate metabolites of cypermethrin biodegradation was observed in gas chromatography mass spectrometry (GC-MS) analysis. Results based on 16S rDNA sequencing, and phylogenetic analysis showed maximum similarity of 2D with Bacillus cereus (Accession ID: MH341691). Stress responsive and catabolic/pesticide degrading proteins were over expressed in the presence of cypermethrin in bacteria. Enzymatic kinetics of laccase was deduced in the test isolate under normal and pesticide stress conditions which suggested that the production of enzyme was induced significantly in pesticide stress (163 μg.μL-1) as compare to normal conditions(29 μg.μL-1) while the Km value was decreased in pesticides stress condition (Km = 10.57 mM) and increases in normal condition (Km = 14.33 mM).Amplification of laccase gene showed a major band of 1200bp. The present study highlights on the potential of 2D bacterial strain i.e., high tolerance level of pesticide, effective biodegradation rate, and presence of laccase gene in bacterial strain 2D, could become a potential biological agent for large-scale treatment of mixture of pesticide (cypermethrin, fipronil, imidacloprid and sulfosulfuron) in natural environment (soil and water)., Competing Interests: The authors have declared that no competing interests exist.

A stochastic model that predicts the maximum specific growth rate (μ max ) of Bacillus cereus sensu lato as a function of temperature was developed. The model integrates the intra-species variability by incorporating distributions of cardinal parameters (T min , T opt , T max ) in the model. Growth rate data were generated for 22 strains, covering 5 major phylogenetic groups of B. cereus, and their cardinal temperatures identified. Published growth rate data were also incorporated in the model fitting, resulting in a set of 33 strains. Based on their cardinal temperatures, we identified clusters of Bacillus cereus strains that show similar response to temperature and these clusters were considered separately in the stochastic model. Interestingly, the μ opt values for psychrotrophic strains were found to be significantly lower than those obtained for mesophilic strains. The model developed within this work takes into account some correlations existing between parameters (μ opt , T min , T opt , T max ). In particular, the relationship highlighted between the b-slope of the Ratkowsky model and T min (doi: https://doi.org/10.3389/fmicb.2017.01890) was adapted to the case of the popular Cardinal Temperature Model. This resulted in a reduced model in which μ opt is replaced by a function of T min , T opt and 2 strain-independent parameters. A correlation between the T min parameter and the experimental minimal growth temperature was also highlighted and integrated in the model for improved predictions near the temperature growth limits. Compared to the classical approach, the model developed in this study leads to improved predictions for temperatures around T min and more realistic tails for the predicted distributions of μ max . It can be useful for describing the variability of the Bacillus cereus Group in Quantitative Microbial Risk Assessment (QMRA). An example of application of the stochastic model to Reconstituted Infant Formulae (RIF) was proposed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacteria survive metal stress by several mechanisms and metal binding is one such mechanism which has been screened in the present study to investigate the survival strategies of metal resistant bacteria. The production of siderophores, a metal chelating agent, was detected by chrome azurol S agar assay. The changes in cell wall studied by analysing the peptidoglycan and teichoic acid content indicated an increase in the cell wall content. Evaluation of morphological and physiological alterations like cell size, granularity analysed by SEM and flow cytometry analysis revealed an increase in cell size and granularity respectively. The transformation of phosphates monitored by 31 P NMR analysis indicated the presence of inorganic phosphate. Based on the cell wall changes and the 31 P NMR analysis, the surface charge of the organism was studied by zeta potential which displayed a difference at pH7., (© 2020 The Society for Applied Microbiology.)

Aims: Bacillus cereus sensu lato is a complex group of closely related bacteria, which are commonly present in the natural environment and food products. These organisms may cause food poisoning and spoilage as well as opportunistic infections. Thus far, their resistance to selected antibiotics has been explored only in part, especially in the context of strain source. Therefore, our goal was to compare the resistance of B. cereus sl from milk (environment with the potential impact of antibiotics) with strains from soil and pepper (environment without contact with antibiotics) in relation to their origin, toxicity and phylogenetic relationships., Methods and Results: Antibiotic resistance of B. cereus sl was assessed by determining their minimal inhibitory concentrations (MICs) followed by statistical analyses. The phylogeny of the bacteria was investigated by multilocus sequence typing, and toxicity was determined with quantitative reverse-transcription real-time PCR. We found that the isolates from milk were more often multiresistant and exhibited a common resistance pattern to β-lactams but a varied sensitivity to the tested macrolides, clindamycin, tetracycline and vancomycin. Moreover they displayed often significantly higher average MICs; however, their resistance did not correlate with phylogeny, toxicity, or in most cases, with taxonomic affiliation., Conclusion: We conclude that mainly food matrices may serve as an important reservoir of resistant isolates of B. cereus sl and that the use of antibiotics for the treatment of animal diseases must be carefully monitored as it strongly promotes natural selection for multiresistant strains, even among opportunist pathogens., Significance and Impact of the Study: The fact that compared to the isolates from natural habitats, nonpathogenic B. cereus sl isolated from food acquire antibiotic resistance faster, should increase producers and consumers awareness and result in protection of public health., (© 2020 The Society for Applied Microbiology.)

This study addresses the biodiversity of Bacillus cereus group population present along the value chain of milk for consumption. The B. cereus population did not grow and remained mainly unaltered during storage of milk at 4 °C while storage at a suboptimal temperature at 8 °C (representative of a broken cold chain) caused a major shift in its composition. Mesophilic strains dominated the B. cereus population in raw milk and after storage at 4 °C, while psycrotrophic strains dominated after storage at 8 °C. All psycrotrophic and mesophilic isolates (n = 368) demonstrated high spoilage potentials of the milk components. Fifteen out of 20 mesophilic isolates but only two out of 40 psychrotrophic isolates, exhibited vero cell toxicity. No genes encoding the emetic toxin cereulide were detected in the genomes of 100 milk isolates while 14 of them harbored the enterotoxin genes cytK1/cytK2. Both psycrotrophic and mesophilic isolates carried the enterotoxin genes nheA and hblA. Together, the results provide insight into the composition and properties, of the B. cereus population present in milk along the value chain and during storage at optimal refrigerated temperature and at suboptimal temperature. This knowledge is useful in the dairy industry's work to assure high quality products and for risk assessment., (Copyright © 2020. Published by Elsevier Ltd.)

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 10 3 cfu/mL, with a dynamic range of detection at 10 3 to 10 7 cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 10 1 cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Bacillus thuringiensis bacteria share similar genetic, physiological, and biochemical characteristics with other members of the Bacillus cereus group. Their diversity and entomopathogenic origin are related to their mobile genetic elements. However, the effects of wide-spread application of B. thuringiensis-based pesticides on genetically related B. cereus group populations present in the environment remain poorly understood., Results: We first identified pBMB76 from B. thuringiensis tenebrionis as a new conjugative plasmid. Mixed mating experiments suggested that pBMB76 may compete with pHT73, another known conjugative plasmid. Applications of single (tenebrionis 4AA1 and kurstaki HD73 carrying pBMB76 and pHT73, respectively) and mixed (4AA1 + HD73) B. thuringiensis strains were performed in confined plot habitats (soil and leaf) over two planting seasons. In total, 684 B. cereus group isolates were randomly selected from different treatment sets, and the transmissibility and occurrence rate of potential conjugative plasmids were surveyed. Results showed that the percentage of isolates with plasmid mobility was markedly enhanced in the B. thuringiensis-sprayed groups. Furthermore, we performed multi-locus sequence typing (MLST) for a subset of 291 isolates, which indicated that the dominant sequence types in the treated habitats were identical or related to the corresponding sprayed formulations., Conclusions: The application of B. thuringiensis strains with conjugal and mobilizing capability drove gene transmissibility within the B. cereus group populations in confined habitats and potentially modified the population structure.

Gram-positive, spore-forming members of the Bacillus cereus group species complex are widespread in natural environments and display various degrees of pathogenicity. Recently, B. cereus group strain Bacillus mycoides Flugge ATCC 21929 was found to represent a novel lineage within the species complex, sharing a relatively low degree of genomic similarity with all B. cereus group genomes (average nucleotide identity [ANI] < 88). ATCC 21929 has been previously associated with the production of a patented antibiotic, antibiotic 60-6 (i.e., cerexin A); however, the virulence potential and growth characteristics of this lineage have never been assessed. Here, we provide an extensive genomic and phenotypic characterization of ATCC 21929, and we assess its pathogenic potential in vitro. ATCC 21929 most closely resembles Bacillus paramycoides NH24A2 T (ANI and in silico DNA-DNA hybridization values of 86.70 and 34.10%, respectively). Phenotypically, ATCC 21929 does not possess cytochrome c oxidase activity and is able to grow at a range of temperatures between 15 and 43°C and a range of pH between 6 and 9. At 32°C, ATCC 21929 shows weak production of diarrheal enterotoxin hemolysin BL (Hbl) but no production of nonhemolytic enterotoxin (Nhe); at 37°C, neither Hbl nor Nhe is produced. Additionally, at 37°C, ATCC 21929 does not exhibit cytotoxic effects toward HeLa cells. With regard to fatty acid composition, ATCC 21929 has iso-C17:0 present in highest abundance. Based on the characterization provided here, ATCC 21929 T (= PS00077A T = PS00077B T = PSU-0922 T = BHP T ) represents a novel effective B. cereus group species, which we propose as effective species " Bacillus clarus " IMPORTANCE The B. cereus group comprises numerous closely related lineages with various degrees of pathogenic potential and industrial relevance. Species-level taxonomic classification of B. cereus group strains is important for risk evaluation and communication but remains challenging. Biochemical and phenotypic assays are often used to assign B. cereus group strains to species but are insufficient for accurate taxonomic classification on a genomic scale. Here, we show that antibiotic-producing ATCC 21929 represents a novel lineage within the B. cereus group that, by all metrics used to delineate prokaryotic species, exemplifies a novel effective species. Furthermore, we show that ATCC 21929 is incapable of producing enterotoxins Hbl and Nhe or exhibiting cytotoxic effects on HeLa cells at human body temperature in vitro These results provide greater insight into the genomic and phenotypic diversity of the B. cereus group and may be leveraged to inform future public health and food safety efforts., (Copyright © 2020 Méndez Acevedo et al.)

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of this study was to optimize the culture conditions for production, recovery and characterization of polyhydroxyalkanoate (PHA) from potential Bacillus cereus strain BNPI-92. Cost effective carbon sources such as crude oil (CO), rice bran (RB), sugar cane molasses (SCM) and wheat bran (WB) were evaluated for PHA production using the isolate. Higher cell dry weight (CDW) (g/L), PHA concentration (P conc.) (g/L) PHA contents (PC) (%) and PHA synthesis rate (PSR) were obtained from Glucose (60.67% w/v) as a carbon source at 37 °C and pH 7 using Taguchi DOE methods. The polymer was characterized by FTIR and its functional groups (C=O) (1719.41 cm -1 wavenumber) was a characteristic feature of PHB polymer. For 1 HNMR, signals of methyne (CH) (5.28 ppm), methylene (-CH2-) (2.45 ppm) and methyl (CH3) (1.27 ppm) and copolymers were predicted. Copolymers such as P (3HB-co-3 HV) and P(3HB-co-3HHX) i.e. characteristic feature of PHB were obtained from a sample of glucose, WB, and RB, respectively. XRD pattern also confirmed the presence of PHA. The major compounds obtained from GC/MS analysis of the polymer recovered from the isolate BNP-92 were 2-butenoic acid, methyl ester (8.6%), ethyl cyclopropanecarboxylate (45.99%), 1-Undecanol (10.18%) which corresponds to and have produced from PHA after thermal degradation., (Copyright © 2019. Published by Elsevier B.V.)

Introduction. Bacillus cereus harbouring Ba813 , a specific chromosomal marker of Bacillus anthtacis, is found in patients with severe manifestations and causes nosocomial outbreaks. Aim. We assessed the genetic characteristics and virulence of Ba813 (+) B. cereus in a hospital setting. Methodology. Three neutropenic patients with haematological malignancy developed B. cereus bacteraemia within a short period. Fifteen B. cereus were isolated from different sites in a haematology ward. A total of 18 isolates were evaluated for Ba813 - and B. anthracis -related virulence, food poisoning-related virulence, genetic diversity, bacteria motility and biofilm formation. Results. Ba813 (+) B. cereus was detected in 33 % (1/3) of patients and 66 % (9/15) of the hospital environment. The 18 strains were divided into 2 major clusters (clade 1 and clade 2), and 14 strains were classified into clade 1. All Ba813 (+) strains, including four sequence types, were classified into clade 1/the cereus III lineage, which is most closely related to the anthracis lineage. Two strains belonging to clade 1/non-cereus III carried the B. anthracis -associated cap gene, but not Ba813. B. cereus , including Ba813 (+) strains, had significantly lower prevalence of enterotoxin genes than clade 2 strains. In clade 1, B. cereus , Ba813 (+) strains showed significantly higher swimming motility and biofilm formation ability than Ba813 (-) strains. Conclusion. Ba813 (+) B. cereus , which are genetically closely related to B. anthracis , were abundant in a haematological ward. Ba813 (+) B. cereus with high motility and biofilm formation abilities may spread easily in hospital environments, and could become a hospital-acquired infection.

Bacillus cereus sensu lato comprises Gram-positive spore-forming bacteria producing toxins associated with foodborne diseases. Three pore-forming enterotoxins, nonhemolytic enterotoxin (Nhe), hemolysin BL (Hbl), and cytotoxin K (CytK), are considered the primary factors in B. cereus sensu lato diarrhea. The aim of this study was to determine the potential risk of enterotoxicity among soil B. cereus sensu lato isolates representing diverse phylogroups and originated from different geographic locations with various climates (Burkina Faso, Kenya, Argentina, Kazakhstan, and Poland). While nheA - and hblA -positive isolates were present among all B. cereus sensu lato populations and distributed across all phylogenetic groups, cytK-2 -positive strains predominated in geographic regions with an arid hot climate (Africa) and clustered together on a phylogenetic tree mainly within mesophilic groups III and IV. The highest in vitro cytotoxicity to Caco-2 and HeLa cells was demonstrated by the strains clustered within phylogroups II and IV. Overall, our results suggest that B. cereus sensu lato pathogenicity is a comprehensive process conditioned by many intracellular factors and diverse environmental conditions. IMPORTANCE This research offers a new route for a wider understanding of the dependency between pathogenicity and phylogeny of a natural bacterial population, specifically within Bacillus cereus sensu lato , that is widely distributed around the world and easily transferred into food products. Our study indicates differences in the phylogenetic and geographical distributions of potential enterotoxigenic B. cereus sensu lato strains. Hence, these bacilli possess a risk for human health, and rapid testing methods for their identification are greatly needed. In particular, the detection of the CytK enterotoxin should be a supporting strategy for the identification of pathogenic B. cereus sensu lato ., (Copyright © 2020 Drewnowska et al.)

Members of the Bacillus cereus group are common contaminants of vegetables. One potential source of contamination is the application of B. thuringiensis based biopesticides. Although evidence of the presence of biopesticidal strains on food products is scarce, this information is essential for assessing potential risks associated with the application of these biopesticides. In order to contribute to knowledge about the presence of biopesticidal B. thuringiensis strains in foodstuffs, we investigated the occurrence of B. thuringiensis on tomatoes and bell pepper. We analyzed 99 samples of fresh bell pepper for B. cereus group members, while 426 samples of tomatoes were tested by the competent food control laboratories of the federal states in Germany. The isolates recovered from these samples were further characterized in terms of their capability to produce parasporal crystals as well as enterotoxins. A possible correlation between the B. thuringiensis isolates and biopesticidal strains was investigated by multilocus sequence typing (MLST) and whole genome Single Nucleotide Polymorphism (wgSNP) analyses. The prevalence of B. cereus group members was 41% for bell pepper and 28% for tomato samples. Isolates recovered from these samples were dominated by B. thuringiensis (93% and 99%, respectively). All B. thuringiensis isolates carried the enterotoxin genes nheA, hblD and cytK-2. In a subset of 83 B. thuringiensis isolates analyzed by MLST, 99% of the isolates matched the sequence types (ST) 8 and 15, which are also shared by the biopesticidal strains B. thuringiensis kurstaki ABTS-351 and B. thuringiensis aizawai ABTS-1857. Of the 82 isolates assigned to ST 8 or ST 15, a selection of 42 isolates was further characterized by wgSNP analysis. Of these, seven isolates differed from strain ABTS-351 by ≤4 core SNPs and 18 isolates differed from strain ABTS-1857 by ≤2 core SNPs, indicating a relationship of these isolates with the respective biopesticidal strain. These isolates originated from samples with maximum colony counts of 5.3 × 10 3  cfu/g for bell pepper and 1.0 × 10 5  cfu/g for tomatoes., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

A salt-tolerant microbe strain JYZ-SD2 was investigated to develop biological soil amendments to stimulate salix growth and acclimation in costal salt-affected soils. The salt tolerance mechanism of strain JYZ-SD2 was investigated by detecting the salt-tolerant growth characteristics, biofilm formation, ion distribution, secondary metabolites, and zymogram profiling. The strain was identified by physiological and biochemical characteristics (Biolog), 16S rDNA sequencing, and cry1/7/9 gene expressing. With increasing of NaCl concentration, strain JYZ-SD2 adapted to the increased osmotic pressure by prolonging the retardation period, slowing down the growth rate of the logarithmic phase, increasing spo0A gene expression, increasing biofilm formation, reducing Na + uptake, and changing the expression of metabolites and intracellular soluble proteins. The results showed that strain JYZ-SD2 could be assigned to Bacillus cereus.

Replacing agrochemicals with plant growth promoting bacteria (PGPB) may offset some of the environmental impacts of food production. The objectives of this study were to (1) isolate and characterize bacterial strains from tomato rhizosphere, including root, shoot and leaf, (2) select and identify the most promising PGPB strains, (3) verify the phytostimulatory activity and mineral uptake potential of selected strains. Bacterial strains isolated from tomato rhizosphere, were screened for phosphorous (P) solubilization, production of indole acetic acid (IAA), amylase activity, antibiotic resistance, and quick test strip (QTS) for biochemical characterization. The tested strains, positive for all five of these assays were selected for molecular identification and subjected to greenhouse growth trails with tomato and mung bean. Two strains were selected and identified as Bacillus cereus (B. cereus) isolated from rhizosphere and Klebsiella variicola (K. variicola) isolated from root endosphere using 16s rRNA sequences. Both strains produced IAA, gibberellic acid (GA3) and kinetin, however B. cereus showed potential GA3 and IAA production as compared to K. variicola. In tomato, only one growth variable (shoot length) was increased over the control by one of the selected bacterial strains (B. cereus). In mung bean, inoculation with either strain B. cereus or K. variicola increased shoot length and dry weight. Moreover, our results showed that the use of PGPB significantly increased plant growth and Fe, Zn, Ca, Mg, Cu, Na and K contents of plants. It seems that evaluated strains had a higher ability in boosting plant growth and higher yield., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Bacillus cereus group comprises numerous closely related species, including bioterrorism agent B. anthracis , foodborne pathogen B. cereus , and biopesticide B. thuringiensis Differentiating organisms capable of causing illness or death from those used in industry is essential for risk assessment and outbreak preparedness. However, current species definitions facilitate species-phenotype incongruences, particularly when horizontally acquired genes are responsible for a phenotype. Using all publicly available B. cereus group genomes ( n  = 2,231), we show that current species definitions lead to overlapping genomospecies clusters, in which 66.2% of genomes belong to multiple genomospecies at a conventional 95 average nucleotide identity (ANI) genomospecies threshold. A genomospecies threshold of ≈92.5 ANI is shown to reflect a natural gap in genome similarity for the B. cereus group, and medoid genomes identified at this threshold are shown to yield resolvable genomospecies clusters with minimal overlap (six of 2,231 genomes assigned to multiple genomospecies; 0.269%). We thus propose a nomenclatural framework for the B. cereus group which accounts for (i) genomospecies using resolvable genomospecies clusters obtained at ≈92.5 ANI, (ii) established lineages of medical importance using a formal collection of subspecies names, and (iii) heterogeneity of clinically and industrially important phenotypes using a formalized and extended collection of biovar terms. We anticipate that the proposed nomenclature will remain interpretable to clinicians, without sacrificing genomic species definitions, which can in turn aid in pathogen surveillance; early detection of emerging, high-risk genotypes; and outbreak preparedness. IMPORTANCE Historical species definitions for many prokaryotes, including pathogens, have relied on phenotypic characteristics that are inconsistent with genome evolution. This scenario forces microbiologists and clinicians to face a tradeoff between taxonomic rigor and clinical interpretability. Using the Bacillus cereus group as a model, a conceptual framework for the taxonomic delineation of prokaryotes which reconciles genomic definitions of species with clinically and industrially relevant phenotypes is presented. The nomenclatural framework outlined here serves as a model for genomics-based bacterial taxonomy that moves beyond arbitrarily set genomospecies thresholds while maintaining congruence with phenotypes and historically important species names., (Copyright © 2020 Carroll et al.)

Real-time biohazard detectors must be developed to facilitate the rapid implementation of appropriate protective measures against foodborne pathogens. Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the real-time detection of hazardous bacteria (HB) in the field. However, distinguishing among various HBs that exhibit similar C, N, O, H, or trace metal atomic emissions complicates HB detection by LIBS. This paper proposes the use of LIBS and chemometric tools to discriminate Staphylococcus aureus, Bacillus cereus, and Escherichia coli on slide substrates. Principal component analysis (PCA) and the genetic algorithm (GA) were used to select features and reduce the size of spectral data. Several models based on the artificial neural network (ANN) and the support vector machine (SVM) were built using the feature lines as input data. The proposed PCA-GA-ANN and PCA-GA-SVM discrimination approaches exhibited correct classification rates of 97.5% and 100%, respectively.

25-hydroxyvitamin D 3 has attracted considerable attention due to its great medical value and huge market demand in animal husbandry. Microbial production of 25-hydroxyvitamin D 3 has been recognized as an alternative superior to traditional chemical synthesis. In this study, a Gram-positive bacteria zju 4-2 (CCTCC M 2019385) was isolated from the soil using vitamin D 3 as the sole carbon source and was identified as Bacillus cereus according to its physiological characteristics and 16S rRNA analysis, which also showed a relatively high capacity for 25-hydroxyvitamin D 3 production. Through systematic optimization of different catalytic conditions, the optimal solvent system of vitamin D 3 , vitamin D 3 addition time and concentration, temperature, and pH were shown to be propylene glycol/ethanol (v/v = 9:1), early stationary phase, 2 g/L, 37 °C, and pH 7.2, respectively. With these optimal conditions, 796 mg/L of 25-hydroxyvitamin D 3 was achieved after 48 h bioconversion with zju 4-2 at the shake flask level. Finally, up to 830 mg/L 25-hydroxyvitamin D 3 with a yield of 41.5% was obtained in a 5 L fermentation tank. Our developed biotransformation process with this newly isolated strain provides a platform to produce 25-hydroxyvitamin D 3 efficiently at industrialization scale.

SYJ15 is a highly pathogenic Gram-positive Bacillus sp. with top bud spore newly isolated from dying soft shell turtle. 16SrDNA sequencing showed that it is highly homologous to B. cereus, B. thuringiensis and B. anthracis. Biochemical examinations showed that it belongs to B. cereus. To further study the new pathogen, we conducted whole-genome sequencing based on single-molecular sequencing technology from PacBio. Genome assembly analysis showed that the strain has a 5,296,886 bp chromosome, a 218,649 bp plasmid and a 5221 bp plasmid with GC content of 35.51%, 31.91% and 29.75%, respectively. The genome contains 5736 coding sequences and 6 CRISPR systems located in the chromosome as well as 11 genomic islands in the chromosome and the large plasmid. Genome function analyses were annotated by nr database, SwissProt, KEGG, COG, GO, PHI, VFDB, ARDB, Secretory&#95;Protein and T3SS. In addition, 13 gene clusters of secondary metabolism were predicted by antiSMASH. Comparison of SYJ15 with B. subtilis, B. anthracis, B. cereus and B. thuringiensis identified 1031 core genes of the five strains and 816 genes specific to SYJ15. In addition, SYJ15 had the most common core genes with B. thuringiensis, and the least with B. subtilis. Phylogenetic analysis demonstrated that SYJ15 is between B. thuringiensis and B. cereus, suggesting that SYJ15 belongs to Bacillus cereus group. We designed a specific primer pair to distinguish SYJ15 from B. pumilus, B. licheniformis, B.subtilis, B. thuringiensis and B. cereus. In conclusion, information of SYJ15 genome will help to enhance our understanding of pathogenesis of SYJ15 and find effective treatment.

Bacillus cereus is a microorganism associated with food poisoning. It has been found in products, such as milk and dairy products. The aim of this study was to isolate and identify B. cereus group strains in artisan cheeses sold in southwestern Mexico, as well as its toxigenic profile, its psychrophilic ability, and its biofilm production. B. cereus isolation was performed on Mannitol Yolk Polymyxin (MYP) agar and this was molecularly confirmed by the amplification of the gyrB gene. Polymerase chain reaction was used to determine the toxigenic profile, amplifying conserved regions of hblABD and nheABC operons, which code for the subunits of Hbl and Nhe toxins, respectively, as well as ges and cytK genes, which code for toxin cereulide and cytotoxin K (Cytk). Frequency of B. cereus contamination in artisan cheeses was 29.48% (23/78), and the bacterium was isolated in similar quantities in all types of products. All strains were amylase positive, and 60.86% (14/23) were able to produce biofilm; 91.30% (21/23) of the strains were psychrophilic. In most of the strains, at least one gene related to enterotoxins was identified (21/23). B. cereus strains in this study have a high toxigenic potential, which increases the risk of food poisoning due to the consumption of artisan cheeses made in Mexico.

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett-Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20-80 °C and pH 5-10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn 2+ , Zn 2+ , and Cu 2+ ). The enzyme kinetics revealed K m and V max values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI-TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively., Competing Interests: The authors declare no conflicts of interest.

Background: The complete genome sequences of 44 Bacillus cereus group isolates collected from diverse sources in Japan were analyzed to determine their genetic backgrounds and diversity levels in Japan. Multilocus sequence typing (MLST) and core-genome single-nucleotide polymorphism (SNP) typing data from whole-genome sequences were analyzed to determine genetic diversity levels. Virulence-associated gene profiles were also used to evaluate the genetic backgrounds and relationships among the isolates., Results: The 44 B. cereus group isolates, including soil- and animal-derived isolates and isolates recovered from hospitalized patients and food poisoning cases, were genotyped by MLST and core-genome SNP typing. Genetic variation among the isolates was identified by the MLST and core-genome SNP phylogeny comparison against reference strains from countries outside of Japan. Exploratory principal component analysis and nonmetric multidimensional scaling (NMDS) analyses were used to assess the genetic similarities among the isolates using gene presence and absence information and isolate origins as the metadata. A significant correlation was seen between the principal components and the presence of genes encoding hemolysin BL and emetic genetic determinants in B. cereus, and the capsule proteins in B. anthracis. NMDS showed that the cluster of soil isolates overlapped with the cluster comprising animal-derived and clinical isolates., Conclusions: Molecular and epidemiological analyses of B. cereus group isolates in Japan suggest that the soil- and animal-derived bacteria from our study are not a significant risk to human health. However, because several of the clinical isolates share close genetic relationships with the environmental isolates, both molecular and epidemiological surveillance studies could be used effectively to estimate virulence in these important pathogens.

Bacillus cereus isolates causing an outbreak in the neonatal intensive care unit were investigated using whole-genome sequencing. The outbreak coincided with construction work performed adjacent to the neonatal intensive care unit and ceased after strict sealing of the construction area. We found the outbreak to be polyclonal, however, the clonality did not correlate with the virulence in vivo. Genotypically similar isolates were associated with both lethal/severe infection and colonization/environmental contamination. Environmental bacterial load may be a major determinant of infection, especially in high-risk patients. Clinicians should be alert to unusual increase in B. cereus isolations from clinical cultures to facilitate early recognition and investigations of Bacillus outbreaks and pseudo-outbreaks. The integration of genomics into the classical infectious disease work can augment our understanding of pathogen transmission and virulence, and can rapidly assist our response to unusual disease trends.

A repetitive sequence-based polymerase chain reaction (rep-PCR) technique utilizing a semiautomated system, namely DiversiLab, was applied to determine the genotypes of Staphylococcus aureus and Bacillus cereus obtained from slaughterhouses. Twenty-four S. aureus and 16 B. cereus isolates from pigs and Hanwoo cattle from three slaughterhouses were used to create a DNA fingerprint library with the system software. Scatterplots demonstrated that rep-PCR groupings of S. aureus isolates were in good agreement with their origins. Specifically, linked rep-PCR profiles were observed for S. aureus isolates recovered from the same slaughterhouse, and higher genetic similarities were found among strains isolated from adjacent regions. All S. aureus isolates except one (ID: A-Hanwoo-9) from slaughterhouse "A" clustered with the three S. aureus reference strains, Korea Culture Center of Microorganisms (KCCM) 41291, KCCM 12214, and Culture Collection of Antimicrobial Resistant Microbes (CCARM) 3A007 (similarity values >95%). Moreover, most isolates obtained from slaughterhouse "B" clustered with S. aureus KCCM 11335 and KCCM 41331, and two isolates from slaughterhouse "C" clustered with CCARM 0027. Therefore, for this species, genotypic characteristics of regional isolates can be used to track the pathway of contamination. In contrast, B. cereus isolates showed high genetic diversity and could not be clustered with any specific group. Collectively, this system is useful for analyzing genetic diversity and is a rapid and reproducible typing method; however, there is a need to develop rep-PCR libraries for its use as a rapid identification method.

Bacillus cereus not only has adverse effects on the nutrition and shelf life of dairy products but also seriously endanger people's health. This study was conducted to reveal the prevalence and genetic diversity of B. cereus strains isolated from raw milk and cattle farm environments. A total 56 of B. cereus strains were detected from 300 environmental samples (soil, water, fodder, air, milk pails, milking machines, cowsheds, bedding, excrement, cow surfaces, udders, overalls, soles, and staff hand samples) and 50 raw milk samples, and divided into 18 sequence types (STs) using multilocus sequence typing method. These STs included ST27, ST61, ST92, ST142, ST168, ST208, ST378, ST427, ST766, ST 857, ST1098, ST1140, ST1194, ST1236, ST1336, ST1339, ST1341, and ST1348, among them, ST857 (7/56, 12.5%) was the dominant ST, and were detected from air, cowsheds, bedding, excrement, and raw milk samples. Our findings could reveal the distribution and genetic diversity of B. cereus strains in raw milk and cattle farm environments, and provide a theoretical basis for controlling the potential harm of this pathogenic bacteria in dairy products.

Neutral proteases have broad application as additives in modern laundry detergents and therefore, thermostability is an integral parameter for effective production of protein crystals. To improve thermostability, the contribution of individual residues of Bacillus cereus neutral protease was examined by site-directed mutagenesis. The Lys11Arg and Lys211Arg mutants clearly possessed improved thermostabilities (T m were 63 and 61 °C respectively) compared to the wild-type (T m was 60 °C). MD simulations further revealed that the mutants had low RMSD and RMSF values compared to wild-type BCN indicating increased stability of the protein structure. Lys11Arg mutant particularly possessed the lowest RMSD values due to increased residue interactions, which resulted in enhanced thermostability. The mutants also displayed strong stability to most inhibitors, organic solvents and surfactants after incubation for 1 h. This study demonstrated Lys-Arg mutation enhanced thermostability of BCN and thus provides insight for engineering stabilizing mutations with improved thermostability for related proteins.

Background: Despite the importance of the B. cereus group as major foodborne pathogens that may cause diarrheal and/or emetic syndrome(s), no study in Tunisia has been conducted in order to characterize the pathogenic potential of the B. cereus group. The aim of this study was to assess the sanitary potential risks of 174 B. cereus group strains isolated from different foodstuffs by detecting and profiling virulence genes (hblA, hblB, hblC, hblD, nheA, nheB, nheC, cytK, bceT and ces), testing the isolates cytotoxic activity on Caco-2 cells and antimicrobial susceptibility towards 11 antibiotics., Results: The entertoxin genes detected among B. cereus isolates were, in decreasing order, nheA (98.9%), nheC (97.7%) and nheB (86.8%) versus hblC (54.6%), hblD (54.6%), hblA (29.9%) and hblB (14.9%), respectively encoding for Non-hemolytic enterotoxin (NHE) and Hemolysin BL (HBL). The isolates are multi-toxigenic, harbouring at least one gene of each NHE and HBL complexes associated or not to bceT, cytK-2 and ces genes. Based on the incidence of virulence genes, the strains were separated into 12 toxigenic groups. Isolates positive for cytK (37,9%) harbored the cytK-2 variant. The detection rates of bceT and ces genes were 50.6 and 4%, respectively. When bacteria were incubated in BHI-YE at 30 °C for 18 h and for 5 d, 70.7 and 35% of the strains were shown to be cytotoxic to Caco-2 cells, respectively. The cytotoxicity of B. cereus strains depended on the food source of isolation. The presence of virulence factors is not always consistent with cytotoxicity. However, different combinations of enterotoxin genetic determinants are significantly associated to the cytotoxic potential of the bacteria. All strains were fully sensitive to rifampicin, chloramphenicol, ciprofloxacin, and gentamycin. The majority of the isolates were susceptible to streptomycin, kanamycin, erythromycin, vancomycin and tetracycline but showed resistance to ampicillin and novobiocin., Conclusion: Our results contribute data that are primary to facilitate risk assessments in order to prevent food poisoning due to B. cereus group.

Polyhydroxyalkanoates are a fascinating group of biological polymers, which have attracted extensive attention. An effectual PHB accumulating Bacillus cereus strain VIT-SSR1 was isolated from industrial effluent contaminated site. Plackett-Burman design was used to screen the critical factors affecting PHA production. Response surface method was employed to evaluate the interactive effects between the independent variables. The Central composite design was performed to optimize the concentrations of Molasses, ammonium sulphate and initial pH. A PHA content of (1.42 ± 0.01) g/L and a maximum PHA yield of (40.3 ± 0.77) % was obtained on implementing the optimized conditions. The produced biopolymer was analysed using Fourier transform infrared spectroscopy (FT-IR) and Gas chromatography-mass spectrometry analysis(GC-MS) and the thermal properties of the biopolymer were evaluated by Thermogravimetric analysis (TGA), and from the Nuclear magnetic Resonance (NMR) analysis the biopolymer was identified to be polyhydroxybutyrate (PHB). PHB-chitosan blends were prepared and tested for biocompatibility by MTT assay on L929 mouse fibroblast cell lines. Curcumin, a well-known drug was loaded onto PHB-chitosan films and their drug release pattern was observed. Curcumin showed a sustained mode of drug release. PHB films are biocompatible and can act as a potent polymer matrix for drug release studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

During March 13-June 23, 2018, anthrax-like cutaneous lesions attributed to the Bacillus cereus group of organisms developed in 12 newborns in India. We traced the source of infection to the healthcare kits used for newborn care. We used multilocus sequence typing to characterize the 19 selected strains from various sources in hospital settings, including the healthcare kits. This analysis revealed the existence of a genetically diverse population comprising mostly new sequence types. Phylogenetic analysis clustered most strains into the previously defined clade I, composed primarily of pathogenic bacilli. We suggest that the synergistic interaction of nonhemolytic enterotoxin and sphingomyelinase might have a role in the development of cutaneous lesions. The infection was controlled by removing the healthcare kits and by implementing an ideal housekeeping program. All the newborns recovered after treatment with ciprofloxacin and amikacin.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based pathogen identification relies on the ribosomal protein spectra provided in the proprietary database. Although these mass spectra can discern various pathogens at species level, the spectra-based method still has limitations in identifying closely-related microbial species. In this study, to overcome the limits of the current MALDI-TOF MS identification method using ribosomal protein spectra, we applied MALDI-TOF MS of low-mass profiling to the identification of two genetically related Bacillus species, the food-borne pathogen Bacillus cereus , and the insect pathogen Bacillus thuringiensis . The mass spectra of small molecules from 17 type strains of two bacilli were compared to the morphological, biochemical, and genetic identification methods of pathogens. The specific mass peaks in the low-mass range ( m/z 500- 3,000) successfully identified various closely-related strains belonging to these two reference species. The intensity profiles of the MALDI-TOF mass spectra clearly revealed the differences between the two genetically-related species at strain level. We suggest that small molecules with low molecular weight, 714.2 and 906.5 m/z can be potential mass biomarkers used for reliable identification of B. cereus and B. thuringiensis .

Objectives: Bacillus cereus is associated with a variety of human diseases such as haematological malignancies, tetanus, gas gangrene, food poisoning and pseudomembranous colitis. Our team found and isolated a strain, named B. cereus S66, from radiation-contaminated soil in Xinjiang Uygur Autonomous Region. In this study, the drug resistance and toxicity of B. cereus S66 was systematically studied and analysed., Methods: Genomic DNA of B. cereus S66 was sequenced using an Illumina HiSeq 2000 sequencing system. The generated reads were assembled using SOAPdenovo, and GapCloser was used to fill in the gaps. The draft genome was annotated, and virulence genes and drug resistance genes were identified by comparison with multiclass databases., Results: The genome of B. cereus strain S66 is 5 628 370 bp with a GC content of 34.95% and contains various genes related to toxicity and antimicrobial resistance. The number of toxicity genes identified was 496, some of which are associated with gastrointestinal infection, eye infection and neutrophil-activating protein NapA. Antimicrobial resistance-related genes comprised 3.23% of the total genes and conferred resistance to lincomycin, tetracycline, fosfomycin, aminoglycosides, linezolid, chloramphenicol, rifampicin, macrolides, daunorubicin and mupirocin. Three different β-lactamases were also found in isolate S66., Conclusions: The genome sequence of B. cereus S66 provides a valuable reference to further research on multidrug resistance characteristics and pathogenic system analysis of Bacillus sp. in the clinic., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

Bacillus cereus is associated with foodborne illnesses characterized by vomiting and diarrhea. Although some B. cereus strains that cause severe extraintestinal infections and nosocomial infections are recognized as serious public health threats in healthcare settings, the genetic backgrounds of B. cereus strains causing such infections remain unknown. By conducting pulsed-field gel electrophoresis and multilocus sequence typing, we found that a novel sequence type (ST), newly registered as ST1420, was the dominant ST isolated from the cases of nosocomial infections that occurred in 3 locations in Japan in 2006, 2013, and 2016. Phylogenetic analysis showed that ST1420 strains belonged to the Cereus III lineage, which is much closer to the Anthracis lineage than to other Cereus lineages. Our results suggest that ST1420 is a prevalent ST in B. cereus strains that have caused recent nosocomial infections in Japan.

The Bacillus cereus group includes several Bacillus species with closely related phylogeny. The most well-studied members of the group, B. anthracis , B. cereus , and B. thuringiensis , are known for their pathogenic potential. Here, we present the historical rationale for speciation and discuss shared and unique features of these bacteria. Aspects of cell morphology and physiology, and genome sequence similarity and gene synteny support close evolutionary relationships for these three species. For many strains, distinct differences in virulence factor synthesis provide facile means for species assignment. B. anthracis is the causative agent of anthrax. Some B. cereus strains are commonly recognized as food poisoning agents, but strains can also cause localized wound and eye infections as well as systemic disease. Certain B. thuringiensis strains are entomopathogens and have been commercialized for use as biopesticides, while some strains have been reported to cause infection in immunocompromised individuals. In this article we compare and contrast B. anthracis , B. cereus , and B. thuringiensis , including ecology, cell structure and development, virulence attributes, gene regulation and genetic exchange systems, and experimental models of disease.

Background: Among the Bacillus cereus group, B. thuringiensis, is one of the most extensively used biological control agent. The present study reports the complete genome and four novel plasmid analysis of the type strain B. thuringiensis ATCC 10792., Methods: Complete genome sequencing of Bacillus thuringiensis ATCC 10792, assembled using de-novo (v.3.2.0, assembly name MIRA3), Pac-Bio sequencers and Hierarchical Genome Assembly Process software (version 4.1) and real-time polymerase chain reaction (qPCR) is a consistent technique for quantifying gene expression based on specific biomarkers, in addition the efficiency of the primers were analysed based on artificially spiked food samples on lettuce, kimbab and spinach with B. thuringiensis ATCC 10792., Results: Complete genome annotation was performed, and a total of 6269 proteins with 5427594 bps were identified and four novel plasmid (poh2, poh3, poh4, poh5) a total of 134, 131, 96, 21 proteins with 113294; 92,949; 86488; 11332 bps were identified. Six selective genes (lipoprotein-lipo, methyltransferase-MT, S-layer homology domain protein-BC, flagellar motor protein-motB, transcriptional regulator-XRE, crystal protein-cry2) and associated four novel plasmids were investigated along with the characteristics and expression profiles of two housekeeping genes (chaperonin protein-GroEL and topoisomerase enzyme-gyrB). Although from the assessment of 120 strains, both GroEL and gyrB showed 100% specificity towards detection of both B. thuringiensis in artificially spiked vegetable samples. All the eight genes revealed no specificity towards any of the 9 non- Bacillus strains., Conclusion: In our study based on the complete genome and plasmid sequence of B. thuringiensis ATCC 10792, among the six discriminating genes, specifically GroEL, gyrB and XRE showed promising results in identifying B. thuringiensis ATCC 10792, and there detection limit was 3.0-9.6 log CFU/g in the food samples respectfully. The key role in control of the predatory biological agent., (Copyright © 2019. Published by Elsevier Ltd.)

Mercury-resistant (Hg R ) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 μg/ml against mercuric chloride (HgCl 2 ). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of mer A nucleotide sequence confirmed 51- 100% homology with the corresponding region of the mer A gene of already reported mercuryresistant Gram-positive bacteria. The mer E gene involved in the transportation of elemental mercury (Hg 0 ) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing 30 μg/ml of HgCl 2 was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively ( p < 0.05).

The variability in spore survival during spray drying of 12 Bacillus cereus strains was evaluated. B. cereus spores were inoculated on whole milk (7.2 ± 0.2 log 10  spores/g dry weight) and processed in a spray-dryer. Twelve independent experiments were carried out in triplicate. The spore count was determined before and after each drying process, based on the dry weight of whole milk and powdered milk. Then, the number of decimal reductions (γ) caused by the spray drying process was calculated. B. cereus strains presented γ values ranging from 1.0 to 4.7 log10 spores/g dry weight, with a high coefficient of variation (CV) of 46.1%. Cluster analysis allowed to group B. cereus as sensitive (strains 511, 512, 540, 432 and ATCC 14579), intermediate (strains B18, B63, and B86) and resistant strains (strains B3, B94, B51 and 436). Three strains (one of each group) were selected for further investigation and characterization of their physicochemical and molecular (proteomics) differences. Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) were used to determine physicochemical characteristics and glass transition temperature (T g ), respectively. No differences in signs among the three strains were found in spectra ranging from 900 to 4000 cm -1 . The endothermic peak ranged between 54 and 130 °C for strain 540; between 81 and 163 °C for strain B63; and between 110 and 171 °C for strain 436. However, they showed different T g : 88.82 °C for strain 540; 114.32 °C for strain B63; and 122.70 °C for strain 436. A total of eleven spots were identified by mass spectrometry, with the spore coat protein GerQ, sporulation protein YtfJ (GerW), and peptidyl-prolyl cis-trans isomerase being found in at least two strains. Altogether, the results suggested that the high survival variability of B. cereus spores to the spray drying process seems to be mainly associated with different T g and protein content. The study highlights the importance of quantifying the effects of this unit operation over the target microorganisms. These data may be relevant for the development of effective measures aiming to control the occurrence of B. cereus in milk powder as well as to reduce spoilage or safety issues associated with the presence of this bacterium in foods, particularly those formulated with milk powder., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Clostridioides (Clostridium) difficile and Bacillus cereus infections are frequently reported in human individually. However, co-infection of both pathogens in human is extremely rare. In the present study, we reported a case of human enteric disease caused by co-infection of C. difficile and B. cereus in Guizhou, China. The 16S rDNA sequencing result showed that C. difficile GMU1 and B. cereus GMU2 were most related to C. difficile ATCC 9689 and B. cereus ATCC 14579. The toxin genotype of C. difficile GMU1 and B. cereus GMU2 were tcdA + tcdB + tcdC + and bceT + nheA + nheB + nheC + , respectively. Cytotoxicity assay demonstrated that C. difficile GMU1 produced significantly higher toxin B compare to C. difficile 630 stain. In contrast, B. cereus GMU2 has comparable NheA toxin productivity compare to previous report. The antimicrobial susceptibility test showed that the combination of ampicillin and vancomycin was most efficient to inhibit both C. difficile GMU1 and B. cereus GMU2., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Some Bacillus strains function as predominant plant-growth-promoting rhizobacteria. Bacillus cereus 905 is a rod-shaped Gram-positive bacterium isolated from wheat rhizosphere and is a rhizobacterium that exhibits significant plant-growth-promoting effects. Species belonging to the genus Bacillus are observed in numerous different habitats. Several papers on B. cereus are related to pathogens that causes food-borne illness and industrial applications. However, genomic analysis of plant-associated B. cereus has yet to be reported. Here, we conducted a genomic analysis comparing strain 905 with three other B. cereus strains and investigate the genomic characteristics and evolution traits of the species in different niches. The genome sizes of four B. cereus strains range from 5.38 M to 6.40 M, and the number of protein-coding genes varies in the four strains. Comparisons of the four B. cereus strains reveal 3,998 core genes. The function of strain-specific genes are related to carbohydrate, amino acid and coenzyme metabolism and transcription. Analysis of single nucleotide polymorphisms (SNPs) indicates local diversification of the four strains. SNPs are unevenly distributed throughout the four genomes, and function interpretation of regions with high SNP density coincides with the function of strain-specific genes. Detailed analysis indicates that certain SNPs contribute to the formation of strain-specific genes. By contrast, genes related to plant-growth-promoting traits are highly conserved. This study shows the genomic differences between four strains from different niches and provides an in-depth understanding of the genome architecture of these species, thus facilitating genetic engineering and agricultural applications in the future.

The aim of the present study was to investigate the separation of oil from water using a bench-scale DAF prototype with the addition of biosurfactants isolated from Pseudomonas cepacia CCT6659 and Bacillus cereus UCP1615. The best operating conditions for the DAF prototype were determined using a central composite rotatable design. The results demonstrated that the biosurfactants from P. cepacia and B. cereus increased the oil separation efficiency from 53.74% (using only microbubbles) to 94.11 and 80.01%, respectively. The prediction models for both DAF-biosurfactant systems were validated, showing an increase in the efficiency of the DAF process from 53.74% to 98.55 and 70.87% using the formulated biosurfactants from P. cepacia and B. cereus, respectively. The biosurfactant from P. cepacia was selected as the more promising product and used for the treatment of oily effluent from a thermoelectric plant, achieving removal rates ranging between 75.74 (isolated biosurfactant) and 95.70% (formulated biosurfactant), respectively.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mashed potato powder as well as powdered infant formula (PIF) are frequently contaminated with Bacillus cereus sensu lato (B. cereus s.l.), mainly with its spores. These products have also been implicated in foodborne illnesses. Here, we characterized B. cereus s.l. isolates originating from powdered products based on sporulation assays, toxin gene profiling, and panC typing combined with a SplitsTree analysis. Furthermore, cytotoxicity assays with B. cytotoxicus isolates were performed. 78% of PIF tested positive for B. cereus s.l., whereas 92% of all mashed potato powders were positive. In total, 43 isolates were further characterized. The nhe and cytK2 genes were most frequently detected. Moreover, a cereulide-producer was detected from PIF. Most isolates were assigned to panC group III, but members of group II, IV, V, and VII could also be found. Nine B. cytotoxicus were isolated out of nine mashed potato powders. All panC group VII isolates were positive for cytK1. Cytotoxicity assays of these nine isolates revealed one highly cytotoxic strain, while all other isolates exhibited no detectable cytotoxicity, underpinning that cytotoxicity of a certain B. cereus group strain cannot be deduced from the sole presence or absence of toxin genes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: Cannulated surgical instruments may retain biologic debris after routine cleaning and sterilization. Residual debris after cleaning is assumed to be sterile; however, there is no experimental basis for this assumption. The purpose of this study was to determine the sterility of retained biodebris found within cannulated surgical instruments after autoclave sterilization., Materials and Methods: Fifteen cannulated drill bits were used to drill pig scapulae to create a plug of bone that was exposed to a mixture of Bacillus cereus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus for 60, 120, or 180 minutes prior to sterilization. The drill bits were autoclave sterilized using standard settings. The "sterilized" bone cores were then incubated in solution and streak-plated on blood agar., Results: All 3 positive controls were positive for the experimental bacteria. Two negative controls were positive for contaminant bacteria. A B. cereus strain was recovered from 1 of the experimental group drill bits in the 180-minute group. Pulsed-field gel electrophoresis confirmed that the recovered B. cereus strain was identical to the experimental inoculate., Conclusion: Retained biodebris in cannulated drills may not be sterile after standard autoclave sterilization. In addition, delay of surgical instrument reprocessing may increase the risk of resistant contamination., (Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)