Three novel species of the Bacillus cereus group isolated from clinical samples in Japan.

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-BC004T (=DSM 111102T=JCM 34122T), Bacillus paramobilis sp. nov., with type strain BML-BC017T (=DSM 111100T=JCM 34124T) and Bacillus hominis sp. nov., with type strain BML-BC059T (=DSM 111101T=JCM 34125T).

Comparison of the antibiotic resistance between genetically diverse and toxigenic Bacillus cereus sensu lato from milk, pepper and natural habitats.

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 ß-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.

Chromium binding Bacillus cereus VITSH1-a promising candidate for heavy metal clean up.

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.

The cytotoxic potential of Bacillus cereus strains of various origins.

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.

Characterization of Bacillus cereus sensu lato isolates from milk for consumption; phylogenetic identity, potential for spoilage and disease.

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.

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk.

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 103 cfu/mL, with a dynamic range of detection at 103 to 107 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 101 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.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

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.

Potential Enterotoxicity of Phylogenetically Diverse Bacillus cereus Sensu Lato Soil Isolates from Different Geographical Locations.

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.

Application of Bacillus thuringiensis strains with conjugal and mobilizing capability drives gene transmissibility within Bacillus cereus group populations in confined habitats.

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.

Characterization and phytostimulatory activity of bacteria isolated from tomato (Lycopersicon esculentum Mill.) rhizosphere.

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.

Complete genome sequence of novel isolate SYJ15 of Bacillus cereus group, a highly lethal pathogen isolated from Chinese soft shell turtle (Pelodiscus Sinensis).

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_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.

Efficient biotransformation of vitamin D3 to 25-hydroxyvitamin D3 by a newly isolated Bacillus cereus strain.

25-hydroxyvitamin D3 has attracted considerable attention due to its great medical value and huge market demand in animal husbandry. Microbial production of 25-hydroxyvitamin D3 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 D3 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 D3 production. Through systematic optimization of different catalytic conditions, the optimal solvent system of vitamin D3, vitamin D3 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 D3 was achieved after 48 h bioconversion with zju 4-2 at the shake flask level. Finally, up to 830 mg/L 25-hydroxyvitamin D3 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 D3 efficiently at industrialization scale.

Discrimination of hazardous bacteria with combination laser-induced breakdown spectroscopy and statistical methods.

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.

Salt Tolerance Mechanism and Species Identification of the Plant Rhizosphere Bacterium JYZ-SD2.

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.

Prevalence of the Strains of Bacillus cereus Group in Artisanal Mexican Cheese.

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.

Novel Sequence Type in Bacillus cereus Strains Associated with Nosocomial Infections and Bacteremia, Japan.

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.

Bacillus cereus-Attributable Primary Cutaneous Anthrax-Like Infection in Newborn Infants, India.

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.

Toxigenic potential and antimicrobial susceptibility of Bacillus cereus group bacteria isolated from Tunisian foodstuffs.

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.

Whole-genome sequence-based comparison and profiling of virulence-associated genes of Bacillus cereus group isolates from diverse sources in Japan.

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.

Whole genome sequence of Bacillus thuringiensis ATCC 10792 and improved discrimination of Bacillus thuringiensis from Bacillus cereus group based on novel biomarkers.

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.