New perspectives on Mega plasmid sequence (poh1) in Bacillus thuringiensis ATCC 10792 harbouring antimicrobial, insecticidal and antibiotic resistance genes.

Bacillus thuringiensis promotes the growth of numerous economically important crops. The present study presents the complete genome sequence for a mega plasmid present in the type strain of B. thuringiensis ATCC 10792, a typical spore-forming Gram-positive bacterium with insecticidal activity, and investigates its genetic characteristics. The genome was sequenced and assembled de novo using Pac-Bio sequencers and the Hierarchical Genome Assembly Process, respectively. Further genome annotation was performed, and a total of 489 proteins and a novel mega-plasmid (poh1) with 584,623 bps were identified. The organization of poh1 revealed the genes involved in the insecticidal toxin pathway. The genes responsible for antimicrobial, insecticidal and antibiotic activities were well conserved in poh1, indicating an intimate association with plant hosts. The poh1 plasmid contains the gene encoding a novel crystal protein kinase responsible for production of zeta toxin, which poisons insects and other Gram-negative bacteria through the global inhibition of peptidoglycan synthesis. Lantibiotics are a group of bacteriocins that include the biologically active antimicrobial peptide Paenibacillin. Further, poh1 also contains the genes that encode the gramicidin S prototypical antibiotic peptide and tetracycline resistance protein. In conclusion, the strain-specific genes of B. thuringiensis strain ATCC 10792 were identified through complete genome sequencing and bioinformatics data based on major pathogenic factors that contribute to further studies of the pathogenic mechanism and phenotype analyses.

Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB.

The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups - B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.

Unique biomarkers as a potential predictive tool for differentiation of Bacillus cereus group based on real-time PCR.

The aim of the study was to develop unique biomarkers for qPCR detection of Bacillus cereus group. Clinical and soil isolates were identified by specifically designed biomarkers - Lipoprotein (OPL-114-lipo), Methyltransferase (MT-17) and S-layer homology domain protein (151-1BC). In order to design biomarkers, we used 120 bacterial strains grouped into B. cereus and non-Bacillus group. The B. cereus group was confirmed by 108 strains of B. cereus and B. thuringiensis (30 reference and 78 wild), along with 3 strains of B. mycoides, B. pseudomycoides, and B. weihenstephanensis; while the non-Bacillus group was composed of 9 Gram-positive and Gram-negative strains. Direct analysis of samples revealed specificity towards identification and characterization of B. cereus group. The newly developed markers OPL-114-lipo and MT-17 showed specificity of 95% and 81%, respectively in identification of B. cereus. They are efficient tools to identify contaminated sources and the degree of bacterial contamination. Environmental and food samples do not require band isolation, re-amplification, sequencing or sequence identification. Thus, reducing the time and cost of analysis. Hence, it will be an alternative approach to traditional culture methods. Commercial food processing industries will be able to employ these biomarkers specific for B. cereus group as a detection tool to reduce economic loss due to B. cereus contamination.

Dynabeads protein G antibody conjugates combined with modified brain heart infusion broth for the enrichment and separation of Bacillus cereus in artificially contaminated vegetables.

A modified brain heart infusion (MBHI) broth and a protocol of immunomagnetic separation (IMS) using antibody-coated Dynabeads® protein G were developed for the enrichment and separation of Bacillus cereus in artificially contaminated vegetable samples. The MBHI consisted of BHI and 0.34 g/L magnesium sulfate, 12.08 g/L sodium pyruvate, 1.82 g/L yeast extract, and polymyxin B. The amount of immunomagnetic beads (IMBs) and immunoreaction time were optimized. The capture efficiency was 58.32% with 0.4 mg IMBs when the immunoreaction time was 20 min. Capture of B. cereus by IMBs did not interfere with competing flora. Pre-enrichment IMS was validated with four B. cereus strains in artificially contaminated baby sprouts, bean sprouts, lettuce, and spinach at two levels (∼0.1 and ∼1 CFU/g). We were able to detect and isolate B. cereus in 40/40 samples of vegetables contaminated at 0.1 CFU/g with IMS after 6 h of enrichment in MBHI.