Byung-Jae Park

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.

In vitro and in vivo defensive effect of probiotic LAB against Pseudomonas aeruginosa using Caenorhabditis elegans model

ABSTRACT This study aimed to investigate in vitro and in vivo the probiotic characteristics of lactic acid bacteria (LAB) isolated from Korean traditional fermented foods. Caenorhabditis elegans (C. elegans) was used for analytical assays of fertility, chemotaxis, life-span, worm-killing and bacterial colonization in the intestinal lumen of the worm. All 35 strains of LAB reduced fertility and slowed development in the worms. The worm-killing assay showed that LAB significantly increased the lifespan (P < 0.05) and reduced the susceptibility to virulent PA14; however, the heat-killed LAB did not. The bacterial colonization assay revealed that LAB proliferated and protected the gut of the worm against infection by Pseudomonas aeruginosa PA14. In addition, specific LAB Pediococcus acidilactici(P. acidilactici DM-9), Pediococcus brevis (L. brevis SDL1411), and Pediococcus pentosaceus (P. pentosaceus SDL1409) strains showed acid resistance (66–91%), resistance to pepsin (64–67%) and viability in simulated intestinal fluid (67–73%) based on in vitro probiotic analyses. Taken together, these results suggest that C. elegans may be a tractable model for screening efficient probiotics.

In vivo screening platform for shiga toxin-producing Escherichia coli (STEC) using Caenorhabditis elegans as a model

Shiga toxin-producing Escherichia coli (STEC) strains are the main cause of bacillary dysentery, although STEC strains generally induce milder disease symptoms compared to Shigella species. This study aimed to determine the virulence of STEC using the nematode Caenorhabditis elegans as a model host. Worm killing, fertility and bacterial colonisation assays were performed to examine the potential difference in the virulence of STEC strains compared to that of the control E. coli OP50 strains on which worms were fed. A statistically significant difference in the survival rates of C. elegans was observed in that the STEC strains caused death in 8–10 days and the E. coli OP50 strains caused death in 15 days. STEC strains severely reduced the fertility of the worms. The intestinal load of bacteria in the adult stage nematodes harbouring the E. coli OP50 strains was found to be 3.5 log CFU mL-1. In contrast, the STEC strains E15, E18 and E22 harboured 4.1, 4.2 and 4.7 log CFU ml−1 per nematode, respectively. The heat-killed STEC strains significantly increased the longevity of the worms compared to the non-heated STEC strains. In addition, PCR-based genomic profiling of shiga toxin genes, viz., stx1 and stx2, identified in selected STEC strains revealed that these toxins may be associated with the virulence of the STEC strains. This study demonstrated that C. elegans is an effective model to examine and compare the pathogenicity and virulence variation of STEC strains to that of E. coli OP50 strains.

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.

Highly efficient and specific separation of Staphylococcus aureus from lettuce and milk using Dynabeads protein G conjugates

An immunomagnetic separation method using antibody-coated Dynabeads® Protein G was developed for specific and efficient separation of Staphylococcus aureus in lettuce and whole milk. The amount of immunomagnetic beads (IMBs) and conjugation conditions were optimized. A high capture efficiency was obtained with 0.4 mg of IMBs, an immunoreaction time of 20 min, and a separation time of 1 min without wash. Under optimal conditions, the capture efficiency (CE) for 100-105 CFU/mL of S. aureus was higher than 91.46%. The IMBs showed high specificity even with a high constant number (107 CFU/mL) of Bacillus cereus, Micrococcus luteus, and Lactobacillus plantarum. The CE of IMBs against S. aureus at concentrations from 102 to 105 CFU/mL ranged from 78.70 to 94.77% for lettuce and 60.0 to 73.27% for milk samples. This IMS can be an appropriate selection for combining with bacterial detection method or efficient isolation procedure for S. aureus from foods.

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.