Jung-Beom Kim

Prevalence, genetic diversity, and antibiotic susceptibility of Bacillus cereus strains isolated from rice and cereals collected in Korea.

Incidence and properties of Bacillus cereus strains naturally present in cereals were evaluated by phenotypic characterization, antibiotic susceptibility testing, and pulsed-field gel electrophoresis. Of 293 cereal samples tested, 73 (25%) contained B. cereus strains. Incidence of B. cereus isolates varied with respect to sample; they were found in 15 (37%) of 83 brown rice samples, 23 (37%) of 63 glutinous rice samples, 16 (21%) of 76 barley samples, and 19 (27%) of 71 Jobs tears samples. All B. cereus isolates from cereals were positive for diarrheal toxin genes. The isolates were susceptible to most of the antibiotics tested, but they were highly resistant to ampicillin, cefepime, oxacillin, and penicillin. Of the genes assayed by the PCR technique, a high frequency of nheA (99%) and hblDC (84%) was found in the genomic DNA of cereal-associated isolates, whereas cytK was less common (55%). From the strains carrying the hblDC genes, 93% produced enterotoxin HBL. B. cereus isolates did not have significant genetic homology. The genetic diversity and toxic potential differ among the strains isolated from cereals. These results provide important information on toxin gene profiles of cereal-associated B. cereus for population studies.

Toxin genes profiles and toxin production ability of Bacillus cereus isolated from clinical and food samples.

Bacillus cereus can cause diarrheal and emetic type of food poisoning but little study has been done on the main toxins of food poisoning caused by B. cereus in Korea. The objective of this study is to characterize the toxin gene profiles and toxin-producing ability of 120 B. cereus isolates from clinical and food samples in Korea. The detection rate of nheABC, hblCDA, entFM, and cytK enterotoxin gene among all B. cereus strains was 94.2, 90.0, 65.8, and 52.5%, respectively. The ces gene encoding emetic toxin was not detected in all strains. Bacillus cereus strains carried at least 1 of the 8 enterotoxin genes were classified into 12 groups according to the presence or absence of 8 virulence genes. The 3 major patterns, I (nheABC, hblCDA, entFM, and cytK gene), II (nheABC, hblCDA and entFM gene), and VI (nheABC and hblCDA gene), accounted for 79.2% of all strains (95 out of 120 B. cereus isolates). Non-hemolytic enterotoxin (NHE) and hemolysin BL (HBL) enterotoxins were produced by 107 and 100 strains, respectively. Our finding revealed that NHE and HBL enterotoxins encoded by nhe and hbl genes were the major toxins among B. cereus tested in this study and enterotoxic type of B. cereus was predominant in Korea.

Emetic toxin producing Bacillus cereus Korean isolates contain genes encoding diarrheal-related enterotoxins.

Bacillus cereus can cause the diarrheal and emetic type of food poisoning but the symptoms of emetic food poisoning caused by B. cereus occasionally include emesis and diarrhea. The enterotoxin characteristics of emetic toxin (cereulide) producing B. cereus were needed to be determined. Therefore, forty B. cereus strains isolated from various sources in Korea were investigated for the presence of enterotoxin genes. All strains were confirmed to produce the emetic toxin using HPLC-MS methods. The rates of the nheABC, hblCDA, entFM and cytK genes amongst emetic toxin producing B. cereus strains were 82.5, 7.5, 50.0 and 27.5%, respectively. Pattern III harbored nheABC and entFM genes and pattern V processed entFM gene and were shown to be the major patterns, being present in 55.0% (21 of 40) of the emetic toxin producing B. cereus strains. Our findings revealed that 34 (85.0%) of 40 emetic toxin producing B. cereus strains isolated in Korea have the potential to cause diarrheal and emetic type of food poisoning, simultaneously. Thus, emetic toxin and enterotoxin genes should be constantly screened to provide insight into B. cereus food poisoning.

Food poisoning associated with emetic-type of Bacillus cereus in Korea.

Bacillus cereus can cause diarrheal and emetic types of food poisoning but little study has been done on emetic type of food poisoning in Korea. The objective of this study was to report on the emetic type of food poisoning associated with B. cereus in Korea. The toxin gene profile, toxin production, and antibiotic resistance of B. cereus isolates were investigated in this study. B. cereus was detected in three out of four samples, while the other food poisoning bacteria were not detected. All isolates (KUGH 10, 11, and 12) presented nhe A, B, and C diarrheal toxin genes (755, 743, and 683 bp), detected using NHA, NHB, and NHC primers, and ces emetic toxin gene (1271 bp), detected using CES primer, and produced nonhemolytic enterotoxin and emetic toxin (cereulide), detected using immunochemical assay and high performance liquid chromotography/mass spectrometry (HPLC/MS) analysis. All emetic-associated isolates were resistant to beta-lactam antibiotics. Most important finding in this study was that the risk of emetic-type B. cereus food poisoning has existed in Korea. This suggested that the food poisoning caused by B. cereus producing emetic and diarrheal toxins should be constantly evaluated to prevent misdiagnosis between emetic and diarrheal types of food poisoning.

Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang.

Bacillus amyloliquefancies CH51 isolated from cheonggukjang, a traditional Korean fermented soy food, has strong fibrinolytic activity and produces several fibrinolytic enzymes. Among four different growth media, tryptic soy broth was the best in terms of supporting cell growth and fibrinolytic activity of this strain. A protein with fibrinolytic activity was partially purified from the culture supernatant by CMSephadex and Phenyl Sepharose column chromatographies. Tandem mass spectrometric analysis showed that this protein is a homolog of AprE from B. subtilis and it was accordingly named AprE51. The optimum pH and temperature for partially purified AprE51 activity were 6.0 and 45 degrees , respectively. A gene encoding AprE51, aprE51, was cloned from B. amyloliquefaciens CH51 genomic DNA. The aprE51 gene was overexpressed in heterologous B. subtilis strains deficient in fibrinolytic activity using an E.colo-Bacillus Shuttle vector, pHY300PLK.

Comparison of enterotoxin production and phenotypic characteristics between emetic and enterotoxic Bacillus cereus.

Bacillus cereus was divided into emetic toxin (cereulide)- and enterotoxin-producing strains, but emetic toxin-producing B. cereus is difficult to detect immunochemically. Screening methods for emetic toxin-producing B. cereus are needed. The objectives of this study were to identify and detect emetic toxin-producing B. cereus among 160 B. cereus strains, and to compare enterotoxin production and phenotypic characteristics between the emetic toxin-producing and enterotoxin-producing strains. Forty emetic toxin-producing B. cereus strains were determined with high-pressure liquid chromatography-mass spectrometry analysis. Among the emetic toxin-producing strains (n = 40), 31 (77.5%) and 3 (7.5%) strains produced nonhemolytic enterotoxin (NHE) and hemolysin BL (HBL) enterotoxins, respectively. In addition, 107 (89.2%) and 100 (83.3%) strains produced NHE and HBL enterotoxins among the enterotoxin-producing strains (n = 120). The number of strains positive for starch hydrolysis, salicin fermentation, and hemolysis among the emetic toxin-producing strains were 3 (7.5%), 3 (7.5%), and 26 (65.0%), respectively, and among enterotoxin-producing strains, these numbers were 101 (84.2%), 100 (83.3%), and 111 (92.5%), respectively. In particular, the three emetic toxin-producing B. cereus strains (JNHE 6, JNHE 36, and KNIH 28) produced the HBL and NHE enterotoxins and were capable of starch hydrolysis and salicin fermentation. The absence of HBL enterotoxin and certain phenotypic properties, such as starch hydrolysis and salicin fermentation, indicates that these properties were not critical characteristics of the emetic toxin-producing B. cereus tested in this study.

Enterotoxigenic profiling of emetic toxin- and enterotoxin-producing Bacillus cereus, Isolated from food, environmental, and clinical samples by multiplex PCR.

Bacillus cereus comprises the largest group of endospore-forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin- (8 patterns) and enterotoxin-producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.

Genetic diversity of emetic toxin producing Bacillus cereus Korean strains

Bacillus cereus is divided into emetic and enterotoxin producing strains. Emetic B. cereus showed the low level of genetic diversity and single evolutionary lineage but no specific study of the genotypic characterization of emetic B. cereus Korean strains has been conducted. The objective of this study was to investigate the genotypic diversity of emetic B. cereus Korean strains. A total 39 strains (35 clinical and 4 food isolates) was analyzed for the genotypic characterization. A total of 17 distinct patterns were obtained from the random-amplified polymorphic DNA (RAPD) banding patterns and the majority of clusters belong to group 3. The pulsed-field gel electrophoresis (PFGE) banding patterns were divided into 17 distinct pulsotypes, and groups B and C were dominated. Emetic B. cereus Korean strains showed diverse pulsotypes in contrast with previous studies. All strains were resistant to β-lactam antibiotics such as penicillin and ampicillin. Combining biochemical properties, PFGE types, RAPD types and antibiotic resistance types, a total of 7 composite clusters were found. The majority of composite clusters were consisted with cluster 5 and 6. Enterotoxin producing reference strains belong to composite cluster 7. However, JNHE 6 (Jeollabuk-do Research Institute of Health & Environment; cluster 1) and JNHE 36 (cluster 2) which possessed the ability of starch hydrolysis and saline fermentation showed different composite clusters comparing with most emetic B. cereus. JNHE 7 and JNHE 53 formed composite cluster 3 and 4. Emetic B. cereus Korean strains showed genotypic diversity comparing with the previous studies.

Determination of myristicin in rat serum samples using microbore high performance liquid chromatography with column-switching

SummaryA microbore high-performance liquid chromatographic method with column-switching was developed for the analysis of myristicin from rat serum without prepurification. Deproteinization, fractionation, concentration and separation of analyte were carried out by appropriate switching of columns and using solvent mixtures. The method showed excellent precision, accuracy and speed with a detection limit of 10 ng mL−1 from 25 μL of serum. The total analysis time per sample was 25 min and the coefficients of variation for intra- and inter-assay were less than 1.8%.

Simultaneous determination of parathion and metabolites in serum by HPLC with column switching

SummaryA new high-performance liquid chromatographic method with column-switching has been developed for the simultaneous determination of parathion and its metabolites such asp-nitrophenol and paraoxon in serum. Serum samples were injected onto a precolumn packed with LiChroprep RP-8 after simple dilution with 20% phosphoric acid. Polar plasma components were washed with 0.02 M phosphate buffer (pH 3.0). After valve switching, the concentrated compounds were eluted in back-flush mode and separated on a Ultracarb ODS 30 column with a gradient of acetonitrile −0.01 M phosphate buffer (pH 3.0) as mobile phase. The method showed excellent precision, accuracy and speed with detection limit of 0.1 μg mL−1. Total analysis time per sample was <40 min and coefficients of variation for intra-and inter-assay were <4.5%.