Bacillus 박종현경원대학교식품생물공학과 1. 1.1 연구의배경과중요성.. 3~4% 15%.,,,.,,,.,., 10 5 ~10 6 CFU/g Bacillus cereus group. B. subtilis, B. megaterium, B. cereus, B. mycoides, B. circulans. ( ) 20%. Bacillus,,, Gram 483
,,. B. cereus Gram-positive, spore-forming food pathogen (emesis) (diarrhea). B. cereus enterotoxin. 8~16. 24. Diarrheal enterotoxin toxin valinomycin cereulide([d-o-leu-d-ala-l-o-val-l-val] 3 ). cereulide cyclic dodecadepsipeptide potassium ionophore mitochondria. natural killer cell [1]. Cold-chain B. cereus. B. cereus, food-poisoning B. cereus population structure toxin,. Bacillus Bacillus food-borne disease.,,. Bacillus..,,,,,,,,,,, Bacillus g 10 5, Bacillus,. B. cereus 2005 B. cereus ( : 1g 1,000 ) B. cereus, PCR,. B. cereus Salmonella, Campylobacter. 484
. B. cereus dose 10 2 ~10 8 CFU/g 10 4 ~10 5.. B. cereus pathogenic potential genetic diversity. B. cereus pathogenic potential. PCR diarrheal toxin, emetic toxin gene toxin profile B. cereus toxin, phylogenic analysis, Bacillus. 1.2 B. cereus 와 B. thuringiensis 의독성물질 [14] B. cereus heat-labile enterotoxin. B. cereus cytotoxicity enterotoxin, 8~16,,. B. cereus emetic toxin 5 enterotoxin pathogen. Hemolysin BL(HBL) non-hemolytic enterotoxin(nhe) 3 protein EntFM, CytK BceT one-component toxin. HBL NHE enterotoxin B. cereus diarrhoeal type. B. cereus B. cereus diarrheal poisoning.. HBL complex hbla, hblc, hbld binding component B lytic component L 1, L 2,, hblb. 40kDa, HBL,. Beecher Wong[15] HBL target cell colloid osmotic lysis. HBL B. cereus diarrhoea. Schoeni Wong[16] biological activity, 1:1:1 biological activity. NHE multicomponent enterotoxin complex strain Lund Granum. NHE complex nhea, nheb, nhec NheA, NheB, NheC. 45, 39 105kDa, Bacillus 485
. NheA HBL L 2, NheB L 1. NheC binding protein NheC, NheC target cell, B. anthracis anthrax toxin [11]. 41kDa B. cereus enterotoxin bcet. DNA HBL NHE. in vitro, (cytotoxicity).. diarrheal syndrome B. cereus,. Asano enterotoxin FM. CytK cytotoxin protein B. cereus, C. per f ringens -toxin S. aureus -hemolysin. Cytotoxin K 1998 B. cereus. Lund single cytotoxic protein(cytk) B. cereus. B. cereus B. cereus enterotoxin, cytk. Guinebretiere diarrheal disease B. cereus 73% cytk, 37%. B. cereus diarrheal syndrome B. cereus toxin intoxication. enterotoxin B. cereus, toxico-infection. 10 7 /ml enterotoxin infectious dose 10 3 ~10 4 /ml enterotoxin ph 3.1 20 80%, trypsin chymotrypsin 20 toxin, 12~24 enterotoxin. B. cereus type emetic syndrome fried rice 1970. B. cereus cereulide, B. cereus small cyclic heat-stable peptide cereulide [1]. Emetic toxin, ph,. cereulide, 486
. Cereulide 5-HT 3 receptor (vagus afferent), potassium. in vitro natural killer cell. B. cereus, cereulide B. cereus cereulide B. cereus. Cereulide,. Cereulide heat-stable,. Emetic B. cereus fried boiled rice pasta noodles, emetic toxin cereulide, cereulide. B. cereus, cereulide B. cereus. toxin. enterotoxin, emetic toxin. Cereulide emetic type 0.5~6,,,. B. cereus emetic toxin(cereulide) valinomycin liver toxicity, mitochondrial toxicity, lipophilicity, immunotoxicity. Emetic B. cereus, [5]. Emetic toxin diarrhoeal toxin. B. cereus. Emetic toxin B. cereus diarrhoeal toxin. Enterotoxin emetic toxin B. cereus. enterotoxin S(entS), sphingomyelinase(sph), phosphatidylinositol specific phospholipase(piplc), cereolysin O(clo). cell-bound metalloprotease(camelysin). B. cereus phospholipase C protein(pih, PCH, SM), sphingomyelinase(sm) phosphatidylcholine hydroloase(pch) hemolysin cereolysin AB, phospholipase C Ca 2+ [8]. Phosphatidylinositol hydrase(pih) plasma membrane hemolysis cytolysis. B. cereus phospholipase C sphingomyelinase. Bacillus 487
1.3 연구의목표 100 GDP.. spore-forming bacteria, thermostable Bacillus spp.. Bacillus 2005 Bacillus cereus.. Bacillus. 2. 2.1 쌀밥의총세균수분리및계수 2. 1~2. 25g, buffered peptone water(bpw) 225ml. plate count agar(pca) 10 1~7 37 12~24. 2.2 쌀밥의품질저하 Bacillus 분리와 group 1B 의신속동정 (VITEK 활용 ) Genus Bacillus. Bacillus 30~45 65. pathogenic Bacillus 3 group Group I. Gram(+),. Group 1A lipid globlues B. cereus, B. megaterium, B. thringenesis, B. mycoides, B. antracis Group 1B lipid globlue B. subtilis, B. lichemi f ormis, B. pumilis, B. coaglulans. Group 1A blood agar colony morphology, 488
hemolysis, motility, penicillin susceptability, spore location typing. Group 2B DNA. MYP Bacillus API kit VITEK 2(biomeirue, France) Species. 2.3 품질저하 Bacillus 의효소생화학적인특성분석 Bacillus API kit -amylase, protease, lipase Bacillus species. 2.4 식중독유발 Bacillus cereus group 분리동정 25ml, 225ml 2. 10-2 10-6 10. MYP 0.2ml 5 1ml 30 24 lecithinase. 30 24,, nitrate, VP, -hemolysis,. g yrb gene target primer BC1(5 -ATT GGT ACC GAT CAA ACA-3 ) BC2(5 -TCA TAC GTA TGG TTA TTC-3 ) PCR [10]. B. cereus group(b. cereus, B. anthracis, B. thuringenesis, B. mycoides, B. pseudomycoides, B. weihenstephanesis, B. medusa) B. cereus multiplex PCR [12]. 2.5 Bacillus culture collection 구성 520 Bacillus spp. B. cereus group, B. cereus., Bacillus spp. Bacillus spp. Culture Collection. Bacillus 489
2.6 B. cereus group 의선별동정 B. cereus group B. cereus, B. thuringiensis. g yrb, cry gene primer PCR B. cereus, B. thuringiensis differentiation identification [9]. 2.7 Emetic toxin 과 diarrheal toxin 분비 B. cereus group 분류를위한 PCR B. cereus group 4 diarrheal toxin PCR. Emetic toxin gene 2005 M. Ehling-Schulz cereulide synthetase target primer. PCR identification 3 Ehling-Schluz (2004)[2], Horwood (2004)[3], Ehling-Schluz (2005)[4]. Ehling-Schulz(2005)[7] cesf1/cesr2 emetic B. cereus. cesf1/cesr2 1 The primers used to detect the emetic/enterotoxin genes from B. cereus group with PCR Target gene Primer Oligonucleotide sequence(5-3 ) Use g yrb BT1/BT2r g yrb BC1/BC2 cr y1ab SB-2/U3-18c cr y1ac RB-19/U8-15c ces CesF1/R2 5 -ATCGGTGATACAGATAAGACT-3 /(F) 5 -ATTGGTGACACCGATCAAACA-3 /(F 5 -TCGGAAAATGTGCCCAT-3 /(F) 5 -GGGACTGCAGGAGTGAT-3 /(F) 5 -GGTGACACATTATCATATAAGGTG-3 5 -GTAAGCGAACCTGTCTGTAACAACA-3 hbla HblA1/HblA2 5 -GCTAATGTAGTTTCACCTGTAGCAAC-3 /(F) B. cereus-b.thuringiensis Emetic toxin gene hblc HBLC-N/HBLC-C 5 -AATAGGTACAGATGGAACAGG-3 /(F) HBL enterotoxin gene hbld HBLD-N/HBLD-C 5 -AATCAAGAGCTGTCACGAAT-3 /(F) nhea nhea344s/nheb843a 5 -TACGCTAAGGAGGGGCA 3 /(F) NHE enterotoxin gene bcet ETF/ETR 5 -TTACATTACCAGGACGTGCTT-3 /(F) Enterotoxin T gene entfm ENTA/ENTB 5 -ATGAAAAAAGTAATTTGCAGG-3 /(F) Enterotoxin FM gene cytk F2/R7 5 -AACAGATATCGGTCAAAATGC-3 /(F) CytK enterotoxin gene Cell surface gene CS_F2/R2 5 -GTTAA(AT)GTGCCAACTAGGTGG-3 5 -CGTTTACAGATATCATTCCAGC-3 RADP 490
cereulide synthetase(nonribosomal peptide sysnthetase) target primer. PCR protocol. 2.8 Toxin 검출, haemolysis 및생화학적인특성 Enterotoxin commercial kit. Hbl enterotoxin L2 component BCET-RPLA B. cereus Enterotoxin Test Kit(Oxoid) Nhe enterotoxin Tecra BDE Kit(Tecra Diagnostics) 10 manufactuer. salicin, Claus Berkeley [6]. Haemolysis triptic soy agar(bbl) 5% bovine blood -haemolysis, -haemolysis. 3. 3.1 전국산지별쌀시료의수집 31. 8, 2, 2, 3, 8, 5, 3 12, 4, 7, 1, 7. (9 ). 2 Raw rice samples collected according to the production areas 1 A CY DW 2 B WB DJ 3 C CN CN ( ) 4 D AT BY 5 E CNS CNS 6 F MS MS 7 G ON KN 8 H YJD RK 9 I OC YCN 10 J PJ RK 11 K GYN HN 12 L DMS YS Bacillus 491
2 13 M GCN GYR 14 N DJH MN 15 O CAH OCN 16 P PNJ SSN 17 Q PSO SMG 18 R KSD DYN 19 S THH OCN 20 T SSS GYP 21 U SMO OTG 22 V SMG SMG 23 W SSM KSM 24 X SSMC KSM 25 Y SSMG KSM 26 Z YGN CBN 27 a YGH SSM 28 b MAB PK 29 c JCC MTY 30 d MCO CCG 31 e MNG SSG 1 Production areas of raw rice on the map shown by yellow color. 492
3.2 쌀밥으로부터의총균수와 Bacillus 의 prevalence 분석 1~2 Bacillus. 31 CFU(Colony Forming Unit) 12 10 9. 9/31(30%) 1~3log scale. Bacillus MYP 9/31(30%) prevalence 1~3log scale Bacillus. MYP Bacillus Bacillus. Bacillus spp.(myp), 3. Bacillus. 3 Viable counts of the cooked rices on PCA and MYP media (Unit: CFU/g) Number Total colony count on PCA Bacillus spp. on MYP 1 N.D + 2 2.0 10 2 1.7 10 2 3 2.0 10 1 4.0 10 1 4 5.0 10 3 + 5 N.D + 6 N.D + 7 N.D + 8 N.D + 9 1.0 10 1 + 10 N.D + 11 N.D + 12 N.D 1.0 10 1 13 1.0 10 1 1.0 10 1 14 N.D 1.0 10 1 15 N.D + 16 3.2 10 2 3.3 10 2 17 1.0 10 1 3.0 10 1 18 N.D 1.0 10 1 19 N.D + 20 N.D N.D Bacillus 493
3 Number Total colony count on PCA Bacillus spp. on MYP 21 N.D + 22 N.D N.D 23 N.D + 24 N.D N.D 25 N.D + 26 N.D N.D 27 N.D N.D 28 N.D N.D 29 N.D N.D 30 2.0 10 1 + 31 1.2 10 9 1.2 10 9 N.D : non-detected, + : Detection after enrichment. 3.3 쌀밥으로부터의균분리및동정 MYP colony 37 Bacillus group. API kit CHB50, VITEK 2 Rapid identification system, PCR. MYP Yellow colony API kit CHB50, VITEK2 Pink colony Bacillus cereus group PCR. API kit API web ID% 95%. VITEK2 ID% 85%. PCR B. cereus, B. thringiensis target gene g yrb(topoisomerase ) target primer[13] gene cycler(biorad, Hercules, CA, USA) electrophoresis. 31 37, B. thuringiensis, B. valismortis, B. pumilus, B. coagulans, B. licheni f ormis, B. cereus, Geobacillus stearothermophilus, Brevibacillus laterosporus. 37 50% 20 B. thuringiensis 9 B. cereus. B. cereus group B. lichni f ormis 494
. B. valismortis, B. pumilus, B. licheniformis Bacillus spp. B. licheni f ormis. Bacillus spp.,. 365 bp M 1 2 3 4 5 6 7 8 9 10 M 2 Detection of B. cereus by BC1, BC2r primer. Lane 1, B. cereus KCCM 40935; Lane 2, B. cereus KCTC 1094; Lane 3, B. thuringiensis KCCM 11428; Lane 4, B. thuringiensis KCTC 3452. 368 bp M 1 2 3 4 5 6 7 8 9 10 M 3 Detection of B. thuringiensis by BT1, BT2r primer. Lane 1, B. cereus KCCM 40935; Lane 2, B. cereus KCTC 1094; Lane 3, B. thuringiensis KCCM 11428; Lane 4, B. thuringiensis KCTC 3452. Bacillus 495
4 Identification isolated from boiled rice on MYP media No. strain Result Gram Catalase Hemolysis 7 Bacillus thuringiensis + + + 8 Bacillus thuringiensis + + + 9 Bacillus thuringiensis + + + 10 Bacillus thuringiensis + + + 11 Bacillus thuringiensis + + + 4 Bacillus valismortis + + - 12 Bacillus thuringiensis + + + 13 Bacillus thuringiensis + + + 14 Bacillus thuringiensis + + + 15 Bacillus thuringiensis + + + 17 Bacillus thuringiensis + + + 5 Bacillus valismortis + + - 26 Bacillus thuringiensis + + + 27 Bacillus thuringiensis + + + 28 Bacillus thuringiensis + + + 29 Bacillus thuringiensis + + + 30 Bacillus thuringiensis + + + 31 Bacillus thuringiensis + + + 32 Bacillus thuringiensis + + + 33 Bacillus thuringiensis + + + 34 Bacillus thuringiensis + + + 1 Bacillus pumilus + + + 3 Bacillus coagulans + + - 35 Bacillus thuringiensis + + + 16 Bacillus cereus + + + 6 Bacillus valismortis + + - 2 Bacillus lichenif ormis + + - 18 Bacillus cereus + + + 36 Geobacillus stearothermophilus + + - 37 Brevibacillus laterosporus + + + 19 Bacillus cereus + + + 20 Bacillus cereus + + + 21 Bacillus cereus + + + 22 Bacillus cereus + + + 23 Bacillus cereus + + + 24 Bacillus cereus + + + 25 Bacillus cereus + + + 496
4 Morphological characteristics of isolates on MYP selective media. 5 Classification of identified isolates from boiled rice Isolate identification Isolate number (%) ID method (%ID) B. thuringiensis 20/37 (54) PCR B. cereus 9/37 (27) PCR B. valismortis 3/37 (8) VITEK2 (90) B. pumilus 1/37 (3) VITEK2 (95) B. coagulans 1/37 (3) VITEK2 (85) B. lichemif ormis 1/37 (3) VITEK2 (96) Brevibacillus laterosporus 1/37 (3) VITEK2 (85) Geobacillus stearothermophilus 1/37 (3) API kit (98) Bacillus 497
3.4 분리된 Bacillus spp. 의생화학적특성 37 Bacillus spp. Api kit VITEK2. B. cereus strain number 16. 6 Biochemical characteristics of isolated Bacillus cereus from boiled rice (Result by Api kit CHB50) No. strain Test result Salicin Amidon Glycogen D-Turanose D-Tagatose L-Fucose 16 - + + - - - 18 + - - + + - 19 + - - + - - 20 + - - - - - 21 + - - + - - 22 + - - (+) - - 23 + - - + (+) (+) 24 + - - - - - 25 + - - + - - 7 Biochemical characteristics of isolated Bacillus spp. from boiled rice (Result by VITEK 2 ) Type of strain Test result BXYL AspA LeuA PheA BGAL PyrA AGAL AlaA TyrA BNAG APPA B. pumilus + (+) + + + - + + + + - B. lichenif ormis - - + + + + (-) + + - - B. coagulans - - + + + - (+) + + - (-) B. valismortis1 + (+) + + + + + + + - (-) B. valismortis2 + (-) + + + + + + + - + B. valismortis3 + (-) + + + + + + + - (+) Brevibacillus - - - - - - - - - - (+) Test result AMAN MTE GlyA dman NAG PLE IRHA BGLU PVATE AGLU dtag B. pumilus + - + + (-) - - + + - + B. lichenif ormis - + - + + + - + + + + B. coagulans - + - + + - + - - - - 498
7 AMAN MTE GlyA dman NAG PLE IRHA BGLU PVATE AGLU dtag B. valismortis1 - + - + + + - - + + - B. valismortis2 - (+) - + + (+) - (-) + + - B. valismortis3 - + (+) + + - - (-) + + - Brevibacillus - + - - - - - - - - - Test result CDEX GLYG INO MdG ELLE dtre drib KAN OLD ESC B. pumilus - - - - - + + - + + B. lichenif ormis + + + + + + + - + + B. coagulans - - - - - + + - + + B. valismortis1 - - + - - + + - + + B. valismortis2 - - + - + + + - + + B. valismortis3 - - + - - + + - + - Brevibacillus - - - - - (-) (+) + - (+) 3.5 분리된 B. cereus 의 toxin gene profile 분석 PCR HBL(hemolysin BL), NheA(non-hemolytic enterotoxin protein), BceT(enterotoxic protein T), CytK(cytotoxin K), Ces(emetic toxin) primer. HBL, Nhe toxin Ces toxin(cerulide). B. cereus hbl toxin nhe gene ces gene. B. cereus hbl gene nhe gene. B. cereus 9 7 ces gene. B. thuringesis nhe gene toxin. Bacillus 499
1269 bp M 1 2 3 4 5 6 7 8 9 10 M 5 Detection of ces gene for B. cereus by CesF1, CesR2 primer. Lane 1, B. cereus KCCM 40935 (negative control); Lane 2, B. cereus NCTC 11143 (positive control); Lane 3~10 Bacillus cereus isolates. 8 Toxin gene distribution of Bacillus cereus from boiled rice. No. strain B. cereus KCCM 40935 HBL Complex hblc hbla hbld Toxin gene nhea bcet cytk ces + + + + + + - 16 + + + + + + - 18 - - - + - + + 19 - - - + - + + 20 - - - + - + + 21 - - - + - + + 22 - - - + - + + 23 - - + + - + + 24 - - - + - + - 25 - - + + - + + 500
9 Toxin gene distribution of Bacillus thuringiensis from boiled rice No. strains B. cereus ATCC 11143 B. cereus KCCM 40935 HBL Complex hblc hbla hbld Toxin gene nhea bcet cytk ces Do not apply + + + + + + + - 26 - - - + - - - 27 - - - + - - - 28 - - - + - - - 29 - - - + - - - 30 - - - + - - - 7 - - - + - - - 15 - - - + - - - 31 - - - + - - - 32 - - - + - - - 33 - - - + - - - 8 - - - + - - - 9 - - - + - - - 10 - - - + - - - 17 - - - + - - - 11 - - - + - - - 12 - - - + - - - 13 - - - + - - - 14 - - - + - - - 34 - - - + - - - 35 - - - + - - - 4.,,,., Bacillus Bacillus 501
Clostridium. Clostridium (strict anaerobe). Bacillus spp. Bacillus,. 31, Bacillus spp.,, system, PCR. 9/31(30%) 1~3log scale. Bacillus MYP Bacillus spp. 9/31(30%) prevalence 1~3log scale Bacillus. MYP Bacillus Bacillus. Bacillus spp. Bacillus. 31 37, B. thuringiensis, B. valismortis, B. pumilus, B. coagulans, B. licheni f ormis, B. cereus, Geobacillus stearothermophilus, Brevibacillus laterosporus. 37 50% 20 B. thuringiensis 9 B. cereus. Bacillus profile., B. thuringiensis. B. thuringiensis non-hemolytic toxin gene B. thuringiensis. B. cereus 9. B. cereus (emetic toxin) (enterotoxin), (diarrheal type). Diarrheal syndrome, enterotoxin. Emetic syndrome emetic toxin cereulide, ces gene B. cereus. 9 B. cereus 7 B. cereus poisoning dose. B. cereus 10 5 cell/g, B. cereus 10 2 502
poisoning dose. 2. B. valismortis, B. pumilus, B. licheni f ormis Bacillus spp. B. licheni f ormis. Bacillus spp.. B. cereus B. thuringiensis. 1. Marahiel, M., T. Stachelhaus, and H.D. Mootz. 1997. Modular peptide synthetase involved in nonribosomal peptide synthetase. Chem. Rev. 97: 2651-2673. 2. Ehling-Schulz, M., M. Fricker, and S. Scherer. 2004. Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay. FEMS Microbiol. Lett. 232: 189-195. 3. Hoewood, P.F., G.W. Burgess, and H.J. Oakey. 2004. Evidence for nonribosomal peptide synthetase production of cereulide(emetic toxin) in Bacillus cereus. FEMS Microbiol. Lett. 232: 319-324. 4. Ehling-Schulz, M., Vukov N., Schulz A., Shaheen R., Andersson M., Martlbauer E., and Scherer S. 2005. Identification and partial characterization of the nonribosomal peptide sunthetase gene responsible for cereulide production in emetic Bacillus cereus. Appl. Environ. Microbiol. 71: 105-113. 5. Finlay, W.J.J., N.A. Logan and A.D. Sutherland. 1999. Semiautomated metabolic staining assay for Bacillus cereus emetic toxin. Appl. Environ. Microbiol. 66: 1811-1812. 6. Claus, D., R.C.W. Berkeley. 1986. Genus Bacillus Cohn 1872, 174th. In Bergey s Manual of Systematic Bacteriolog y, Vol 2, pp.1105-1139. P.H.A. Sneath, N.S. Mair, M.E. Sharpe, J.G. Holt (eds.). Baltimore, MD, William and Wilkins. 7. Ehling-Schulz, M., B. Svensson, M.H. Guinebretiere, T. Linback, M. Andersson, A. Schulz, M. Fricker, A. Christiansson, P.E. Granum, E. martlbauer, C. Nguyen, M. Salkinoja, and S. Scherer. 2005. Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains. Microbiology 151: 153-197. 8. Grethe, I.B., Andersen S., Marianne S., Terje L.T., Einar G.T. 2001. Growth and toxin profiles of Bacillus cereus isolated from different food sources. Int. J. Food Microbial. 69: 237-246. 9. Manzano, M., L. Cocolin, C. Cantoni, G. Comi. 2002. Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides differentiation using a PCR-RE technique. Int. J. Food Microbiol. 81: 249-254. 10. Chen, M.L. and H.Y. Tsen. 2002. Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rrna and gyrb gene based PCR primers and sequencing of their annealing sites. J. Appl. Microbiol. 92: 912-919. 11. Cherif, A., L. Brusetti, S. Borin1, A. Rizzi1, A. Boudabous, H. Khyami-Horani and D. Daffonchio. 2003. Genetic relationship in the Bacillus cereus group by rep-pcr fingerprinting and sequencing of a Bacillus anthracisspecific rep-pcr fragment. J. Appl. Microbiol. 94: 1108-1119. Bacillus 503
12. Bavykin, S.G., Y.P. Lysov, Y. Zakhariev, J.J. Kelly, J. Jackman, D.A. Stahl, A. Cherni. 2004. Use of 16S rrna, 23S rrna, and gyrb gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. J. Clin. Microbiol. 42: 3711-3730. 13. Reyes-Ramirez, A., J.E. Ibarra. 2005. Fingerprinting of Bacillus thuringiensis type strains and isolates by using Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR analysis. Appl. Environ. Microbiol. 71: 1346-1355. 14. Hwang, J.H. 2009. Biochemical characteristics and enterotoxin gene distribution of food-borne Bacillus cereus. MS thesis, Kyungwon University. 15. Beecher, D.J., J.L. Schoeni, A.C.L. Wong. 1995. Enterotoxic activity of hemolysin BL from Bacillus cereus. Infect Immun. 63: 4423-4428. 16. Schoeni, J.L., A.C. Wong. 2005. Bacillus cereus food poisoning and its toxin. J. food Prot. 68: 636-648. 504