Prevalence, Genetic Diversity, and Antibiotic Resistance of Bacillus cereus Isolated from Korean Fermented Soybean Products Cheol-Woo Kim, Seung-Hak Cho, Suk-Ho Kang, Yong-Bae Park, Mi-Hye Yoon, Jong-Bok Lee, Wan-Seob No, and Jung-Beom Kim
Bacillus cereus contamination is a major food safety problem for Korean fermented soybean products, but few studies have assessed its potential to cause foodborne illness. The objectives of this study were to investigate the prevalence and characteristics of B. cereus isolated from Korean fermented soybean products. B. cereus was detected in 110 of 162 (67.9%) samples. The highest B. cereus frequency was observed in deonjang (68 of 93 samples, 73.1%) and cheonggukjang (18 of 25, 72.0%); however, nonhemolytic enterotoxin was detected only in 22 of 162 samples (13.6%). Although the tested B. cereus isolates showed diverse pulsotypes according to repetitive sequence-PCR banding patterns, they displayed similar antibiotic sensitivity spectra. The low frequency of enterotoxin detection suggests that the potential risk of B. cereus foodborne illness associated with Korean fermented soybean products is lower than generally presumed. However, considering the prevalence of B. cereus and the high content of fermented soybean products in the Korean diet, it is necessary to constantly monitor the level of contamination with B. cereus and its toxins in such Korean food products.
Abstract:
Introduction Bacillus cereus is a causative agent of diarrheal and emetic food poisoning associated with various categories of food products (EFSA, European Food Safety Authority, 2005). The foodborne diarrhea is caused by heat-labile enterotoxins such as hemolysin BL (HBL), nonhemolytic enterotoxin (NHE), enterotoxin FM (EntFM), and single-protein enterotoxin cytotoxin K (CytK; Schoeni and Wong 1999). The emetic foodborne illness is induced by a small heat- and acid-stable cyclic dodecadepsipeptide toxin cereulide (Agata and others 1994). A severe B. cereus–related foodborne illness, necrotic enteritis, has caused 4 deaths, including the sudden death of a young adult (Lund and others 2000; Naranjo and others 2011). B. cereus has been recognized as an opportunistic foodborne pathogen and is of growing concern to the food industry (Oh and others 2012). Korean fermented soybean products such as deonjang, kochujang, cheonggukjang, and meju can be produced commercially and at home. The factory-made products are obtained by fermentation with Aspergillus species and Bacillus subtilis, while home-made product is prepared by natural fermentation (Jo and others 2011). Meju is soybean koji (Korean soybean block) produced by fermentation with B. subtilis and various molds in industrial settings; however, a home-made product is usually prepared using rice straw, which naturally contains a variety of microorganisms and is commonly contaminated with B. cereus (Lim and Lee 2010). Health benefits of these fermented products are based on unique physicochemical characteristics (Kim and Choi 2003) that
underlie a variety of functional effects (Kang and others 2005), including strong anticancer and antimutagenic activities (Park and others 2003; Jung and others 2006). These fermented products are therefore being recognized as healthy foods. The availability and popularity of Korean fermented soybean products has been constantly increasing worldwide following the registration of deonjang in the Codex Alimentarius as an international food in 2009 (Kwon and others 2010). However, Korean fermented soybean products, especially those made at home, are based on meju and B. cereus contamination is a major problem for the safety of Korean fermented soybean products (Lee and others 2011). In 2006, the Korean government issued regulations regarding B. cereus content (no more than 4 log CFU/g) in deonjang, kochujang, and cheonggukjang (Lee and others 2008). However, few studies have been performed on the pathogenic potential of B. cereus in these food products (Kim and others 2008; Lim and others 2011), underscoring the necessity of comprehensive evaluation of the safety of Korean fermented soybean products and characteristics of B. cereus isolates. The objectives of this study were to determine the incidence of B. cereus and the levels of its enterotoxins in deonjang, kochujang, cheonggukjang, and meju.
Materials and Methods
Samples A total of 162 products were tested, comprising 93 deonjang, 32 kochujang, 25 cheonggukjang, and 12 meju samples. All samples were divided into factory- and home-made products. The 56 factory samples were purchased from local markets, and the 106 home-made samples were obtained nationwide from 2010 to MS 20140401 Submitted 3/11/2014, Accepted 10/15/2014. Authors C.-W. Kim and No are with Dept. of Food Science & Biotechnology, Dongguk Univ., 30, 2012. Pildong-ro 1-gil, Joong-gu, Seoul, Republic of Korea. Author Cho are with Div. of Enteric Bacterial Infections, Center for Infectious Diseases, Korea Natl. Inst. of Health, Isolation and identification of B. cereus Osong, Republic of Korea. Authors Kang, Park, Yoon, Lee and J.-B. Kim are with Each sample (25 g) was mixed with 225 mL buffered peptone Div. of Health Research, Gyeonggi-do Research Inst. of Health & Environment, 95, Pajangchen-ro, Suwon, Gyeonggi-do, Republic of Korea. Direct inquiries to author solution (Oxoid Ltd., Basingstoke, U.K.) and homogenized by a Kim (E-mail: [email protected]). stomacher (BagMixer 400; Interscience, Paris, France) for 5 min.
The homogenized mixture (0.1 mL) was spread onto B. cereus rapid R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12720 Further reproduction without permission is prohibited
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Keywords: antibiotic susceptibility, Bacillus cereus, Korean fermented soybean product, genetic diversity
B. cereus in Korean soybean products . . . agar (BACARA; AES Chemunex, Paris, France) and incubated at 30 °C for 20 h. The colonies that exhibited pink-orange color with lecithinase reaction on BACARA were selected for further culturing on tryptone soya agar (TSA; Oxoid) and 5% sheep blood agar (BA; Komed, Seoul, Korea) at 35 °C for 20 h; the colonies with hemolytic activity on BA were analyzed by Gram staining. The biochemical identification of the isolates was conducted by using API 50 CHB and API 20E test kits (bioM´erieux, Inc., Marcy l’Etoile, France).
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Detection of enterotoxins in Korean fermented soybean products The B. cereus enterotoxin-reversed passive latex agglutination (BCET-RPLA) kit (Oxoid) was used to detect the HBL enterotoxin according to the manufacturer’s instructions. Briefly, 50 g of each sample was placed into a plastic bag containing a lateral filter (BagMixer 400) and homogenized with 50 mL of 0.85% NaCl for 10 min; the filtered mixture was centrifuged for 30 min at 900 × g (Mega 17R, Hanil Science Industrial, Incheon, Korea). The supernatant was filtered using a 0.45-μm membrane filter and used as a sample solution for the HBL enterotoxin test. NHE was detected using the Bacillus diarrheal enterotoxin visual immunoassay (BDE-VIA) kit (Tecra Diagnostics, Reading, U.K.) according to the manufacturer’s instructions. Briefly, 10 g of each sample was placed into a plastic bag containing a lateral filter and blended with 50 mL of 0.25 M Tris-HCl (pH 8.0) for 10 min. The mixture was then centrifuged for 10 min at 1000 × g. The supernatant was filtered through cotton wool inserted in a disposable 10-mL plastic syringe. After pH adjustment to 7 to 8, 2 mL of the supernatant was mixed with 20 μL of sample additive contained in kit and used as a sample solution for the NHE test. Repetitive sequence-PCR DNA fingerprinting The 23 B. cereus isolates tested in this study were randomly chosen with sample sources and types, and composed of 13 deonjang (5 factory and 8 home-made products), 3 kochujang (1 factory and 2 home-made products), 3 cheonggukjang (2 factory and 1 home-made products), and 4 meju (2 factory and 2 home-made products). A total of 23 isolates and B. cereus reference strain (ATCC 11778) were grown at 35 °C for 24 h on TSA. DNA extraction and repetitive sequence-PCR (rep-PCR) were performed as previously described (Chon and others 2012b). Briefly, DNA was extracted using the UltraCleanTM Microbial DNA isolation kit (MoBio Laboratories, Solana Beach, Calif., U.S.A.) and amplified for DNA fingerprinting using the DiversiLab Bacillus kit (bioM´erieux). PCR reactions were carried out R polymerase, in a 25μL volume containing 0.5 μL of AmpliTaq 2.5 μL of 10× GeneAMP PCR buffer І, 2 μL primer mix, 18 μL rep-PCR master mix (all from Applied Biosystems, Foster City, Calif., U.S.A.), and 2 μL of 25 ng/μL DNA template. The thermal cycling conditions were as follows: initial cycle at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 70 °C for 90 s, and a final cycle at 70 °C for 3 min. The rep-PCR products were separated and detected using a microfluidics chip and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif., U.S.A.). The web-based DiversiLabTM software (version 2.1.66; bioM´erieux) was used for the comparative analysis of B. cereus isolates.
Table 1–Detection of Bacillus cereus in Korean fermented soybean products. No. of samples containing B. cereus (%)b Sourcea Deonjang Kochujang Cheonggukjang Meju Total
Factory products 21/25a 6/9a 8/12a 4/10a
(84.0) (66.7) (66.7) (40.0) 39/56a (69.6)
Home products 47/68a 12/23a 10/13a 2/2a
(69.1) (52.2) (76.9) (100) 71/106a (67.0)
Total 68/93 (73.1) 18/32 (56.3) 18/25 (72.0) 6/12 (50.0) 110/162 (67.9)
a Deonjang: Korean fermented soybean paste; Kochujang: Korean fermented red pepper–soybean paste; Cheonggukjang: Korean fermented soybean rapid paste; Meju: Korean fermented soybean block (Korean style soybean koji). b Different letters within a row and line indicate significant differences (P ࣘ 0.05).
Table 2–Detection of enterotoxins in Korean fermented soybean products. No. of samples containing toxins (%) in Korean fermented soybean products Factory products Home products
Total
Sourcea
HBLb
NHEc
HBL
NHE
HBL
NHE
Deonjang Kochujang Cheonggukjang Meju Total
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
5 (20.0) 0 (0.0) 0 (0.0) 0 (0.0) 5 (8.9)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
14 (20.6) 2 (8.7) 1 (7.7) 0 (0.0) 17 (16.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
19 (20.4) 2 (6.3) 1 (4.0) 0 (0.0) 22 (13.6)
a Deonjang: Korean fermented soybean paste; kochujang: Korean fermented red pepper–soybean paste; cheonggukjang: Korean fermented soybean rapid paste; meju: Korean fermented soybean block (Korean style soybean koji). b Bacillus cereus enterotoxin reversed passive latex agglutination (BCET-RPLA) was used to detect hemolysin BL enterotoxin. c Bacillus diarrheal enterotoxin visual immunoassay (BDE-VIA) kit was used to detect nonhemolytic enterotoxin of B. cereus.
and others 2009). The B. cereus isolates were grown in Mueller– Hilton broth (Oxoid) at 35 °C overnight. Cultures were spread on Mueller–Hilton agar and incubated at 35 °C overnight with antibiotic-containing discs placed on the agar surface. The discs contained ampicillin (10 μg), penicillin (10 U), gentamicin (10 μg), tetracycline (30 μg), erythromycin (15 μg), chloramphenicol (30 μg), cefepime (30 μg), cefotetan (30 μg), ciprofloxacin (5 μg), imipenem (10 μg), trimethoprim (1.25 μg), sulfamethoxazole (23.75 μg), oxacillin (1 μg), rifampin (5 μg), clindamycin (2 μg), and vancomycin (30 μg). The criteria to assess B. cereus antibiotic sensitivity have not been issued by the Clinical and Laboratory Standards Inst. (CLSI); therefore, the results of the B. cereus antibiotic test in this study were interpreted according to the standard developed for Staphylococcus aureus (CLSI 2006).
Data analysis For statistical analysis, the SPSS Statistics (Version 18.0, SPSS Inc., Chicago, Ill., U.S.A.) program was used. The number of detection samples of B. cereus on soybean product sources and types were compared using Fisher’s exact test. The P value of was less than 0.05 was considered as significant.
Results and Discussion
Prevalence of B. cereus in Korean fermented soybean products B. cereus was detected in 110 of 162 samples (67.9%); the highest Antibiotic susceptibility testing frequency was observed in deonjang (68 of 93 samples, 73.1%) and To analyze antibiotic susceptibility patterns of the B. cereus cheonggukjang (18 of 25 samples, 72%), which was much higher isolates, we used the Kirby–Bauer disk diffusion method (Park than that in kochujang (56.3%) and meju (50%; Table 1). There were
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B. cereus in Korean soybean products . . . discrepancy in the results may be explained by the use of highly selective chromogenic medium such as BACARA for the detection of B. cereus in this study, whereas a conventional selective media such as mannitol–yolk–polymyxin B agar (MYPA) or polymyxin B–pyruvate–egg yolk–mannitol–bromothymol blue agar (PEMBA) were used in previous studies. Because Korean fermented products commonly have high levels of background microflora, the selective isolation of B. cereus on these conventional media is difficult (Chon and others 2012a), However, the
Figure 1–Repetitive sequence-PCR fingerprinting result (dendrogram and gel-like image) for Bacillus cereus strains isolated from Korean fermented soybean product. The B. cereus ATCC 11778 strain is included for the reference.
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no statistical differences in the detection rate (P > 0.05) among soybean products sources (factory and home products) and types (deonjang, kochujang, cheonggukjang, and meju). Kim and others (2006) reported that B. cereus was found in 48.1% of Sunsik ingredients. The incidence of B. cereus in raw basmati rice and ready-toeat food showed 70% to 100% in Belgium (Samapundo and others 2011). The high incidence rates of B. cereus are in contrast to previous findings that B. cereus contamination of kochujang and deonjang was 24.4% and 60.0%, respectively (Lee and others 2009). The
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B. cereus in Korean soybean products . . . competing flora is usually eliminated on chromogenic media making differentiation of B. cereus much easier (Fricker and others 2008; Chon and others 2012b). Our finding that 67.9% of Korean fermented soybean products are contaminated with B. cereus suggests that the consumption of these products could cause foodborne illness.
Detection of B. cereus toxin in Korean fermented soybean products Foodborne illness caused by B. cereus is related to the production of HBL, NHE, CytK, EntFM, and the emetic toxin cereulide (Bottone 2010). Among these toxins, the primary virulence factors are HBL and NHE (Ngamwongsatit and others 2008). To estimate the potential risk of B. cereus foodborne illness, the presence of these enterotoxins in Korean fermented soybean products was investigated using the BCET-RPLA and BDE-VIA kits (Table 2). HBL enterotoxin was not detected in any of the
fermented products, but NHE was found in 22 of the analyzed 162 samples (13.6%); the highest NHE detection rate was observed in deonjang (19 of 93 samples, 20.4%). The HBL enterotoxin complex, which causes a major diarrheal foodborne illness, consists of binding protein B and 2 lytic components (L1 and L2) encoded by the hblA, hblC, and hblD genes (Schoeni and Wong 1999). The NHE complex consists of NheA, NheB, and NheC subunits, encoded by genes nheA, nheB, and nheC, respectively (Granum and others 1999). Most B. cereus strains isolated from different sources contain NHE-encoding genes (Guinebretiere and others 2002). NHE was shown to be the dominant enterotoxin produced in 92% to 100% of the B. cereus isolates (Stenfos-Arnesen and others 2008). A much lower detection rate of NHE (13.6%) compared to that of B. cereus prevalence (67.9%) may be attributed to relatively low number of B. cereus counts: only 10 out of the 162 samples (6.2%) had 105 bacterial cells/g (data not shown). B. cereus usually secretes toxins from the exponential to stationary
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Figure 2–Antibiotic susceptibility of Bacillus cereus isolates from factory-made (A) and home-made (B) Korean fermented soybean products. AM, ampicillin (10 µg); FEP, cefepime (30 µg); CTT, cefotetan (30 µg); CIP, ciprofloxacin (5 µg); C, chloramphenicol (30 µg); DA, clindamycin (2 µg); E, erythromycin (15 µg); CN, gentamicin (10 µg); IPM, imipenem (10 µg); P, penicillin (10 U); RD, rifampin (5 µg); TE, tetracycline (30 µg); SXT, trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg); OX, oxacillin (1 µg); VA, vancomycin (30 µg).
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B. cereus in Korean soybean products . . . (Chen and others 2004) has been observed in previous studies (Park and others 2009). The antibiotic resistance patterns of B. cereus isolates from Korean fermented soybean products are therefore consistent with earlier findings.
Conclusions
Our results suggest that the potential risk of B. cereus foodborne illness related to the consumption of Korean fermented soybean products is lower than the generally considered rate. However, considering the prevalence of B. cereus and fermented soybean product-based diet in Korea, it is necessary to constantly monitor Repetitive sequence-PCR fingerprinting B. cereus contamination and the levels of related toxins in these In total, 24 of B. cereus isolates, including one reference strain Korean fermented products. (B. cereus ATCC 11778), were characterized by rep-PCR to assess the similarity of B. cereus isolates. The 23 B. cereus isolates References randomly chosen with sample sources and types were obtained Agata N, Mori M, Ohta M, Suwan S, Ohtani I, Isobe M. 1994. A novel duodeca-depsipeptide, emetic toxin, isolated from Bacillus cereus cause vacuole formation in Hep-2 cells. FEMS from 13 deonjang (factory: F10013, F10014, F10016, F10028, Microbiol Lett 121:31–4. and F10029; home: F10051, F10052, F10053, F10054, F11068, Bottone EJ. 2010. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23:382–98. Y, Tenover FC, Koehler TM. 2004. β-Lactamase gene expression in a penicillin-resistant F11069, F11121, and F11122), 3 kochujang (factory: F10011; Chen Bacillus anthracis strain. Antimicrob Agents Chemother 48:4873–7. home: F10039 and F11110), 3 cheonggukjang (factory: F10008 and Cherif A, Brusetti L, Borin S, Rizzi A, Boundabous A, Khyami-Horani H, Daffonchio D. 2003. Genetic relationship in the ‘Bacillus cereus group’ by rep-PCR fingerprinting and sequencing F10015; home: F11087), and 4 meju (factory: F10002 and F10003; of Bacillus anthracis-specific rep-PCR fragment. J Appl Microbiol 94:1108–9. home: F11126 and 11128) samples. Several molecular fingerprint- Chon JW, Hyeon JY, Park JH, Song KY, Kim JH, Seo KH. 2012a. Improvement of mannitol– yolk–polymyxin B agar by supplementing with trimethoprim for quantitative detection of ing methods can be used for distinguishing foodborne pathogens. Bacillus cereus in foods. J Food Protect 75:1342–5. Although pulsed-field gel electrophoresis (PFGE) is recognized as Chon JW, Kim JH, Lee SJ, Hyeon JY, Seo KH. 2012b. Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik. Food Microbiol a gold standard method for molecular typing (Hyeon and others 32:217–22. 2011), rep-PCR fingerprinting is a useful tool to analyze the ge- Clinical and Laboratory Standards Inst. 2006. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline. Wayne, netic diversity of B. cereus strains (Cherif and others 2003) because Pennsylvania: Clinical and Laboratory Standards Inst. p. 16. it provides high reproducibility of band patterns (Healy and others ESFA (European Food Safety Authority). 2005. Bacillus cereus and other Bacillus spp. in foodstuffs. EFSA J 175:1–48. 2008). The dendrogram and computer-generated virtual gel Fricker M, Reissbrodt R, Ehling-Schulz M. 2008. Evaluation of standard and new chromogenic images are presented in Figure 1. Four cases of rep-PCR banding selective plating media for isolation and identification of Bacillus cereus. Intl J Food Microbiol 121:27–34. patterns were related with similarities level of 99%. First case PE, O’Sullivan K, Lund T. 1999. The sequence of the non-hemolytic enterotoxin was between F10053 (home-made deonjang isolate) and F10016 Granum operon from Bacillus cereus. FEMS Microbiol Lett 177:225–29. (factory-made deonjang isolate). Second case was between F11122 Guinebretiere M-H, Broussolle V, Nguyen-The C. 2002. Enterotoxigenic profiles of foodpoisoning and food-borne Bacillus cereus strains. J Clin Microbiol 40:3053–6. and F11121, which were isolated from home-made deonjang Healy B, Mullane N, Collin V, Mailler S, Iversen C, Chatellier S, Storrs M, Fanning S. 2008. but the sampling sites were different. Third case was between Evaluation of an automated repetitive sequence-base PCR system for subtyping Enterobacter sakazakii. J Food Protect 71:1372–8. F10054 (home-made deonjang isolate) and F10003 (factory-made Hyeon JY, Chon JW, Hwang IG, Kwak HS, Kim MS, Kim SK, Choi IS, Song CS, Park C, meju isolate). Fourth case was between F11110 (home-made Seo KH. 2011. Prevalence, antibiotic resistance, and molecular characterization of Salmonella serovars in retail meat products. J Food Protect 74:161–6. kochujang isolate) and F10029 (factory-made deonjang isolate). Jo YJ, Cho IH, Song CK, Shin HW, Kim YS. 2011. Comparison of fermented soybean paste However, B. cereus isolates tested in this study showed diverse (Deonjang) prepared by different methods based on profiling of volatile compounds. J Food Sci 76:C368–79. pulsotypes, indicating high genetic diversity according to the Jung OK, Park SY, Park KY. 2006. Longer aging time increased anticancer and antimetastatic rep-PCR banding patterns. These results are in accordance with properties of deonjang. Nutrition 22:539–45. a previous study that classified cereal-isolated B. cereus strains into Kang SE, Rhee JH, Park C, Sung MH, Lee IH. 2005. Distribution of poly-γ -glutamate (γ PGA) producer in Korean fermented foods, cheonggukjang, deonjang, and kochujang. Food Sci various pulsotypes based on the PFGE banding patterns (Park Technol 14:704–8. and others 2009); environmentally isolated B. cereus strains also Kim DH, Choi HJ. 2003. Physicochemical properties of kochujang prepared by Bacillus sp. Koji. J Food Sci Biotechnol 35:1174–81. demonstrated a high degree of genetic diversity (Cherif and others KimKorean SH, Kim JS, Choi JP, Park JH. 2006. Prevalence and frequency of foodborne pathogens on unprocessed agricultural and marine products. Korean J Food Sci Biotechnol 38:594–8. 2003). The rep-PCR fingerprinting patterns of B. cereus isolated YS, Ahn YS, Jeong DY, Shin DH. 2008. Isolation and identification of Bacillus cereus from from Korean fermented soybean products were not associated Kimfermented red pepper-soybean paste (Kochujang), and its heat resistance characteristics. Food with specific product types (deonjang, kochujang, cheonggukjang, or Sci Biotechnol 17:123–9. Kwon DY, Daily JW 3rd, Kim HJ, Park S. 2010. Antidiabetic effects of fermented soybean meju). products on type 2 diabetes. Nutr Res 30:1–13.
Antibiotic susceptibility of B. cereus isolates B. cereus has been recognized as an opportunistic foodborne pathogen (Oh and others 2012) that may cause severe foodborne illness resulting in sudden death (Naranjo and others 2011), underlying the necessity to investigate B. cereus susceptibility to antibiotics. The antibiotic sensitivity spectra of B. cereus isolates are presented in Figure 2(A) and (B). All tested isolates were susceptible to gentamicin, imipenem, ciprofloxacin, tetracycline, and vancomycin but not to β-lactam antibiotics such as ampicillin, cefepime, oxacillin, and penicillin. The resistance of B. cereus to the β-lactam antibiotics provided by the synthesis of β-lactamase
Lee JY, Kwon GH, Park JY, Park CS, Kwon DY, Lim JK, Kim JS, Kim JH. 2011. A RAPD-PCR method for rapid detection of Bacillus cereus. J Microbiol Biotechnol 21:274–6. Lee HT, Kim JH, Lee S. S. 2009. Analysis of microbiological contamination and biogenic amines content in traditional and commercial deonjang. J Food hyg Saf 24:102–9. Lee NK, Park JW, Cho IJ, Kim BY, Kwon KO, Hahm YT. 2008. Isolation of Bacillus spp. from Cheonggukjang and its antagonistic effect against Bacillus cereus. Korean J Food Sci Biotechnol 40:669–73. Lim SI, Lee BY. 2010. Effect of the licorice (Glycyrrhiza glabra) ethanol extract on growth of Bacillus cereus at the meju producing stage. J Korean Soc Appl Biol Chem 53:184–91. Lim SJ, Kim MR, Kim W, Hong KW. 2011. Detection and differentiation of non-emetic and emetic Bacillus cereus strains in food by real-time PCR. J Korean Soc Appl Biol Chem 54:105–111. Lund T, Debuyser ML, Granum PE. 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molec Microbiol 38:254–61. Naranjo M, Denayer S, Botteldoorn N, Delbrassinne L, Veys J, Waegenaere J, Sirtaine N, Driesen RB, Sipido KR, Mahillon J, Dierick K. 2011. Sudden death of a young adult associated with Bacillus cereus food poisoning. J Clin Microbiol 49:4379–81.
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growth phase, and toxin levels sufficient to produce foodborne illness are associated with a B. cereus contamination level of 105 to 108 cells/g (EFSA, 2005). These results suggest that the potential risk of B. cereus foodborne illness related to the consumption of Korean fermented soybean products is lower than presumed thus far. However, given that Korean fermented soybean products are commonly used condiments without additional heat treatment, they could bear risk of foodborne B. cereus illness. Thus, it is necessary that in these Korean fermented products, B. cereus contamination and the level of its toxins should be regularly monitored.
B. cereus in Korean soybean products . . . Ngamwongsatit P, Buasri W, Pianariyanon P, Pulsrikan C, Ohba M, Assavanig A, Panbabgred W. 2008. Broad distribution of enterotoxin genes (hblCDA, nhe ABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers. Intl J Food Microbiol 121:352–6. Oh MH, Ham JS, Cox JM. 2012. Diversity and toxigenicity among members of Bacillus cereus group. Intl J Food Microbiol 152:1–8. Park KY, Jung KO, Rhee SH, Choi YH. 2003. Antimutagenic effects of deonjang (Korean fermented soypaste) and its activity compounds. Mutat Res 523:43–53. Park YB, Kim JB, Shin SW, Kim JC, Cho SH, Lee BK, Ahn JH, Oh DH. 2009. Prevalence, genetic diversity, and antibiotic susceptibility of Bacillus cereus strains isolated from rice and cereals collected in Korea. J Food Prot 72:612–7.
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Samapundo S, Heyndrickx M, Xhaferi R, Devlieghere. 2011. Incidence, diversity and toxin gene characteristics of bacillus cereus group strains isolated from food products marketed in Belgium. Intl J Food Microbiol 150:34–41. Schoeni JL, Wong ACL. 1999. Heterogeneity observed in the components of haemolysin BL, an enterotoxin produced by Bacillus cereus. Intl J Food Microbiol 53: 159–67. Schoeni JL, Wong ACL. 2005. Bacillus cereus food poisoning and its toxins. J Food Prot 68:636–48. Stenfos-Arnesen LP, Fagerlund A, Granum PE, 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32:579–606.
Bacillus cereus contamination is a major food safety problem for Korean fermented soybean products, but few studies have assessed its potential to cause foodborne illness. The objectives of this study were to investigate the prevalence and characteristics of B. cereus isolated from Korean fermented soybean products. B. cereus was detected in 110 of 162 (67.9%) samples. The highest B. cereus frequency was observed in deonjang (68 of 93 samples, 73.1%) and cheonggukjang (18 of 25, 72.0%); however, nonhemolytic enterotoxin was detected only in 22 of 162 samples (13.6%). Although the tested B. cereus isolates showed diverse pulsotypes according to repetitive sequence-PCR banding patterns, they displayed similar antibiotic sensitivity spectra. The low frequency of enterotoxin detection suggests that the potential risk of B. cereus foodborne illness associated with Korean fermented soybean products is lower than generally presumed. However, considering the prevalence of B. cereus and the high content of fermented soybean products in the Korean diet, it is necessary to constantly monitor the level of contamination with B. cereus and its toxins in such Korean food products.
Abstract:
Introduction Bacillus cereus is a causative agent of diarrheal and emetic food poisoning associated with various categories of food products (EFSA, European Food Safety Authority, 2005). The foodborne diarrhea is caused by heat-labile enterotoxins such as hemolysin BL (HBL), nonhemolytic enterotoxin (NHE), enterotoxin FM (EntFM), and single-protein enterotoxin cytotoxin K (CytK; Schoeni and Wong 1999). The emetic foodborne illness is induced by a small heat- and acid-stable cyclic dodecadepsipeptide toxin cereulide (Agata and others 1994). A severe B. cereus–related foodborne illness, necrotic enteritis, has caused 4 deaths, including the sudden death of a young adult (Lund and others 2000; Naranjo and others 2011). B. cereus has been recognized as an opportunistic foodborne pathogen and is of growing concern to the food industry (Oh and others 2012). Korean fermented soybean products such as deonjang, kochujang, cheonggukjang, and meju can be produced commercially and at home. The factory-made products are obtained by fermentation with Aspergillus species and Bacillus subtilis, while home-made product is prepared by natural fermentation (Jo and others 2011). Meju is soybean koji (Korean soybean block) produced by fermentation with B. subtilis and various molds in industrial settings; however, a home-made product is usually prepared using rice straw, which naturally contains a variety of microorganisms and is commonly contaminated with B. cereus (Lim and Lee 2010). Health benefits of these fermented products are based on unique physicochemical characteristics (Kim and Choi 2003) that
underlie a variety of functional effects (Kang and others 2005), including strong anticancer and antimutagenic activities (Park and others 2003; Jung and others 2006). These fermented products are therefore being recognized as healthy foods. The availability and popularity of Korean fermented soybean products has been constantly increasing worldwide following the registration of deonjang in the Codex Alimentarius as an international food in 2009 (Kwon and others 2010). However, Korean fermented soybean products, especially those made at home, are based on meju and B. cereus contamination is a major problem for the safety of Korean fermented soybean products (Lee and others 2011). In 2006, the Korean government issued regulations regarding B. cereus content (no more than 4 log CFU/g) in deonjang, kochujang, and cheonggukjang (Lee and others 2008). However, few studies have been performed on the pathogenic potential of B. cereus in these food products (Kim and others 2008; Lim and others 2011), underscoring the necessity of comprehensive evaluation of the safety of Korean fermented soybean products and characteristics of B. cereus isolates. The objectives of this study were to determine the incidence of B. cereus and the levels of its enterotoxins in deonjang, kochujang, cheonggukjang, and meju.
Materials and Methods
Samples A total of 162 products were tested, comprising 93 deonjang, 32 kochujang, 25 cheonggukjang, and 12 meju samples. All samples were divided into factory- and home-made products. The 56 factory samples were purchased from local markets, and the 106 home-made samples were obtained nationwide from 2010 to MS 20140401 Submitted 3/11/2014, Accepted 10/15/2014. Authors C.-W. Kim and No are with Dept. of Food Science & Biotechnology, Dongguk Univ., 30, 2012. Pildong-ro 1-gil, Joong-gu, Seoul, Republic of Korea. Author Cho are with Div. of Enteric Bacterial Infections, Center for Infectious Diseases, Korea Natl. Inst. of Health, Isolation and identification of B. cereus Osong, Republic of Korea. Authors Kang, Park, Yoon, Lee and J.-B. Kim are with Each sample (25 g) was mixed with 225 mL buffered peptone Div. of Health Research, Gyeonggi-do Research Inst. of Health & Environment, 95, Pajangchen-ro, Suwon, Gyeonggi-do, Republic of Korea. Direct inquiries to author solution (Oxoid Ltd., Basingstoke, U.K.) and homogenized by a Kim (E-mail: [email protected]). stomacher (BagMixer 400; Interscience, Paris, France) for 5 min.
The homogenized mixture (0.1 mL) was spread onto B. cereus rapid R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12720 Further reproduction without permission is prohibited
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Keywords: antibiotic susceptibility, Bacillus cereus, Korean fermented soybean product, genetic diversity
B. cereus in Korean soybean products . . . agar (BACARA; AES Chemunex, Paris, France) and incubated at 30 °C for 20 h. The colonies that exhibited pink-orange color with lecithinase reaction on BACARA were selected for further culturing on tryptone soya agar (TSA; Oxoid) and 5% sheep blood agar (BA; Komed, Seoul, Korea) at 35 °C for 20 h; the colonies with hemolytic activity on BA were analyzed by Gram staining. The biochemical identification of the isolates was conducted by using API 50 CHB and API 20E test kits (bioM´erieux, Inc., Marcy l’Etoile, France).
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Detection of enterotoxins in Korean fermented soybean products The B. cereus enterotoxin-reversed passive latex agglutination (BCET-RPLA) kit (Oxoid) was used to detect the HBL enterotoxin according to the manufacturer’s instructions. Briefly, 50 g of each sample was placed into a plastic bag containing a lateral filter (BagMixer 400) and homogenized with 50 mL of 0.85% NaCl for 10 min; the filtered mixture was centrifuged for 30 min at 900 × g (Mega 17R, Hanil Science Industrial, Incheon, Korea). The supernatant was filtered using a 0.45-μm membrane filter and used as a sample solution for the HBL enterotoxin test. NHE was detected using the Bacillus diarrheal enterotoxin visual immunoassay (BDE-VIA) kit (Tecra Diagnostics, Reading, U.K.) according to the manufacturer’s instructions. Briefly, 10 g of each sample was placed into a plastic bag containing a lateral filter and blended with 50 mL of 0.25 M Tris-HCl (pH 8.0) for 10 min. The mixture was then centrifuged for 10 min at 1000 × g. The supernatant was filtered through cotton wool inserted in a disposable 10-mL plastic syringe. After pH adjustment to 7 to 8, 2 mL of the supernatant was mixed with 20 μL of sample additive contained in kit and used as a sample solution for the NHE test. Repetitive sequence-PCR DNA fingerprinting The 23 B. cereus isolates tested in this study were randomly chosen with sample sources and types, and composed of 13 deonjang (5 factory and 8 home-made products), 3 kochujang (1 factory and 2 home-made products), 3 cheonggukjang (2 factory and 1 home-made products), and 4 meju (2 factory and 2 home-made products). A total of 23 isolates and B. cereus reference strain (ATCC 11778) were grown at 35 °C for 24 h on TSA. DNA extraction and repetitive sequence-PCR (rep-PCR) were performed as previously described (Chon and others 2012b). Briefly, DNA was extracted using the UltraCleanTM Microbial DNA isolation kit (MoBio Laboratories, Solana Beach, Calif., U.S.A.) and amplified for DNA fingerprinting using the DiversiLab Bacillus kit (bioM´erieux). PCR reactions were carried out R polymerase, in a 25μL volume containing 0.5 μL of AmpliTaq 2.5 μL of 10× GeneAMP PCR buffer І, 2 μL primer mix, 18 μL rep-PCR master mix (all from Applied Biosystems, Foster City, Calif., U.S.A.), and 2 μL of 25 ng/μL DNA template. The thermal cycling conditions were as follows: initial cycle at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 70 °C for 90 s, and a final cycle at 70 °C for 3 min. The rep-PCR products were separated and detected using a microfluidics chip and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif., U.S.A.). The web-based DiversiLabTM software (version 2.1.66; bioM´erieux) was used for the comparative analysis of B. cereus isolates.
Table 1–Detection of Bacillus cereus in Korean fermented soybean products. No. of samples containing B. cereus (%)b Sourcea Deonjang Kochujang Cheonggukjang Meju Total
Factory products 21/25a 6/9a 8/12a 4/10a
(84.0) (66.7) (66.7) (40.0) 39/56a (69.6)
Home products 47/68a 12/23a 10/13a 2/2a
(69.1) (52.2) (76.9) (100) 71/106a (67.0)
Total 68/93 (73.1) 18/32 (56.3) 18/25 (72.0) 6/12 (50.0) 110/162 (67.9)
a Deonjang: Korean fermented soybean paste; Kochujang: Korean fermented red pepper–soybean paste; Cheonggukjang: Korean fermented soybean rapid paste; Meju: Korean fermented soybean block (Korean style soybean koji). b Different letters within a row and line indicate significant differences (P ࣘ 0.05).
Table 2–Detection of enterotoxins in Korean fermented soybean products. No. of samples containing toxins (%) in Korean fermented soybean products Factory products Home products
Total
Sourcea
HBLb
NHEc
HBL
NHE
HBL
NHE
Deonjang Kochujang Cheonggukjang Meju Total
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
5 (20.0) 0 (0.0) 0 (0.0) 0 (0.0) 5 (8.9)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
14 (20.6) 2 (8.7) 1 (7.7) 0 (0.0) 17 (16.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
19 (20.4) 2 (6.3) 1 (4.0) 0 (0.0) 22 (13.6)
a Deonjang: Korean fermented soybean paste; kochujang: Korean fermented red pepper–soybean paste; cheonggukjang: Korean fermented soybean rapid paste; meju: Korean fermented soybean block (Korean style soybean koji). b Bacillus cereus enterotoxin reversed passive latex agglutination (BCET-RPLA) was used to detect hemolysin BL enterotoxin. c Bacillus diarrheal enterotoxin visual immunoassay (BDE-VIA) kit was used to detect nonhemolytic enterotoxin of B. cereus.
and others 2009). The B. cereus isolates were grown in Mueller– Hilton broth (Oxoid) at 35 °C overnight. Cultures were spread on Mueller–Hilton agar and incubated at 35 °C overnight with antibiotic-containing discs placed on the agar surface. The discs contained ampicillin (10 μg), penicillin (10 U), gentamicin (10 μg), tetracycline (30 μg), erythromycin (15 μg), chloramphenicol (30 μg), cefepime (30 μg), cefotetan (30 μg), ciprofloxacin (5 μg), imipenem (10 μg), trimethoprim (1.25 μg), sulfamethoxazole (23.75 μg), oxacillin (1 μg), rifampin (5 μg), clindamycin (2 μg), and vancomycin (30 μg). The criteria to assess B. cereus antibiotic sensitivity have not been issued by the Clinical and Laboratory Standards Inst. (CLSI); therefore, the results of the B. cereus antibiotic test in this study were interpreted according to the standard developed for Staphylococcus aureus (CLSI 2006).
Data analysis For statistical analysis, the SPSS Statistics (Version 18.0, SPSS Inc., Chicago, Ill., U.S.A.) program was used. The number of detection samples of B. cereus on soybean product sources and types were compared using Fisher’s exact test. The P value of was less than 0.05 was considered as significant.
Results and Discussion
Prevalence of B. cereus in Korean fermented soybean products B. cereus was detected in 110 of 162 samples (67.9%); the highest Antibiotic susceptibility testing frequency was observed in deonjang (68 of 93 samples, 73.1%) and To analyze antibiotic susceptibility patterns of the B. cereus cheonggukjang (18 of 25 samples, 72%), which was much higher isolates, we used the Kirby–Bauer disk diffusion method (Park than that in kochujang (56.3%) and meju (50%; Table 1). There were
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B. cereus in Korean soybean products . . . discrepancy in the results may be explained by the use of highly selective chromogenic medium such as BACARA for the detection of B. cereus in this study, whereas a conventional selective media such as mannitol–yolk–polymyxin B agar (MYPA) or polymyxin B–pyruvate–egg yolk–mannitol–bromothymol blue agar (PEMBA) were used in previous studies. Because Korean fermented products commonly have high levels of background microflora, the selective isolation of B. cereus on these conventional media is difficult (Chon and others 2012a), However, the
Figure 1–Repetitive sequence-PCR fingerprinting result (dendrogram and gel-like image) for Bacillus cereus strains isolated from Korean fermented soybean product. The B. cereus ATCC 11778 strain is included for the reference.
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no statistical differences in the detection rate (P > 0.05) among soybean products sources (factory and home products) and types (deonjang, kochujang, cheonggukjang, and meju). Kim and others (2006) reported that B. cereus was found in 48.1% of Sunsik ingredients. The incidence of B. cereus in raw basmati rice and ready-toeat food showed 70% to 100% in Belgium (Samapundo and others 2011). The high incidence rates of B. cereus are in contrast to previous findings that B. cereus contamination of kochujang and deonjang was 24.4% and 60.0%, respectively (Lee and others 2009). The
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B. cereus in Korean soybean products . . . competing flora is usually eliminated on chromogenic media making differentiation of B. cereus much easier (Fricker and others 2008; Chon and others 2012b). Our finding that 67.9% of Korean fermented soybean products are contaminated with B. cereus suggests that the consumption of these products could cause foodborne illness.
Detection of B. cereus toxin in Korean fermented soybean products Foodborne illness caused by B. cereus is related to the production of HBL, NHE, CytK, EntFM, and the emetic toxin cereulide (Bottone 2010). Among these toxins, the primary virulence factors are HBL and NHE (Ngamwongsatit and others 2008). To estimate the potential risk of B. cereus foodborne illness, the presence of these enterotoxins in Korean fermented soybean products was investigated using the BCET-RPLA and BDE-VIA kits (Table 2). HBL enterotoxin was not detected in any of the
fermented products, but NHE was found in 22 of the analyzed 162 samples (13.6%); the highest NHE detection rate was observed in deonjang (19 of 93 samples, 20.4%). The HBL enterotoxin complex, which causes a major diarrheal foodborne illness, consists of binding protein B and 2 lytic components (L1 and L2) encoded by the hblA, hblC, and hblD genes (Schoeni and Wong 1999). The NHE complex consists of NheA, NheB, and NheC subunits, encoded by genes nheA, nheB, and nheC, respectively (Granum and others 1999). Most B. cereus strains isolated from different sources contain NHE-encoding genes (Guinebretiere and others 2002). NHE was shown to be the dominant enterotoxin produced in 92% to 100% of the B. cereus isolates (Stenfos-Arnesen and others 2008). A much lower detection rate of NHE (13.6%) compared to that of B. cereus prevalence (67.9%) may be attributed to relatively low number of B. cereus counts: only 10 out of the 162 samples (6.2%) had 105 bacterial cells/g (data not shown). B. cereus usually secretes toxins from the exponential to stationary
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Figure 2–Antibiotic susceptibility of Bacillus cereus isolates from factory-made (A) and home-made (B) Korean fermented soybean products. AM, ampicillin (10 µg); FEP, cefepime (30 µg); CTT, cefotetan (30 µg); CIP, ciprofloxacin (5 µg); C, chloramphenicol (30 µg); DA, clindamycin (2 µg); E, erythromycin (15 µg); CN, gentamicin (10 µg); IPM, imipenem (10 µg); P, penicillin (10 U); RD, rifampin (5 µg); TE, tetracycline (30 µg); SXT, trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg); OX, oxacillin (1 µg); VA, vancomycin (30 µg).
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B. cereus in Korean soybean products . . . (Chen and others 2004) has been observed in previous studies (Park and others 2009). The antibiotic resistance patterns of B. cereus isolates from Korean fermented soybean products are therefore consistent with earlier findings.
Conclusions
Our results suggest that the potential risk of B. cereus foodborne illness related to the consumption of Korean fermented soybean products is lower than the generally considered rate. However, considering the prevalence of B. cereus and fermented soybean product-based diet in Korea, it is necessary to constantly monitor Repetitive sequence-PCR fingerprinting B. cereus contamination and the levels of related toxins in these In total, 24 of B. cereus isolates, including one reference strain Korean fermented products. (B. cereus ATCC 11778), were characterized by rep-PCR to assess the similarity of B. cereus isolates. The 23 B. cereus isolates References randomly chosen with sample sources and types were obtained Agata N, Mori M, Ohta M, Suwan S, Ohtani I, Isobe M. 1994. A novel duodeca-depsipeptide, emetic toxin, isolated from Bacillus cereus cause vacuole formation in Hep-2 cells. FEMS from 13 deonjang (factory: F10013, F10014, F10016, F10028, Microbiol Lett 121:31–4. and F10029; home: F10051, F10052, F10053, F10054, F11068, Bottone EJ. 2010. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23:382–98. Y, Tenover FC, Koehler TM. 2004. β-Lactamase gene expression in a penicillin-resistant F11069, F11121, and F11122), 3 kochujang (factory: F10011; Chen Bacillus anthracis strain. Antimicrob Agents Chemother 48:4873–7. home: F10039 and F11110), 3 cheonggukjang (factory: F10008 and Cherif A, Brusetti L, Borin S, Rizzi A, Boundabous A, Khyami-Horani H, Daffonchio D. 2003. Genetic relationship in the ‘Bacillus cereus group’ by rep-PCR fingerprinting and sequencing F10015; home: F11087), and 4 meju (factory: F10002 and F10003; of Bacillus anthracis-specific rep-PCR fragment. J Appl Microbiol 94:1108–9. home: F11126 and 11128) samples. Several molecular fingerprint- Chon JW, Hyeon JY, Park JH, Song KY, Kim JH, Seo KH. 2012a. Improvement of mannitol– yolk–polymyxin B agar by supplementing with trimethoprim for quantitative detection of ing methods can be used for distinguishing foodborne pathogens. Bacillus cereus in foods. J Food Protect 75:1342–5. Although pulsed-field gel electrophoresis (PFGE) is recognized as Chon JW, Kim JH, Lee SJ, Hyeon JY, Seo KH. 2012b. Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik. Food Microbiol a gold standard method for molecular typing (Hyeon and others 32:217–22. 2011), rep-PCR fingerprinting is a useful tool to analyze the ge- Clinical and Laboratory Standards Inst. 2006. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline. Wayne, netic diversity of B. cereus strains (Cherif and others 2003) because Pennsylvania: Clinical and Laboratory Standards Inst. p. 16. it provides high reproducibility of band patterns (Healy and others ESFA (European Food Safety Authority). 2005. Bacillus cereus and other Bacillus spp. in foodstuffs. EFSA J 175:1–48. 2008). The dendrogram and computer-generated virtual gel Fricker M, Reissbrodt R, Ehling-Schulz M. 2008. Evaluation of standard and new chromogenic images are presented in Figure 1. Four cases of rep-PCR banding selective plating media for isolation and identification of Bacillus cereus. Intl J Food Microbiol 121:27–34. patterns were related with similarities level of 99%. First case PE, O’Sullivan K, Lund T. 1999. The sequence of the non-hemolytic enterotoxin was between F10053 (home-made deonjang isolate) and F10016 Granum operon from Bacillus cereus. FEMS Microbiol Lett 177:225–29. (factory-made deonjang isolate). Second case was between F11122 Guinebretiere M-H, Broussolle V, Nguyen-The C. 2002. Enterotoxigenic profiles of foodpoisoning and food-borne Bacillus cereus strains. J Clin Microbiol 40:3053–6. and F11121, which were isolated from home-made deonjang Healy B, Mullane N, Collin V, Mailler S, Iversen C, Chatellier S, Storrs M, Fanning S. 2008. but the sampling sites were different. Third case was between Evaluation of an automated repetitive sequence-base PCR system for subtyping Enterobacter sakazakii. J Food Protect 71:1372–8. F10054 (home-made deonjang isolate) and F10003 (factory-made Hyeon JY, Chon JW, Hwang IG, Kwak HS, Kim MS, Kim SK, Choi IS, Song CS, Park C, meju isolate). Fourth case was between F11110 (home-made Seo KH. 2011. Prevalence, antibiotic resistance, and molecular characterization of Salmonella serovars in retail meat products. J Food Protect 74:161–6. kochujang isolate) and F10029 (factory-made deonjang isolate). Jo YJ, Cho IH, Song CK, Shin HW, Kim YS. 2011. Comparison of fermented soybean paste However, B. cereus isolates tested in this study showed diverse (Deonjang) prepared by different methods based on profiling of volatile compounds. J Food Sci 76:C368–79. pulsotypes, indicating high genetic diversity according to the Jung OK, Park SY, Park KY. 2006. Longer aging time increased anticancer and antimetastatic rep-PCR banding patterns. These results are in accordance with properties of deonjang. Nutrition 22:539–45. a previous study that classified cereal-isolated B. cereus strains into Kang SE, Rhee JH, Park C, Sung MH, Lee IH. 2005. Distribution of poly-γ -glutamate (γ PGA) producer in Korean fermented foods, cheonggukjang, deonjang, and kochujang. Food Sci various pulsotypes based on the PFGE banding patterns (Park Technol 14:704–8. and others 2009); environmentally isolated B. cereus strains also Kim DH, Choi HJ. 2003. Physicochemical properties of kochujang prepared by Bacillus sp. Koji. J Food Sci Biotechnol 35:1174–81. demonstrated a high degree of genetic diversity (Cherif and others KimKorean SH, Kim JS, Choi JP, Park JH. 2006. Prevalence and frequency of foodborne pathogens on unprocessed agricultural and marine products. Korean J Food Sci Biotechnol 38:594–8. 2003). The rep-PCR fingerprinting patterns of B. cereus isolated YS, Ahn YS, Jeong DY, Shin DH. 2008. Isolation and identification of Bacillus cereus from from Korean fermented soybean products were not associated Kimfermented red pepper-soybean paste (Kochujang), and its heat resistance characteristics. Food with specific product types (deonjang, kochujang, cheonggukjang, or Sci Biotechnol 17:123–9. Kwon DY, Daily JW 3rd, Kim HJ, Park S. 2010. Antidiabetic effects of fermented soybean meju). products on type 2 diabetes. Nutr Res 30:1–13.
Antibiotic susceptibility of B. cereus isolates B. cereus has been recognized as an opportunistic foodborne pathogen (Oh and others 2012) that may cause severe foodborne illness resulting in sudden death (Naranjo and others 2011), underlying the necessity to investigate B. cereus susceptibility to antibiotics. The antibiotic sensitivity spectra of B. cereus isolates are presented in Figure 2(A) and (B). All tested isolates were susceptible to gentamicin, imipenem, ciprofloxacin, tetracycline, and vancomycin but not to β-lactam antibiotics such as ampicillin, cefepime, oxacillin, and penicillin. The resistance of B. cereus to the β-lactam antibiotics provided by the synthesis of β-lactamase
Lee JY, Kwon GH, Park JY, Park CS, Kwon DY, Lim JK, Kim JS, Kim JH. 2011. A RAPD-PCR method for rapid detection of Bacillus cereus. J Microbiol Biotechnol 21:274–6. Lee HT, Kim JH, Lee S. S. 2009. Analysis of microbiological contamination and biogenic amines content in traditional and commercial deonjang. J Food hyg Saf 24:102–9. Lee NK, Park JW, Cho IJ, Kim BY, Kwon KO, Hahm YT. 2008. Isolation of Bacillus spp. from Cheonggukjang and its antagonistic effect against Bacillus cereus. Korean J Food Sci Biotechnol 40:669–73. Lim SI, Lee BY. 2010. Effect of the licorice (Glycyrrhiza glabra) ethanol extract on growth of Bacillus cereus at the meju producing stage. J Korean Soc Appl Biol Chem 53:184–91. Lim SJ, Kim MR, Kim W, Hong KW. 2011. Detection and differentiation of non-emetic and emetic Bacillus cereus strains in food by real-time PCR. J Korean Soc Appl Biol Chem 54:105–111. Lund T, Debuyser ML, Granum PE. 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molec Microbiol 38:254–61. Naranjo M, Denayer S, Botteldoorn N, Delbrassinne L, Veys J, Waegenaere J, Sirtaine N, Driesen RB, Sipido KR, Mahillon J, Dierick K. 2011. Sudden death of a young adult associated with Bacillus cereus food poisoning. J Clin Microbiol 49:4379–81.
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growth phase, and toxin levels sufficient to produce foodborne illness are associated with a B. cereus contamination level of 105 to 108 cells/g (EFSA, 2005). These results suggest that the potential risk of B. cereus foodborne illness related to the consumption of Korean fermented soybean products is lower than presumed thus far. However, given that Korean fermented soybean products are commonly used condiments without additional heat treatment, they could bear risk of foodborne B. cereus illness. Thus, it is necessary that in these Korean fermented products, B. cereus contamination and the level of its toxins should be regularly monitored.
B. cereus in Korean soybean products . . . Ngamwongsatit P, Buasri W, Pianariyanon P, Pulsrikan C, Ohba M, Assavanig A, Panbabgred W. 2008. Broad distribution of enterotoxin genes (hblCDA, nhe ABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers. Intl J Food Microbiol 121:352–6. Oh MH, Ham JS, Cox JM. 2012. Diversity and toxigenicity among members of Bacillus cereus group. Intl J Food Microbiol 152:1–8. Park KY, Jung KO, Rhee SH, Choi YH. 2003. Antimutagenic effects of deonjang (Korean fermented soypaste) and its activity compounds. Mutat Res 523:43–53. Park YB, Kim JB, Shin SW, Kim JC, Cho SH, Lee BK, Ahn JH, Oh DH. 2009. Prevalence, genetic diversity, and antibiotic susceptibility of Bacillus cereus strains isolated from rice and cereals collected in Korea. J Food Prot 72:612–7.
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Samapundo S, Heyndrickx M, Xhaferi R, Devlieghere. 2011. Incidence, diversity and toxin gene characteristics of bacillus cereus group strains isolated from food products marketed in Belgium. Intl J Food Microbiol 150:34–41. Schoeni JL, Wong ACL. 1999. Heterogeneity observed in the components of haemolysin BL, an enterotoxin produced by Bacillus cereus. Intl J Food Microbiol 53: 159–67. Schoeni JL, Wong ACL. 2005. Bacillus cereus food poisoning and its toxins. J Food Prot 68:636–48. Stenfos-Arnesen LP, Fagerlund A, Granum PE, 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32:579–606.
