A study on toxin genes and cytotoxicity levels of Bacillus cereus in various ready-to-eat foods and pastry products in Turkey

Yıl 2022, Cilt: 6 Sayı: 3, 152 - 159, 31.12.2022

Başak Gökçe Çöl , Harun Aksu

https://doi.org/10.30704/http-www-jivs-net.1198813

Cited By: 2

Öz

Bacillus cereus is a spore-forming and toxin-producing gram-positive bacteria widely isolated from soils, meat, milk, and vegetables. It is recognized as one of the pathogenic bacteria that can lead to food poisoning and food spoilage in food service systems due to its ease of contamination of foods and lack of guarantee of elimination by pasteurization and sanitation practices. B. cereus causes two types of diseases mainly characterized by diarrhea and vomiting type syndrome with the toxins it produces. Toxins produced by B. cereus are mainly heat-stable emetic toxin and three different heat-labile enterotoxins. Foodborne illnesses of the diarrheal type are caused by the single protein toxin; cytotoxin K (CytK), and both tripartite toxins; hemolysin BL (Hbl), and the non-hemolytic enterotoxin (Nhe), whereas the emetic type, is caused by an emetic toxin cereulide. In this study, 225 ready-to-eat foods and pastry products were analyzed for B. cereus, its toxin profiles, and cytotoxicity effects. Multiplex PCR is used to identify the presence of the Hbl, CytK, and emetic toxin encoding genes. Component-specific antibody-based ELISA tests were utilized to determine the Hbl-L2 and NheB components. Cytotoxic activity of the B. cereus isolates on Vero cells was also identified. In total, B. cereus was detected in 37 out of 225 (16.4%) food samples. From the positive 37 B. cereus isolates, the ces gene was not identified, whereas 91.9% (34) Nhe, 56.8% (21) Hbl, and 8.1% (3) CytK encoding genes revealed positive results on PCR analysis. PCR results were also compatible with ELISA and Cytotoxicity tests. In a nutshell, 16.4% prevalence of B. cereus in foods is insufficient, and the presence or absence of toxin genes may not yield reliable results. It is critical to detect pathogenic B. cereus toxin gene profiles as well as toxin production ability at the same time. This study presents for the first time, data from a cell culture cytotoxicity test using specific monoclonal antibody-based sandwich ELISA and multiplex PCR for ready-to-eat foods and pastry products in Turkey

Anahtar Kelimeler

Bacillus cereus, enzyme immunoassay, non-hemolytic enterotoxin, ready-to-eat foods

Kaynakça

• Agata, N., Ohta, M., Mori, M., & Isobe, M. (1995). A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS microbiology letters, 129(1), 17-19.
• Aksu, H. ve Ergün, Ö. (1996). Çeşitli Pasta Ürünlerinde Ve Sütlü Tatlılarda Bacillus Cereus'un Varlığı. Gıda Sanayii, 46, 25-27.
• Ankolekar, C., Rahmati, T., & Labbé, R. G. (2009). Detection of toxigenic Bacillus cereus and Bacillus thuringiensis spores in US rice. International journal of food microbiology, 128(3), 460-466.
• Beecher, D. J., & Wong, A. C. (1994). Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar. Applied and environmental microbiology, 60(5), 1646-1651.
• Bonerba, E., Di Pinto, A., Novello, L., Montemurro, F., Terio, V., Colao, V., ... & Tantillo, G. (2010). Detection of potentially enterotoxigenic food‐related Bacillus cereus by PCR analysis. International journal of food science & technology, 45(6), 1310-1315.
• Çadırcı, Ö., Gücükoğlu, A., Terzi, G., Kevenk, T., & Alişarli, M. (2013). Determination of enterotoxigenic gene profiles of Bacillus cereus strains isolated from dairy desserts by multiplex PCR. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 19(5), 869-874.
• Clair, G., Roussi, S., Armengaud, J., & Duport, C. (2010). Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions. Molecular & Cellular Proteomics, 9(7), 1486-1498.
• Didier, A., & Dietrich, R. (2016). Mä rtlbauer E. Antibody binding studies reveal conformational flexibility of the Bacillus cereus nonhemolytic enterotoxin (Nhe) A-component. PLoS One, 11(10), e0165135.
• Dietrich, R., Fella, C., Strich, S., & Martlbauer, E. (1999). Production and characterization of monoclonal antibodies against the hemolysin BL enterotoxin complex produced by Bacillus cereus. Applied and Environmental Microbiology, 65(10), 4470-4474.
• Dietrich, R., Moravek, M., Bürk, C., Granum, P. E., & Märtlbauer, E. (2005). Production and characterization of antibodies against each of the three subunits of the Bacillus cereus nonhemolytic enterotoxin complex. Applied and environmental microbiology, 71(12), 8214-8220.
• Dietrich, R., Jessberger, N., Ehling-Schulz, M., Märtlbauer, E., & Granum, P. E. (2021). The food poisoning toxins of Bacillus cereus. Toxins, 13(2), 98. https://doi.org/10.3390/toxins13020098
• EFSA Panel on Biological Hazards (BIOHAZ). (2016). Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs. EFSA Journal, 14(7), e04524.
• Ehling-Schulz, M., Fricker, M., & Scherer, S. (2004). Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay. FEMS microbiology letters, 232(2), 189-195 Ehling-Schulz, M., Svensson, B., Guinebretiere, M.-H., Lindbäck, T.,Andersson, M., Schulz, A., et al. (2005a). Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains. Microbiology 151, 183–197.
• Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Ma¨rtlbauer, E., Scherer, S., (2005b). Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Applied and Environmental Microbiology 71 (1), 1105–1113
• Ehling-Schulz, M., Guinebretiere, M. H., Monthán, A., Berge, O., Fricker, M., & Svensson, B. (2006). Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS microbiology letters, 260(2), 232-240.
• Fagerlund, A., Ween, O., Lund, T., Hardy, S. P., & Granum, P. E. (2004). Genetic and functional analysis of the cytK family of genes in Bacillus cereus. Microbiology, 150(8), 2689-2697.
• Gaulin, C., Viger, Y. B., & Fillion, L. (2002). An outbreak of Bacillus cereus implicating a part-time banquet caterer. Canadian journal of public health, 93(5), 353-355.
• Granum, P. E., Brynestad, S., & Kramer, J. M. (1993). Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections. International journal of food microbiology, 17(4), 269-279.
• Granum, P. E., O'sullivan, K., & Lund, T. (1999). The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus. FEMS microbiology letters, 177(2), 225-229.
• Guinebretière, M. H., Broussolle, V., & Nguyen-The, C. (2002). Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. Journal of Clinical Microbiology, 40(8), 3053-3056.
• Hansen, B. M., & Hendriksen, N. B. (2001). Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Applied and environmental Microbiology, 67(1), 185-189.
• Hansen, B. M., Høiby, P. E., Jensen, G. B., & Hendriksen, N. B. (2003). The Bacillus cereus bceT enterotoxin sequence reappraised. FEMS Microbiology Letters, 223(1), 21-24.
• Hwang, J. Y., & Park, J. H. (2015). Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods. Journal of Dairy Science, 98(3), 1652-1660.
• In't Veld, P. H., W. S. Ritmeester, E. H. M. DelfgouvanAsch, J. B. Dufrenne, K. Wernars, E. Smit, and F. M. vanLeusden. (2001). Detection of genes encoding for enterotoxins and determination of the production of enterotoxins by HBL blood plates and immunoassays of psychrotrophic strains of Bacillus cereus isolated from pasteurised milk. International Journal of Food Microbiology. 64:63-70
• Jeßberger, N., Rademacher, C., Krey, V. M., Dietrich, R., Mohr, A. K., Böhm, M. E., ... & Märtlbauer, E. (2017). Simulating intestinal growth conditions enhances toxin production of enteropathogenic Bacillus cereus. Frontiers in Microbiology, 8, 627.
• Jung, S. M., Kim, N. O., Cha, I., Na, H. Y., Chung, G. T., Kawk, H. S., & Hong, S. (2017). Surveillance of Bacillus cereus Isolates in Korea from 2012 to 2014. Osong public health and research perspectives, 8(1), 71–77.
• Lindbäck, T., Hardy, S. P., Dietrich, R., Sødring, M., Didier, A., Moravek, M., ... & Märtlbauer, E. (2010). Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components. Infection and immunity, 78(9), 3813-3821.
• Logan, N. A., & Rodrigez-Diaz, M. (2006). Bacillus spp. and related genera. Principles and practice of clinical bacteriology, 2, 139-158.
• Lund, T., & Granum, P. E. (1996). Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS microbiology letters, 141(2-3), 151-156.
• Lund, T., De Buyser, M. L., & Granum, P. E. (2000). A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular microbiology, 38(2), 254-261.
• Marxen, S., Stark, T. D., Rütschle, A., Lücking, G., Frenzel, E., Scherer, S., ... & Hofmann, T. (2015). Multiparametric quantitation of the Bacillus cereus toxins cereulide and isocereulides A–G in foods. Journal of agricultural and food chemistry, 63(37), 8307-8313.
• Moravek, M., Wegscheider, M., Schulz, A., Dietrich, R., Bürk, C., & Märtlbauer, E. (2004). Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus. FEMS microbiology letters, 238(1), 107–113.
• Moravek, M., Dietrich, R., Buerk, C., Broussolle, V., Guinebretière, M. H., Granum, P. E., ... & Märtlbauer, E. (2006). Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses. FEMS microbiology letters, 257(2), 293-298.
• Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA (eds)(2007) Manual of clinical microbiology, 9th edn. American Society of Microbiology Press, Washington DC
• Ouoba, L. I. I., Thorsen, L., & Varnam, A. H. (2008). Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments. International journal of food microbiology, 124(3), 224-230.
• Schoeni, J. L., & LEE WONG, A. C. (2005). Bacillus cereus food poisoning and its toxins. Journal of food protection, 68(3), 636-648.
• Sastalla, I., Fattah, R., Coppage, N., Nandy, P., Crown, D., Pomerantsev, A. P., & Leppla, S. H. (2013). The Bacillus cereus Hbl and Nhe tripartite enterotoxin components assemble sequentially on the surface of target cells and are not interchangeable. PLoS One, 8(10), e76955.
• Schwenk, V., Riegg, J., Lacroix, M., Märtlbauer, E., & Jessberger, N. (2020). Enteropathogenic potential of Bacillus thuringiensis isolates from soil, animals, food and biopesticides. Foods, 9(10), 1484.
• Stenfors Arnesen, L. P., Fagerlund, A., & Granum, P. E. (2008). From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS microbiology reviews, 32(4), 579-606.
• Tewari, A., & Abdullah, S. (2015). Bacillus cereus food poisoning: international and Indian perspective. Journal of food science and technology, 52(5), 2500-2511.
• Wijnands, L. M., Dufrenne, J. B., & Rombouts, F. M. In’t Veld, PH, Van Leusden, FM (2006). Prevalence of potentially pathogenic Bacillus cereus in food commodities in the Netherlands. J Food Prot, 69, 2587-2594.
• Wijnands, L. M., Dufrenne, J. B., Van Leusden, F. M., & Abee, T. (2007). Germination of Bacillus cereus spores is induced by germinants from differentiated Caco-2 cells, a human cell line mimicking the epithelial cells of the small intestine. Applied and Environmental Microbiology, 73(15), 5052-5054.
• Valero, M., Hernandez-Herrero, L. A., & Giner, M. J. (2007). Survival, isolation and characterization of a psychrotrophic Bacillus cereus strain from a mayonnaise-based ready-to-eat vegetable salad. Food Microbiology, 24(7-8), 671-677.
• Van Netten, P., van De Moosdijk, A., Van Hoensel, P., Mossel, D. A. A., & Perales, I. (1990). Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology, 69(1), 73-79.
• Yang, I. C., Shih, D. Y. C., Huang, T. P., Huang, Y. P., Wang, J. Y., & Pan, T. M. (2005). Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group. Journal of food protection, 68(10), 2123-2130.
• Yibar, A., Cetinkaya, F., Soyutemiz, E., & Yaman, G. (2017). Prevalence, enterotoxin production and antibiotic resistance of Bacillus cereus isolated from milk and cheese. Kafkas Univ. Vet. Fak. Derg, 23, 635-642.