Assay of Bacillus cereus Emetic toxin produced in orange squash

Keywords: Bacillus cereus, emetic toxin, chicken liver mitochondria, orange squash, vacuolar assay, antibodies

Abstract

The contamination of squash by B. cereus, an enterotoxin producer, was found to range between 7.5×104  and 1.8×104 CFU/g in orange squash (during storage), that is hazardous. Orange squash is widely produced and consumed in India, but has a low rating of 3 on the scale of 10 (on feedback), mostly due to high sugars, not preferred these days. It can be preserved for >9 months due to added sugars and preservatives. During processing squash, if juice is not quickly cooled and/or squash is kept for long at temperatures <48 °C after processing, it can be a source of food poisoning. Reason, a large number of toxins can be produced by B. cereus. B. cereus strains, isolated from squash, produce heat stable toxin. Vacuolar assay confirmed them as emetic toxins, produced in squash. The toxin behaved like an ionophore in assay using mitochondria, extracted from liver cells of chicken with potassium ions in buffer. The toxicity of toxin by assay was 3200 IU/ng (BC IV strain) and 800 IU/ng (BC X strain). By the vacuolar expansions of mitochondria in assay, toxins of B. cereus demonstrated a toxic effect, in the range of 20.93 to 60.94 % by BC IV toxin and 43.28 to 45.02 % by BC X toxin, on the 3rd day growth of B. cereus in squash and toxin extraction for assay. It was also possible to produce antibodies against the B. cereus whole cell and toxin of BC IV, as an attempt to detect B. cereus contaminations in foods, by Ouchterlony’s immune-diffusion test

Downloads

Download data is not yet available.

Author Biographies

Sunita Singh, Indian Agricultural Research Institute

Division of Food Science and Post-harvest Technology

Prachi Lad, Indegene Private Limited

Department of Promotional & Medical Review Solutions (for AstraZeneca, USA.)

References

Newell, D. G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H. et. al. (2010). Food-borne diseases – The challenges of 20years ago still persist while new ones continue to emerge. International Journal of Food Microbiology, 139, S3–S15. doi: https://doi.org/10.1016/j.ijfoodmicro.2010.01.021

Van Netten, P., van de Moosdijk, A., van Hoensel, P., Mossel, D. A. A., Perales, I. (1990). Psychrotrophic strains of Bacillus cereusproducing enterotoxin. Journal of Applied Bacteriology, 69 (1), 73–79. doi: https://doi.org/10.1111/j.1365-2672.1990.tb02913.x

Gilbert, R. J., Parry, J. M. (1977). Serotypes of Bacillus cereus from outbreaks of food poisoning and from routine foods. Journal of Hygiene, 78 (1), 69–74. doi: https://doi.org/10.1017/s0022172400055947

Yokoyama, K., Ito, M., Agata, N., Isobe, M., Shibayama, K., Horii, T., Ohta, M. (1999). Pathological effect of synthetic cereulide, an emetic toxin ofBacillus cereus, is reversible in mice. FEMS Immunology & Medical Microbiology, 24 (1), 115–120. doi: https://doi.org/10.1111/j.1574-695x.1999.tb01272.x

Kramer, J. M., Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In: Food borne Bacterial Pathogens. New York, 21–70.

Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess, M., Andersson, M. et. al. (2002). Inhibition of human natural killer cell activity by cereulide, an emetic toxin from Bacillus cereus. Clinical & Experimental Immunology, 129 (3), 420–428. doi: https://doi.org/10.1046/j.1365-2249.2002.01898.x

Thwaite, J. E., Atkins, H. S. (2012). Bacillus. Medical Microbiology, 237–244. doi: https://doi.org/10.1016/b978-0-7020-4089-4.00036-6

Bryan, F. L., Bartleson, C. A., Christopherson, N. (1981). Hazard Analyses, in Reference to Bacillus cereus, of Boiled and Fried Rice in Cantonese-Style Restaurants. Journal of Food Protection, 44 (7), 500–512. doi: https://doi.org/10.4315/0362-028x-44.7.500

Hughes, S., Bartholomew, B., Hardy, J. C., Kramer, J. M. (1988). Potential application of a HEp-2 cell assay in the investigation of Bacillus cereusemetic-syndrome food poisoning. FEMS Microbiology Letters, 52 (1-2), 7–11. doi: https://doi.org/10.1111/j.1574-6968.1988.tb02563.x

Turnbull, P. C., Kramer, J. M., Jørgensen, K., Gilbert, R. J., Melling, J. (1979). Properties and production characteristics of vomiting, diarrheal, and necrotizing toxins of Bacillus cereus. The American Journal of Clinical Nutrition, 32 (1), 219–228. doi: https://doi.org/10.1093/ajcn/32.1.219

Ahmed, A. A.-H., Moustafa, M. K., Marth, E. H. (1983). Incidence of Bacillus cereus in Milk and Some Milk Products. Journal of Food Protection, 46 (2), 126–128. doi: https://doi.org/10.4315/0362-028x-46.2.126

Rodriquez, M. H., Barrett, E. L. (1986). Changes in Microbial Population and Growth of Bacillus cereus During Storage of Reconstituted Dry Milk. Journal of Food Protection, 49 (9), 680–686. doi: https://doi.org/10.4315/0362-028x-49.9.680

Sooltan, J. R. A., Mead, G. C., Norris, A. P. (1987). Incidence and growth potential of Bacillus cereus in poultrymeat products. Food Microbiology, 4 (4), 347–351. doi: https://doi.org/10.1016/s0740-0020(87)80009-6

Harmon, S. M., Kautter, D. A. (1991). Incidence and Growth Potential of Bacillus cereus in Ready-to-Serve Foods. Journal of Food Protection, 54 (5), 372–374. doi: https://doi.org/10.4315/0362-028x-54.5.372

Sunita, S. (2006). Final Report of the Project No. 502 “Establish/ modify assay for detection of Bacillus cereus and its toxin”. Institute code No: P1-2004/4-IAE-Q 02 (ICAR code No. CIAE/APD/APD/2004-4. Available at: https://www.researchgate.net/project/Establish-modify-assay-for-detection-of-Bacillus-cereus-and-its-toxin-ICAR-Code-No-PI-2004-4-IAE-Q-02-Institute-Code-No-CIAE-APD-2004-4-502-as-PI

Melling, J., Capel, B. J. (1978). Characteristics of Bacillus cereusemetic toxin. FEMS Microbiology Letters, 4 (3), 133–135. doi: https://doi.org/10.1111/j.1574-6968.1978.tb02849.x

Granum, P. E., Lund, T. (2006). Bacillus cereus and its food poisoning toxins. FEMS Microbiology Letters, 157 (2), 223–228. doi: https://doi.org/10.1111/j.1574-6968.1997.tb12776.x

Van Netten, P., Kramer, J. M. (1992). Media for the detection and enumeration of Bacillus cereus in foods: a review. International Journal of Food Microbiology, 17 (2), 85–99. doi: https://doi.org/10.1016/0168-1605(92)90108-f

Beattie, S. H., Williams, A. G. (1999). BACILLUS | Detection of Toxins. Encyclopedia of Food Microbiology, 141–149. doi: https://doi.org/10.1006/rwfm.1999.0125

Jääskeläinen, E. L., Teplova, V., Andersson, M. A., Andersson, L. C., Tammela, P., Andersson, M. C. et. al. (2003). In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus. Toxicology in Vitro, 17 (5-6), 737–744. doi: https://doi.org/10.1016/s0887-2333(03)00096-1

Agata, N., Ohta, M., Yokoyama, K. (2002). Production of Bacillus cereus emetic toxin (cereulide) in various foods. International Journal of Food Microbiology, 73 (1), 23–27. doi: https://doi.org/10.1016/s0168-1605(01)00692-4

Shinagawa, K., Otake, S., Matsusaka, N., Sugii, S. (1992). Production of the Vacuolation Factor of Bacillus cereus Isolated from Vomiting-Type Food Poisoning. The Journal of Veterinary Medical Science, 54 (3), 443–446. doi: https://doi.org/10.1292/jvms.54.443

Agata, N., Mori, M., Ohta, M., Suwan, S., Ohtani, I., Isobe, M. (1994). A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereuscauses vacuole formation in HEp-2 cells. FEMS Microbiology Letters, 121 (1), 31–34. doi: https://doi.org/10.1111/j.1574-6968.1994.tb07071.x

Suwan, S., Isobe, M., Ohtani, I., Agata, N., Mori, M., Ohta, M. (1995). Structure of cereulide, a cyclic dodecadepsipeptide toxin from Bacillus cereus and studies on NMR characteristics of its alkali metal complexes including a conformational structure of the K+ complex. Journal of the Chemical Society, Perkin Transactions 1, (7), 765. doi: https://doi.org/10.1039/p19950000765

Carlin, F., Fricker, M., Pielaat, A., Heisterkamp, S., Shaheen, R., Salkinojasalonen, M. et. al. (2006). Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group. International Journal of Food Microbiology, 109 (1-2), 132–138. doi: https://doi.org/10.1016/j.ijfoodmicro.2006.01.022

Mikkola, R., Saris, N.-E. L., Grigoriev, P. A., Andersson, M. A., Salkinoja-Salonen, M. S. (1999). Ionophoretic properties and mitochondrial effects of cereulide. The emetic toxin of B. cereus. European Journal of Biochemistry, 263 (1), 112–117. doi: https://doi.org/10.1046/j.1432-1327.1999.00476.x

Ranganna, S. (1986). Handbook of analysis and quality control for fruit and vegetable products. New Delhi: Tata McGraw-Hill. Available at: https://www.worldcat.org/title/handbook-of-analysis-and-quality-control-for-fruit-and-vegetable-products/oclc/692163756

Holbrook, R., Anderson, J. M. (1980). An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Canadian Journal of Microbiology, 26 (7), 753–759. doi: https://doi.org/10.1139/m80-131

Collins, C. H., Lyne, P. M. (1976). Microbiological Methods. Butterworths Pub., 521.

Lindbäck, T., Granum, P. E. (2013). Bacillus cereus. Guide to Foodborne Pathogens, 75–81. doi: https://doi.org/10.1002/9781118684856.ch4

Andrews, W. H. (1984). A Perspective Review of the Development of AOAC Microbiological Methods. Journal of AOAC INTERNATIONAL, 67 (4), 661–673. doi: https://doi.org/10.1093/jaoac/67.4.661

Grutsch, A. A., Nimmer, P. S., Pittsley, R. H., McKillip, J. L. (2018). Bacillus spp. as Pathogens in the Dairy Industry. Foodborne Diseases, 193–211. doi: https://doi.org/10.1016/b978-0-12-811444-5.00007-5

Harrigan, W. F., McCance, M. F. (1976). Laboratory Methods in Food and Dairy Microbiology. Academic Press, 452.

Varadaraj, M. C. (1993). Methods for detection and enumeration of food borne bacterial pathogens: A critical evaluation. Journal of Food Science and Technology, 30 (1), 1–13.

JNU (1984). Biochemistry Practicals - A Handbook. School of Life Sciences, JNU, New Delhi, 48–49.

Szabo, R. A., Speirs, J. I., Akhtar, M. (1991). Cell Culture Detection and Conditions for Production of a Bacillus cereus Heat-Stable Toxin. Journal of Food Protection, 54 (4), 272–276. doi: https://doi.org/10.4315/0362-028x-54.4.272

Finlay, W. J. J., Logan, N. A., Sutherland, A. D. (1999). Semiautomated Metabolic Staining Assay for Bacillus cereus Emetic Toxin. Applied and Environmental Microbiology, 65 (4), 1811–1812. doi: https://doi.org/10.1128/aem.65.4.1811-1812.1999

Pressman, B. C. (1965). Induced active transport of ions in mitochondria. Proceedings of the National Academy of Sciences, 53 (5), 1076–1083. doi: https://doi.org/10.1073/pnas.53.5.1076

Scheffler, I. E. (2001). A century of mitochondrial research: achievements and perspectives. Mitochondrion, 1 (1), 3–31. doi: https://doi.org/10.1016/s1567-7249(00)00002-7

Spector, I., Palfrey, C., Littauer, U. Z. (1975). Enhancement of the electrical excitability of neuroblastoma cells by valinomycin. Nature, 254 (5496), 121–124. doi: https://doi.org/10.1038/254121a0

Einar, G. P. (1997). Bacillus cereus. In: Fundamentals in Food Microbiology. American Society for Microbiology, Washington DC., 327–336.

El-Arabi, T. F., Griffiths, M. W. (2013). Bacillus cereus. Foodborne Infections and Intoxications, 401–407. doi: https://doi.org/10.1016/b978-0-12-416041-5.00029-9

Isobe, M., Ishikawa, T., Suwan, S., Agata, N., Ohta, M. (1995). Synthesis and activity of cereulide, a cyclic dodecadepsipeptide ionophore as emetic toxin from Bacillus cereus. Bioorganic & Medicinal Chemistry Letters, 5 (23), 2855–2858. doi: https://doi.org/10.1016/0960-894x(95)00503-l

Product Review- Kissan Orange Squash. Available at: https://foodnetindia.in/product-review-kissan-orange-squash/

Guinebretière, M.-H., Thompson, F. L., Sorokin, A., Normand, P., Dawyndt, P., Ehling-Schulz, M. et. al. (2008). Ecological diversification in the Bacillus cereus Group. Environmental Microbiology, 10 (4), 851–865. doi: https://doi.org/10.1111/j.1462-2920.2007.01495.x

Mitscherlich, E., Marth, E. H. (1984). Microbial Survival in the Environment. Bacteria and Rickettsiae Important in Human and Animal Health. Springer, 803. doi: https://doi.org/10.1007/978-3-642-69974-0

Häggblom, M. M., Apetroaie, C., Andersson, M. A., Salkinoja-Salonen, M. S. (2002). Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions. Applied and Environmental Microbiology, 68 (5), 2479–2483. doi: https://doi.org/10.1128/aem.68.5.2479-2483.2002

Te Giffel, M. C., Beumer, R. R., Leijendekkers, S., Rombouts, F. M. (1996). Incidence of Bacillus cereus and Bacillus subtilis in foods in the Netherlands. Food Microbiology, 13 (1), 53–58. doi: https://doi.org/10.1006/fmic.1996.0007

Goepfert, J. M., Spira, W. M., Kim, H. U. (1972). Bacillus cereus: food poisoning organism. A review. Journal of Milk and Food Technology, 35 (4), 213–227. doi: https://doi.org/10.4315/0022-2747-35.4.213

Johnson, K. M. (1984). An Update. Journal of Food Protection, 47 (2), 145–153. doi: https://doi.org/10.4315/0362-028x-47.2.145

Benedict, R. C., Partridge, T., Wells, D., Buchanan, R. L. (1993). Bacillus cereus: Aerobic Growth Kinetics. Journal of Food Protection, 56 (3), 211–214. doi: https://doi.org/10.4315/0362-028x-56.3.211

Kimanya, M. E., Mamiro, P. R. S., Van Camp, J., Devlieghere, F., Opsomer, A., Kolsteren, P., Debevere, J. (2003). Growth of Staphylococcus aureus and Bacillus cereus during germination and drying of finger millet and kidney beans. International Journal of Food Science and Technology, 38 (2), 119–125. doi: https://doi.org/10.1046/j.1365-2621.2003.00652.x

Kamat, A. S., Nerkar, D. P., Nair, P. M. (1989). Bacillus cereus in some indian foods, incidence and antibiotic, heat and radiation resistance. Journal of Food Safety, 10 (1), 31–41. doi: https://doi.org/10.1111/j.1745-4565.1989.tb00005.x

Kanda, K., Yasuda, Y., Tochikubo, K. (1991). Germination response of Bacillus subtilis PCI219 Spores to Caramelized Sugar and l-Asparagine. Journal of Food Science, 56 (5), 1399–1403. doi: https://doi.org/10.1111/j.1365-2621.1991.tb04783.x

Lattuada, C. P., McClain, D. (1998). Examination of meat and poultry products for Bacillus cereus. USDA/FSIS. Microbiology Laboratory Guidebook. Available at: http://docshare04.docshare.tips/files/10169/101693424.pdf

Eley, A. R. (1992). Toxic bacterial food poisoning. Microbial Food Poisoning, 37–55. doi: https://doi.org/10.1007/978-1-4899-3121-4_3

Byrne, B., Dunne, G., Bolton, D. J. (2006). Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll. Food Microbiology, 23 (8), 803–808. doi: https://doi.org/10.1016/j.fm.2006.02.002

Machaiah, M. I., Krishnan, M. H. (2014). Immunodetection of Bacillus cereus haemolytic enterotoxin (HBL) in food samples. Analytical Methods, 6 (6), 1841. doi: https://doi.org/10.1039/c3ay41737a

Notermans, S., Tatini, S. (1993). Characterization of Bacillus cereus in relation to toxin production. Nederlands melk en Zuiveltijdschrift, 47 (2), 71–77. Available at: http://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3916662

Andersson, M. A., Mikkola, R., Helin, J., Andersson, M. C., Salkinoja-Salonen, M. (1998). A Novel Sensitive Bioassay for Detection ofBacillus cereus Emetic Toxin and Related Depsipeptide Ionophores. Applied and Environmental Microbiology, 64 (4), 1338–1343. doi: https://doi.org/10.1128/aem.64.4.1338-1343.1998

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. doi: https://doi.org/10.1016/s0378-1097(04)00066-7

Marxen, S., Stark, T. D., Frenzel, E., Rütschle, A., Lücking, G., Pürstinger, G. et. al. (2015). Chemo-diversity of cereulide, the emetic toxin of Bacillus cereus. Analytical and Bioanalytical Chemistry, 407 (9), 2439–2453. doi: https://doi.org/10.1007/s00216-015-8511-y

Mahler, H., Pasi, A., Kramer, J. M., Schulte, P., Scoging, A. C., Bär, W., Krähenbühl, S. (1997). Fulminant Liver Failure in Association with the Emetic Toxin of Bacillus cereus. New England Journal of Medicine, 336 (16), 1142–1148. doi: https://doi.org/10.1056/nejm199704173361604

Sakurai, N., Koike, K. A., Irie, Y., Hayashi, H. (1994). The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell. Microbiology and Immunology, 38 (5), 337–343. doi: https://doi.org/10.1111/j.1348-0421.1994.tb01788.x

Kopel, A. C., Carvounis, P. E., Holz, E. R. (2008). Bacillus Cereus Endophthalmitis Following Intravitreous Bevacizumab Injection. Ophthalmic Surgery, Lasers, and Imaging, 39 (2), 153–154. doi: https://doi.org/10.3928/15428877-20080301-10

Moyer, A. L., Ramadan, R. T., Novosad, B. D., Astley, R., Callegan, M. C. (2009). Bacillus cereus–Induced Permeability of the Blood–Ocular Barrier during Experimental Endophthalmitis. Investigative Opthalmology & Visual Science, 50 (8), 3783. doi: https://doi.org/10.1167/iovs.08-3051


👁 43
⬇ 21
Published
2021-04-01
How to Cite
Singh, S., & Lad, P. (2021). Assay of Bacillus cereus Emetic toxin produced in orange squash. EUREKA: Life Sciences, (2), 41-55. https://doi.org/10.21303/2504-5695.2021.001753
Section
Food Science and Technology