Molecular Detection of Bacillus cereus in Milk by Polymerase Chain Reaction

Öz

It is a valuable and good food source with the ingredients it contains in milk, but if hygienic conditions are not ensured during milking, storage and processing, diseases related to milk and dairy products are likely to occur. More than 90% of these diseases are of bacterial origin. B. cereus, found in the Bacillaceae family, is a significant source of contamination in milk and dairy products due to the presence of psychrotrophic strains. In recent years, it has been reported that milk and milk products contaminated with B. cereus have adverse effects on human health, and toxic substances are formed during the storage of contaminated milk and milk products. This study, it is aimed to molecularly identify the B. cereus bacteria species that cause adverse effects such as food poisoning and loss of quality in milk. In this study, the DNA of Bacillus cereus, one of the microorganisms found in raw milk, was isolated and toxic gene regions were determined at the molecular level by PCR method using specific primers to the gene regions Bacillus cereus. Six primers specific for nheA, nheB, nheC, hblA, hblC, hblD and 16S rRNA gene regions were used for molecular detection of B. cereus in milk samples. At the end of the study, Bacillus cereus bacteria were detected in 8 of the 13 milk samples. It has been revealed that B. cereus can be detected quickly and reliably in milk and dairy products by molecular species identification using specific primers.

Anahtar Kelimeler

• Bacillus cereus
• non-hemolytic enterotoxin (Nhe)
• milk
• molecular species detection
• PCR

Kaynakça

1. 1. Burdova, O., et al., Hygiene of pasteurized milk depending on psychrotrophic microorganisms. Bulletin-Veterinary Institute In Pulawy, 2002. 46(2): p. 325-330.
2. 2. Berthold-Pluta, A., Pluta, A., and Garbowska, M., The effect of selected factors on the survival of Bacillus cereus in the human gastrointestinal tract. Microbial pathogenesis, 2015. 82: p. 7-14.
3. 3. Fagerlund, A., Lindbäck, T., and Granum, P.E., Bacillus cereus cytotoxins Hbl, Nhe and CytK are secreted via the Sec translocation pathway. BMC microbiology, 2010. 10(1): 1-8.
4. 4. Bonerba, E., et al., Detection of potentially enterotoxigenic food‐related Bacillus cereus by PCR analysis. International journal of food science & technology, 2010. 45(6): p. 1310-1315.
5. 5. Granum, P.E., Bacillus cereus and its toxins. Journal of Applied Bacteriology, 1994. 76: p. 61-66.
6. 6. Faille, C., et al., Occurrence of Bacillus cereus spores with a damaged exosporium: consequences on the spore adhesion on surfaces of food processing lines. Journal of food protection, 2007. 70(10): p. 2346-2353.
7. 7. Lindbäck, T., and Granum, P.E., Bacillus cereus. Food Microbiology: Fundamentals And Frontiers, 2019. p. 541-554.
8. 8. Garrido, A., et al., A new multiplex real-time PCR developed method for Salmonella spp. and Listeria monocytogenes detection in food and environmental samples. Food Control, 2013. 30(1): p. 76-85.

9. 9. Zhang, Z., et al., Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control. Food control, 2014. 35(1): p. 401-406.
10. 10. Wehrle, E., et al., Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus. Journal of microbiological methods, 2009. 78(3): p. 265-270.
11. 11. Nilsson, J., et al., A RAPD-PCR method for large-scale typing of Bacillus cereus. Letters in applied microbiology, 1998. 27(3): p. 168-172.
12. 12. Hansen, B.M., Leser, T.D., and Hendriksen, N.B., Polymerase chain reaction assay for the detection of Bacillus cereus group cells. FEMS Microbiology Letters, 2001. 202(2): p. 209-213.
13. 13. Park, S. H., et al., Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR. Journal of microbiology and biotechnology, 2007. 17(7): p. 1177-1182.
14. 14. Razei, A., et al., Presenting a rapid method for detection of Bacillus cereus, Listeria monocytogenes and Campylobacter jejuni in food samples. Iranian journal of basic medical sciences, 2017. 20(9): p. 1050.
15. 15. Banykó, J., and Vyletělová, M., Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt. Letters in applied microbiology, 2009. 48(3): p. 318-323.
16. 16. Lesley, M.B., et al., Detection of Bacillus cereus in formula milk and ultra high temperature (UHT) treated milk products. International Food Research Journal, 2017. 24(3).
17. 17. Meena, S.C., et al., Isolation and Identification of Bacillus cereus from Milk and Milk Products in Udaipur, Rajasthan, India. Int. J. Curr. Microbiol. App. Sci, 2019. 8(9): p. 2783-2787.
18. 18. Adame-Gómez, R., et al., Prevalence of the strains of Bacillus cereus group in artisanal Mexican cheese. Foodborne pathogens and disease, 2020. 17(1): p. 8-14.
19. 19. Fricker, M., et al., Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Applied and environmental microbiology, 2007. 73(6): p. 1892-1898.
20. 20. Moravek, M., et al., Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus. FEMS microbiology letters, 2004. 238(1): p. 107-113.
21. 21. Zhang, Z., et al., Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control. Food control, 2014. 35(1): p. 401-406.
22. 22. Dierick, K., et al., Fatal family outbreak of Bacillus cereus-associated food poisoning. Journal of clinical microbiology, 2005. 43(8): p. 4277-4279.
23. 23. Dieffenbach, C.W., Lowe, T.M.J., and Dveksler, G.S., General concepts for PCR primer design. General Concepts for PCR Primer Design Parameters Used in Basic PCR Primer Design. Cold Spring Harb Lab Press May. 2008.
24. 24. Sachse, K., Specificity and performance of PCR detection assays for microbial pathogens. Molecular biotechnology, 2004. 26(1): p. 61-79.
25. 25. Ogawa, H., et al., A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species. PLoS One, 2015. 10(3): p e0122004.
26. 26. Ehling-Schulz, M., et al. Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS microbiology letters, 2006, 260(2): p. 232-240.
27. 27. Beecher, D.J., and MacMillan, J.D. A novel bicomponent hemolysin from Bacillus cereus. Infection and immunity, 1990. 58(7): p. 2220-2227.
28. 28. Molayi Kohneshahri S, et al., Detection of hblA and bal Genes in Bacillus cereus Isolates From Cheese Samples Using the Polymerase Chain Reaction. Avicenna J Clin Microbiol Infect. 2016.
29. 29. Yang, I.C., et al., Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group. Journal of food protection, 2005. 68(10): p. 2123-2130.
30. 30. Ngamwongsatit, P., et al., Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers. International journal of food microbiology, 2008. 121(3): p. 352-356.
31. 31. Zhang, Z., et al., Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR. Journal of dairy science, 2016. 99(2): p. 1047-1055.