Malatya ilinde üretilen peynirlerden izole edilen Bacillus cereus suşlarında enterotoksin kodlayan genler ile antibiyotik direncinin araştırılması

Öz

Bu çalışmada, Malatya ilinde üretilen ve halk tarafından yaygın olarak tüketilen yöresel köy tipi peynir örneklerinde, i) Bacillus cereus’un varlığının belirlenmesi, ii) izolatlarda enterotoksin genlerinin polimeraz zincir reaksiyonu (PCR) ile saptanması ve iii) antibiyotik direncinin belirlenmesi amaçlandı. Analize alınan toplam 75 adet peynir örneğinin 55’inde (%73,3) B. cereus saptandı. Yapılan moleküler analizler sonucunda, toplam 55 adet izolat B. cereus yönünden hemolizin geni hedef alınarak doğrulandı. Bu izolatlardan 3’ünün sadece nhe genini, 1’inin ise hem nhe hem de cytK genlerini taşıdığı tespit edilirken, ancak izolatların hiçbirisinde hbl genine rastlanılmadı. Antibiyotik duyarlılık testi sonucunda, izolatların tamamı (%100) sefoksitine karşı dirençli olarak bulundu. Bunu sırasıyla, %94,5 ile rifampin, %87,2 ile penisilin, %58,1 ile eritromisin, %56,3 ile gentamisin ve %12,7 ile kloramfenikol izledi. İzolatların %94,5’inin çoklu antibiyotik direnci gösterdiği belirlendi. Gıda güvenliği ve halk sağlığı kapsamında değerlendirildiğinde, çalışmadan elde edilen bulgular, sağlıklı peynir üretimi için gerekli teknolojik ve hijyenik önlemlerin alınması yönünde hem süt endüstrisine hem de halka önemli bilgiler sunmaktadır. Ayrıca, izolatların yüksek düzeyde çoklu antibiyotik direnci göstermesi, bu konuda alınacak önlemlere bağlı olarak halk sağlığının korunmasına katkı sağlayacaktır.

Anahtar Kelimeler

• Bacillus cereus
• peynir
• antibiyotik
• enterotoksin
• gen

Investigation of enterotoxin-encoding genes and antibiotic resistance in Bacillus cereus strains isolated from cheese produced in Malatya province

Abstract

In this study, it was aimed, i) to determine the presence of Bacillus cereus in local village type cheese samples produced in Malatya and consumed widely by the people of Malatya, ii) detection of enterotoxin genes in the isolates by polymerase chain reaction (PCR), and iii) determination of antibiotic resistance. B. cereus was detected in 55 (73.3%) of 75 cheese samples in total. After molecular analysis, a total of 55 isolates were confirmed as B. cereus by targeting the hemolysin gene. It was determined that 3 of these isolates carried only the nhe gene, and 1 of them carried both nhe and cytK genes. However, no hbl gene was found in any of the isolates. As a result of the antibiotic susceptibility test, all of the isolates (100 %) were resistant to cefoxitin, followed by resistance to rifampin (94.5%), penicillin (87.2%), erythromycin (58.1%), gentamicin (56.3%), and chloramphenicol (12.7%). It was determined that 94.5% of the isolates showed multiple antibiotic resistance. Within the scope of food safety and public health, these findings can give important information to the both dairy industry and public in order to take the necessary technological and hygienic measures for healthy cheese production. In addition, determination of very high level of multiple antibiotic resistance among the isolates will contribute to the protection of public health depending on the measures to be taken in this regard.

Keywords

• Bacillus cereus
• cheese
• antibiotic
• enterotoxin
• gene

Kaynakça

1. Abraha A., Bikila T., Alemu S., Muktar Y. Bacillus cereus isolation and load from raw cow milk sold in markets of Haramaya District, eastern Ethiopia. International Journal of Food Contamination 2017; 4: 15.
2. Adame-Gómez R., Muñoz-Barrios S., Castro-Alarcón N., Leyva-Vázquez MA., Toribio-Jiménez J., Ramírez-Peralta A. Prevalence of the strains of Bacillus cereus group in artisanal Mexican cheese. Foodborne Pathogens and Disease 2020; 17(1): 8-14.
3. Ağaoğlu S., Alemdar S., Ekici K., Güdücüoğlu H. Çiğ süt ve bazı süt ürünlerinde Bacillus cereus’un varlığının araştırılması. Yüzüncü Yıl Üniversitesi, Ziraat Fakültesi Tarım Bilimleri Dergisi 1999; 9(1): 5-7.
4. Ahmed AA., Moustafa MK., Marth EH. Incidence of Bacillus cereus in milk and some milk products. Journal of Food Protection 1983; 46(2): 126-128.
5. Becker H., Schaller G., von Wiese W., Terplan G. Bacillus cereus in infant foods and dried milk products. International Journal of Food Microbiology 1994; 23: 1-15.
6. Berthold-Pluta A., Pluta A., Garbowska M., Stefańska I. Prevalence and toxicity characterization of Bacillus cereus in food products from Poland. Foods 2019; 8(7): 269.
7. Can HY., Elmalı M., Karagöz A., Dişli HB. Psychrotrophic properties, toxigenic characteristics, and PFGE profiles of Bacillus cereus isolated from different foods and spices. Ciência Rural 2022; 52(4):1-11.
8. CLSI, Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement, M100-S25.

9. Ehling-Schulz M., Guinebretiere MH., Monthán A., Berge O., Fricker M., Svensson B. Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS Microbiology Letters 2006; 260(2): 232-40.
10. Gao T., Ding Y., Wu Q., Wang J., Zhang J., Yu S., Yu P., Liu C., Kong L., Feng Z., Chen M., Wu S., Zeng H., Wu H. Prevalence, virulence genes, antimicrobial susceptibility, and genetic diversity of Bacillus cereus isolated from pasteurized milk in China. Frontiers in Microbiology 2018; 9: 533.
11. Gdoura-Ben Amor M., Siala M., Zayani M., Grosset N., Smaoui S., Messadi-Akrout F., Baron F., Jan S., Gautier M., Gdoura R. Isolation, identification, prevalence, and genetic diversity of Bacillus cereus group bacteria from different foodstuffs in Tunisia. Frontiers in Microbiology 2018; 9: 447.
12. Gundogan N., Avci E. Occurrence and antibiotic resistance of Escherichia coli, Staphylococcus aureus and Bacillus cereus in raw milk and dairy products in Turkey. International Journal of Dairy Technology 2014; 67(4): 562-569.
13. Hammad AM., Eltahan A., Khalifa E., Abbas NH., Shimamoto T. Toxigenic potential of Bacillus cereus strains isolated from retail dairy products in Egypt. Foodborne Pathogens and Disease 2021; 18(9): 655-660.
14. Kotiranta A., Lounatmaa K., Haapasalo M. Epidemiology and pathogenesis of Bacillus cereus infections. Microbes and Infection 2000; 2: 189-198.
15. Larsen HD., Jørgensen K. The occurrence of Bacillus cereus in Danish pasteurized milk. International Journal of Food Microbiology 1997; 34(2): 179-86.
16. Liu XY., Hu Q., Xu F., Ding SY., Zhu K. Characterization of Bacillus cereus in dairy products in China. Toxins (Basel), 2020; 12(7): 454.
17. Logan NA. Bacillus and relatives in foodborne illness. Journal of Applied Microbiology 2012; 112(3): 417-29.
18. Magnusson M., Christiansson A., Svensson B. Bacillus cereus spores during housing of dairy cows: Factors affecting contamination of raw milk. Journal of Dairy Science 2007; 90 (6): 2745-2754.
19. McKillip JL. Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review. Antonie Van Leeuwenhoek 2000; 77: 393-399.
20. Owusu-Kwarteng J., Wuni A., Akabanda F., Tano-Debrah K., Jespersen L. Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato isolated from dairy farms and traditional dairy products. BMC Microbiology 2017;17(1): 65.
21. Özdemir H. Pastörize sütlerde Bacillus cereus'un varlığı. Gıda 2003; 28 (6): 611-614.
22. Pei X., Yang S., Zhan L., Zhu J., Song X., Hu X., Liu G., Ma G., Li N., Yang D. Prevalence of Bacillus cereus in powdered infant and powdered follow-up formula in China. Food Control 2018; 93: 101-105.
23. Pretorius C., Buys EM. Extended shelf life milk processing: Effect of simulated cleaning in place on the germination and attachment of Bacillus cereus spores. International Journal of Dairy Technology 2021; 74(1): 75-83.
24. Schiefer B., Macdonald KR., Klavano GG., van Dreumel AA. Pathology of Bacillus cereus mastitis in dairy cows. Canadian Veterinary Journal 1976; 17(9): 239-43.
25. Stenfors Arnesen LP., Fagerlund A., Granum PE. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiolology Letters 2008; 32(4): 579-606.
26. Tallent SM., Kotewicz KM., Strain EA., Bennett RW. Efficient isolation and identification of Bacillus cereus group. Journal of AOAC International 2012; 95(2): 446-51.
27. Te Giffel MCT., Beumer RR., Granum PE., Rombouts FM. Isolation and characterisation of Bacillus cereus from pasteurised milk in household refrigerators in the Netherlands. International Journal of Food Microbiology1997; 34 (3): 307-318.
28. Tekinşen KK. Microbiological and chemical quality of herb cheese manufactured in Hakkari. Eurasian Journal of Veterinary Sciences 2004; 20(2): 79-85.
29. Tewari A., Abdullah S. Bacillus cereus food poisoning: international and Indian perspective. Journal of Food Science and Technology 2015; 52(5): 2500-11.
30. Uraz A., Arslan S., Gündoğan N. Çiğ süt, pastörize süt ve beyaz peynir örneklerinden izole edilen ve iyodometrik test yöntemiyle beta-laktamaz varlığı saptanan Bacillus türleri. Gıda 1996; 21(4): 275-280.
31. Walker-York-Moore L., Moore SC., Fox EM. Characterization of enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus isolates and associated enterotoxin production dynamics in milk or meat-based broth. Toxins (Basel) 2017; 9(7): 225.
32. Wang RF., Cao WW., Cerniglia CE. A universal protocol for PCR detection of 13 species of foodborne pathogens in foods. Journal of Applied Microbiology 1997; 83 (6): 727-736.
33. WHO, Dünya Sağlık Örgütü. 2020. Antibiotic resistance. https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance. Erişim tarihi: 25.10.2022.
34. WHO, Dünya Sağlık Örgütü. 2021. Antimicrobial resistance. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance. Erişim tarihi: 25.10.2022.
35. Wijnands LM., Dufrenne JB., Rombouts FM., in 't Veld PH., van Leusden FM. Prevalence of potentially pathogenic Bacillus cereus in food commodities in The Netherlands. Journal of Food Protection 2006; 69(11): 2587-94.
36. Wong HC., Chang MH., Fan JY. Incidence and characterization of Bacillus cereus isolates contaminating dairy products. Applied and Environmental Microbiology 1988; 54(3): 699-702.
37. Yıbar A., Çetinkaya F., Soyutemiz E., Yaman G. Prevalence, enterotoxin production and antibiotic resistance of Bacillus cereus isolated from milk and cheese. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 2017; 23 (4): 635-642.