TULUM PEYNİRİNDEN Escherichia coli İZOLASYONU, PATOTİPLENDİRMESİ VE ANTİBİYOTİK DİRENÇ PROFİLLERİNİN BELİRLENMESİ

Abstract

Çalışma kapsamında Isparta ve Konya illerindeki yerel pazarlardan temin edilen toplam 70 Tulum peyniri örneğinde Escherichia coli varlığı, patotip özellikleri ve antibiyotik direnç profilleri araştırılmıştır. Örneklerden kromojenik koliform agar kullanılarak toplam 25 muhtemel E. coli izolatı elde edilmiştir. İzolatların morfolojik tanısında Gram boyama, indol ve katalaz testleri uygulanmış; tüm izolatların Gram negatif çubuk morfolojisinde olduğu, indol ve katalaz pozitif reaksiyon verdiği belirlenmiştir. Moleküler doğrulama amacıyla gerçekleştirilen 16S rRNA temelli PCR analizlerinde, tüm izolatlarda E. coli’ye özgü 544 bç büyüklüğündeki hedef bölge tespit edilmiş ve peynir örneklerinin %35.71’inde E. coli varlığı doğrulanmıştır. Patotip araştırmalarında ise incelenen suşların hiçbirinde hedeflenen virülans genleri saptanmamış, bu nedenle izolatların kommensal veya patojenik olmayan E. coli olarak değerlendirilebileceği sonucuna ulaşılmıştır. Antibiyotik dirençlilik testlerinde izolatların tamamının eravasiklin, trimetoprim-sülfametoksazol, meropenem, aztreonam, amoksisilin-klavulanik asit, nitrofurantoin, sefoksitin, imipenem ve ampisiline duyarlı olduğu belirlenmiştir. Buna karşın en yüksek direnç oranı norfloksasine (%64) karşı saptanmış, bunu gentamisin (%52), amikasin (%36), seftriakson (%28) ve siprofloksasin (%28) izlemiştir. İzolatların önemli bir kısmının 2–5 antibiyotiğe dirençli olduğu belirlenmiştir. Çoklu antibiyotik direnç (MAR) indeks değerlerinin 0.000–0.312 arasında değişmesi, bazı suşların yoğun antibiyotik baskısına maruz kalmış yüksek riskli kaynaklardan köken almış olabileceğini göstermiştir. Elde edilen sonuçlar, geleneksel Tulum peynirlerinin hijyenik kalitesinin sürekli olarak izlenmesi gerektiğini ve antibiyotik dirençli E. coli suşlarının halk sağlığı açısından potansiyel bir risk oluşturabileceğini ortaya koymaktadır.

Keywords

• Tulum peyniri
• Escherichia coli
• patotiplendirme
• antibiyotik direnç
• MAR indeksi

ISOLATION of Escherichia coli FROM TULUM CHEESE, PATHOTYPING, AND DETERMINATION OF ANTIBIOTIC RESISTANCE PROFILES

Abstract

Within the scope of this study, the presence of Escherichia coli, its pathotype characteristics, and antimicrobial resistance profiles were investigated in a total of 70 Tulum cheese samples obtained from local markets in the provinces of Isparta and Konya, Türkiye. A total of 25 presumptive E. coli isolates were recovered using Chromogenic Coliform agar. Morphological characterization of the isolates was performed by Gram staining, indole, and catalase tests, revealing that all isolates exhibited Gram-negative rod morphology and were positive for both indole and catalase reactions. For molecular confirmation, 16S rRNA gene-based PCR analyses were conducted, and the E. coli-specific 544 bp target fragment was detected in all isolates. Consequently, E. coli was detected in 35.71% of the cheese samples examined. In the pathotype analyses, none of the investigated isolates carried the targeted virulence genes, indicating that these strains could be considered commensal or non-pathogenic E. coli. Antimicrobial resistance testing demonstrated that all isolates were susceptible to eravacycline, trimethoprim-sulfamethoxazole, meropenem, aztreonam, amoxicillin-clavulanic acid, nitrofurantoin, cefoxitin, imipenem, and ampicillin. In contrast, the highest resistance rate was observed against norfloxacin (64%), followed by gentamicin (52%), amikacin (36%), ceftriaxone, and ciprofloxacin (28% each). A considerable proportion of the isolates exhibited resistance to two to five antibiotics. Multiple antibiotic resistance (MAR) index values ranged from 0.0 to 0.312, suggesting that some strains may have originated from high-risk sources exposed to intensive antibiotic pressure. Overall, these findings highlight the need for continuous monitoring of traditional Tulum cheeses with respect to hygienic quality and indicate that the presence of antibiotic-resistant E. coli strains may represent a potential public health concern.

Keywords

• Tulum cheese
• Escherichia coli
• pathotyping
• antibiotic resistance
• MAR index

References

1. Adzitey, F., Huda, N., Shariff, A.H.M. (2021). Phenotypic antimicrobial susceptibility of Escherichia coli from raw meats, and their related samples in one health context. Microorganisms, 9(2): 326. https://doi.org/10.3390/microorganisms9020326
2. Alhadlaq, M.A., Aljurayyad, O.I., Almansour, A. Al-Akeel SI, Alzahrani KO, Alsalman SA, Yahya R, Al-Hindi RR, Hakami MA, Alshahrani SD, Alhumeed NA, Al Moneea AM, Al-Seghayer MS, AlHarbi AL, Al-Reshoodi FM, Alajel S. (2024). Overview of pathogenic Escherichia coli, with a focus on Shiga toxin-producing serotypes, global outbreaks (1982–2024) and food safety criteria. Gut Pathogens, 16, 57. https://doi.org/10.1186/s13099-024-00641-9.
3. Ayandele, A.A., Oladipo, E.K., Oyebisi, O., Kaka, M.O. (2020). Prevalence of multi-antibiotic resistant Escherichia coli and Klebsiella species obtained from a tertiary medical institution in Oyo State, Nigeria. Qatar Medical Journal, 1:1–6. https://doi.org/10.5339/QMJ.2020.9.
4. Bingöl, E.B., Çetin, Ö., Çolak, H., Hampikyan, H. (2012). Presence of enterotoxin and verotoxin in Turkish cheeses sold in İstanbul. Turkish Journal of Veterinary and Animal Sciences. 36: 4. https://doi.org/10.3906/vet-1105-5 Blount ZD. (2015). The unexhausted potential of E. coli. Elife. doi: 10.7554/eLife.05826. PMID: 25807083; PMCID: PMC4373459.
5. Bonyadian M, Moshtaghi H, Akhavan Taheri M. (2014). Molecular characterization and antibiotic resistance of enterotoxigenic and entero-aggregative Escherichia coli isolated from raw milk and unpasteurized cheeses. Veterinary Research Forum, 5(1):29-34. PMID: 25568689; PMCID: PMC4279659.
6. Cancilla, M.R., Powell, L.B., Hillier, A.J., Davidson, B.E. (1992). Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels. Applied and Environmental Microbiology, 58, 1772-1775.
7. Chandra, M., Cheng, P., Rondeau, G., Porwollik, S., McClelland, M. (2013). A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella. International Journal of Medical Microbiology, 303(4):210-6. doi: 10.1016/j.ijmm.2013.02.013. Epub 2013 Mar 5. PMID: 23562277.
8. Condor, S., Duma, M., Crăciun, S., Mihaiu, M., Cîmpean, R., Crisan-Reget, O.L., Dan, S.D., Condor, L., Ionica, C.-N., Tabaran, A. (2025). Quantification of total Staphylococci and Escherichia coli in milk and dairy products from small ruminants and characterization of the antimicrobial resistance profiles of isolated pathogenic strains. Microorganisms 13, 2756. https://doi.org/10.3390/microorganisms13122756.

9. Crippa C, De Cesare A, Lucchi A, Parisi A, Manfreda G, Pasquali F. (2024). Occurrence and genomic characterization of antimicrobial-resistant and potential pathogenic Escherichia coli from Italian artisanal food productions of animal origin. Italian Journal Food Safety, doi: 10.4081/ijfs.2024.12205.
10. Çakır, Ç. A., Demirkapı, F. (2023). Urfa peynirinin fiziksel, kimyasal ve mikrobiyolojik özellikleri üzerine farklı tuz konsantrasyonlarının etkisi. ISPEC Journal of Agricultural Sciences, 7(3), 623-633. https://doi.org/10.18016/10.5281/zenodo.8354331
11. de Campos ACLP, Puño-Sarmiento JJ, Medeiros LP, Gazal LES, Maluta RP, Navarro A, Kobayashi RKT, Fagan EP, Nakazato G. (2018). Virulence genes and antimicrobial resistance in Escherichia coli from cheese made from unpasteurized milk in Brazil. Foodborne Pathogen and Disease. 15(2):94-100. doi: 10.1089/fpd.2017.2345
12. EFSA (European Food Safety Authority) and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). (2025a). The European Union One Health 2024 Zoonoses Report. EFSA Journal, 23(12), e9759. https://doi.org/10.2903/j.efsa.2025.9759
13. EFSA (European Food Safety Authority) and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). (2025b). The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2022–2023. EFSA Journal, 23(3), e9237. https://doi.org/10.2903/j.efsa.2025.9237
14. EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). (2026). The European Union Summary Report on Antimicrobial Resistance in zoonotic and indicator bacteria from humans, animals and food in 2023–2024. EFSA Journal, 24(2), e9887. https://doi.org/10.2903/j.efsa.2026.9887
15. EUCAST. (2025). European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters Version 15.0, valid from 2025-01-01.
16. Farrukh, M., Munawar, A., Nawaz, Z., Hussain, N., Hafeez, AB., Szweda, P. (2025). Antibiotic resistance and preventive strategies in foodborne pathogenic bacteria: a comprehensive review. Food Science and Biotechnology, 34(10), 2101-2129. doi: 10.1007/s10068-024-01767-x.
17. Giri, S., Kudva, V., Shetty, K., Shetty, V. (2021). Prevalence and characterization of extended-spectrum ß-lactamase-producing antibiotic resistant Escherichia coli and Klebsiella pneumoniae in ready-to-eat street foods. Antibiotics, 10 (7): 850. doi: 10.3390/antibiotics10070850.
18. Hu, X.J., Chen, Y.Y., Xu, H., Qiao, J., Ge, H.Y., Liu, R.S., Zheng, B.W. (2023). Genomic epidemiology and transmission characteristics of mcr1-positive colistin-resistant Escherichia coli strains circulating at natural environment. Science of the Total Environment, 882, 163600. https://doi.org/10.1016/j.scitotenv.2023.163600
19. Hussein, N.D., Hassan, J.W., Osman, M., El-Omari, K., Kharroubi, S.A., Toufeili, I., Kassem, I.I. (2023). Assessment of the microbiological acceptability of White Cheese (Akkawi) in Lebanon and the antimicrobial resistance profiles of associated Escherichia coli. Antibiotics, 2023, 12, 610. https://doi.org/10.3390/antibiotics12030610.
20. Imre, K., Ban-Cucerzan, A., Herman, V., Sallam, K. I., Cristina, R. T., Abd-Elghany, S. M., Morar, A. (2022). Occurrence, pathogenic potential and antimicrobial resistance of Escherichia coli isolated from raw milk cheese commercialized in Banat Region, Romania. Antibiotics, 11(6), 721. https://doi.org/10.3390/antibiotics11060721.
21. Jones, G., Lefèvre, S., Donguy, M.-P., Nisavanh, A., Terpant, G., Fougère, E., Vaissière, E., Guinard, A., Mailles, A., de Valk, H. (2019). Outbreak of Shiga toxin-producing Escherichia coli (STEC) O26 paediatric haemolytic uraemic syndrome (HUS) cases associated with the consumption of soft raw cow’s milk cheeses, France, March to May 2019. Eurosurveillance, 24, 1900305. https://doi.org/10.2807/1560-7917.es.2019.24.22.1900305.
22. Kara, R., Akkaya, L. (2015). Afyon Tulum peynirinin mikrobiyolojik ve fiziko-kimyasal özellikleri ile laktik asit bakteri dağılımlarının belirlenmesi. Afyon Kocatepe Üniversitesi Fen ve Mühendislik Bilimleri Dergisi, 15:1-6. doi: 10.5578/fmbd.8717.
23. Khanom, H., Nath, C., Mshelbwala, P.P., Pasha, M.R. (2025). Epidemiology and molecular characterization of multidrug-resistant Escherichia coli isolated from chiken meat. Plos One, 20 (5): e0323909. https://doi.org/10.1371/journal.pone.0323909.
24. Krumperman, P.H. (1983). Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Applied Environmental Microbiology, 46(1):165-70. https://doi.org/10.1128/aem.46.1.165-170.
25. Liu, C., Sun, S., Sun, Y., Li, X., Gu, W., Luo, Y., Wang, N., Wang, Q. (2024). Antibiotic resistance of Escherichia coli isolated from food and clinical environment in China from 2001 to 2020. Science of The Total Environment, Volume 939, 2024, 173498, ISSN 0048-9697, https://doi.org/10.1016/j.scitotenv.2024.173498.
26. Mangroliya, D., Adhyaru, H., Kabariya, J., Rmani, V. (2025). Genomic insights into plasmid mediated AMR genes, virulence factors and mobile genetic elements in raw milk Escherichia coli from Gujarat, India. Scientific Reports, 15, 6320. https://doi.org/10.1038/s41598-025-90761-2
27. Menge, C., 2020. The role of Escherichia coli Shiga toxins in STEC colonization of cattle. Toxins, 12(9), 607. https://doi.org/10.3390/toxins12090607
28. Metz, M., Sheehan, J., Feng, P.C.H. (2020). Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses – A literature review. Food Microbiology, Volume 85,103283, ISSN 0740-0020, https://doi.org/10.1016/j.fm.2019.103283.
29. Morandi, S., Silvetti, T., Bonazza, F., Brasca, M. (2024). Occurrence and diversity of Shiga toxin-producing Escherichia coli (STEC) in Italian Alpine raw milk cheeses and their development in the earlier stages of different cheese-making processes, LWT, Volume 213, https://doi.org/10.1016/j.lwt.2024.117029.
30. Mudau, K.L., Ntobeng, L.R., Kalu, C.M., Tekere, M. (2025). Pathogenicity and virulence factors of Escherichia coli discovered using next generation sequencing technologies and proteomics. Frontier Bacteriology, 4:1677775. doi: 10.3389/fbrio.2025.1677775
31. Naidoo, N., Zishiri, O.T. (2025). Presence, pathogenicity, antibiotic resistance, and virulence factors of Escherichia coli: A Review. Bacteria, 4, 16. https://doi.org/10.3390/bacteria4010016.
32. Nasrollahian, S., Graham, J. P., & Halaji, M. (2024). A review of the mechanisms that confer antibiotic resistance in pathotypes of E. coli. Frontiers in Cellular and Infection Microbiology, 14, 1387497. https://doi.org/10.3389/fcimb.2024.1387497.
33. Ombarak, R.A., Hinenoya, A., Elbagory, A.-R.M., Yamasaki, S. (2018). Prevalence and Molecular characterization of cntimicrobial cesistance in Escherichia coli isolated from raw milk and raw milk cheese in Egypt. Journal of Food Protection, 81:2 (226-232). https://doi.org/10.4315/0362-028X.JFP-17-277.
34. Pakbin, B., Brück, W. M., Rossen, J. W. (2021). Virulence factors of enteric pathogenic Escherichia coli: A review. International Journal of Molecular Sciences, 22(18), 9922. https://doi.org/10.3390/ijms22189922.
35. Patır, B., Güngören, A., Kayaoğlu, E. (2025). Bingöl’de halk pazarında satılan salamura beyaz peynirlerin mikrobiyolojik ve kimyasal kalitesi. Dicle Üniversitesi Veterinerlik Fakültesi Dergisi, 18(2):141-148. https://doi.org/10.47027/duvetfd.1685688.
36. Penna, A. L. B., Gigante, M. L., Todorov, S. D. (2021). Artisanal Brazilian cheeses—history, marketing, technological and microbiological aspects. Foods, 10(7), 1562. https://doi.org/10.3390/foods10071562.
37. Poirel, L., Madec, J.Y., Lupo, A., Schink, A.K., Kieffer, N., Nordmann, P., Schwarz, S., (2018). Antimicrobial resistance in Escherichia coli. Microbiology Spectrum 6, doi: 10.1128/microbiolspec.ARBA-0026-2017.
38. Praça, J., Furtado, R., Coelho, A., Correia, C. B., Borges, V., Gomes, J. P., ... & Batista, R., 2023. Listeria monocytogenes, Escherichia coli and coagulase positive staphylococci in cured raw milk cheese from Alentejo region, Portugal. Microorganisms, 11(2), 322. https://doi.org/10.3390/microorganisms11020322.
39. Rasheed, M. U., Thajuddin, N., Ahamed, P., Teklemariam, Z., Jamil, K. (2014). Antimicrobial drug resistance in strains of Escherichia coli isolated from food sources. Revista do Instituto de Medicina Tropical de São Paulo, 56(4), 341-346. https://doi.org/10.1590/S0036-46652014000400012.
40. Ribeiro, L.F. Rossi, G.A.M. Sato, R.A. de Souza Pollo, A. Cardozo, M.V. Amaral, L.A.D., Fairbrother, J.M. (2024). Epidemiology, virulence and antimicrobial resistance of Escherichia coli isolated from small Brazilian farms producers of raw milk fresh cheese. Microorganisms, 12, 1739. https://doi.org/10.3390/microorganisms12081739.
41. Rodrigues, L. F. S., Melo, R. A. d., Borges, N. M., Aragão, A. C. S., Araújo, M. O. d., dos Santos, R. D., Bilac, C. A., Gomes, K. O., do Prado, B. A., Sá Barreto, L. C. L. d., Silva, I. C. R. d., Orsi, D. C. (2025). Antimicrobial resistance phenotypes and genotypes of Escherichia coli isolates from artisanal Minas Frescal cheeses from the Federal District, Brazil. Antibiotics, 14 (11): 1101. https://doi.org/10.3390/antibiotics14111101
42. Sabat G., Rose P., Hickey W.J., Harkin, J.M. (2000). Selective and sensitive method for PCR amplification of Escherichia coli 16S rRNA genes in soil. Applied and Environmental Microbiology. 66(2):844-849. DOI: 10.1128/aem.66.2.844-849.2000. PMID: 10653763; PMCID: PMC91908.
43. Tenaillon, O., Skurnik, D., Picard, B., Denamur, E. (2010). The population genetics of commensal Escherichia coli. Nature Reviews Microbiology, 8(3), 207-217.  https://doi.org/10.1038/nrmicro2298
44. Zarei Ahmady, A., Aliyan Aliabadi, R., Amin, M., Ameri, A., Montazeri, E.A. (2023). Occurrence of diarrheagenic Escherichia coli pathotypes from raw milk and unpasteurized buttermilk by culture and multiplex polymerase chain reaction in southwest Iran. Molecular Biology Reports, 50: 3661–3667. https://doi.org/10.1007/s11033-023-08261-3.