Mastitisli İneklerin Süt Örneklerinden Staphylococcus epidermidis İzolasyonu ve Biyofilm Oluşturma Yeteneğinin İncelenmesi

Öz

Mastitis, süt sığırlarında görülen en önemli enfeksiyöz hastalıklardan biridir ve önemli ekonomik kayıplarla ilişkilidir. Etkenler arasında yer alan Staphylococcus epidermidis, biyofilm oluşturma yeteneği önemli bir virülens faktörü olarak kabul edilen fırsatçı bir patojendir. Bu çalışmanın amacı, sığır mastitisli süt örneklerinden elde edilen S. epidermidis izolatlarının biyofilm oluşturma kapasitesini fenotipik ve genotipik yöntemler kullanarak değerlendirmektir. Bu amaçla, İzmir ve Aydın illerindeki farklı işletmelerde bulunan klinik mastitisli 150 farklı süt sığırından toplam 150 süt örneği toplanmıştır. Örnekler %5 koyun kanlı agara ekilmiş ve izolatlar konvansiyonel yöntemler ile VITEK® 2 Compact sistemi kullanılarak tanımlanmıştır. Fenotipik biyofilm üretimi Kongo Red Agar (CRA) yöntemi ile değerlendirilmiş, icaA ve icaD genlerinin varlığı ise moleküler olarak incelenmiştir. Bakteriyolojik inceleme sonucunda 13 izolat S. epidermidis olarak tanımlanmıştır. Bunların 11’i (%84,6) CRA yöntemi ile biyofilm pozitif, 2’si (%15,4) ise biyofilm negatif olarak değerlendirilmiştir. Moleküler analiz sonucunda ise izolatların hiçbirinde icaA veya icaD geni saptanmamıştır. Sonuç olarak, mastitisli süt örneklerinden elde edilen S. epidermidis izolatlarının büyük çoğunluğu, araştırılan klasik biyofilm ile ilişkili genlerin yokluğuna rağmen fenotipik olarak biyofilm oluşturma yeteneği göstermiştir. Bu bulgular, sığırlara ait S. epidermidis izolatlarında biyofilm oluşumunun alternatif moleküler mekanizmalar içerebileceğini ve fenotipik sonuçların genotipik analizlerle birlikte değerlendirilmesi gerektiğini düşündürmektedir.

Anahtar Kelimeler

• Biyofilm oluşumu
• Mastitis
• Sığır
• Staphylococcus epidermidis
• Süt

Isolation of Staphylococcus epidermidis from Milk Samples of Cows with Mastitis and Investigation of Its Biofilm Formation Ability

Öz

Mastitis is one of the most important infectious diseases in dairy cattle and is associated with considerable economic losses. Among the causative agents, Staphylococcus epidermidis is an opportunistic pathogen whose ability to form biofilm is considered an important virulence factor. The aim of this study was to evaluate the biofilm-forming capacity of S. epidermidis isolates recovered from bovine mastitic milk samples using phenotypic and genotypic methods. For this purpose, a total of 150 milk samples were collected from 150 different dairy cows with clinical mastitis from different farms in the provinces of İzmir and Aydın. The samples were cultured on 5% sheep blood agar, and the isolates were identified by conventional procedures and the VITEK® 2 Compact system. Phenotypic biofilm production was evaluated by the Congo Red Agar (CRA) method, and the presence of the icaA and icaD genes was investigated molecularly. As a result of bacteriological examination, 13 isolates were identified as S. epidermidis. Of these, 11 (84.6%) were found to be biofilm-positive by the CRA method, whereas 2 (15.4%) were biofilm-negative. Molecular analysis revealed that none of the isolates carried the icaA or icaD genes. In conclusion, most S. epidermidis isolates obtained from mastitic milk samples showed phenotypic biofilm-forming ability despite the absence of the investigated classical biofilm-associated genes. These findings suggest that biofilm formation in bovine S. epidermidis isolates may involve alternative molecular mechanisms and that phenotypic results should be interpreted together with genotypic analyses.

Anahtar Kelimeler

• Biofilm formation
• Cattle
• Mastitis
• Milk
• Staphylococcus epidermidis

Kaynakça

1. Sharun K, Dhama K, Tiwari R et al. (2021). Advances in therapeutic and managemental approaches of bovine mastitis: A comprehensive review. Vet Q., 41(1):107–136.
2. Stanek P, Zawiślak J, Różańska-Zawieja J (2024). A Review on Mastitis in Dairy Cows Research. Agriculture, 14(8):1292.
3. Costa A, Bovenhuis H, Egger-Danner C et al. (2025). Mastitis has a cumulative and lasting effect on milk yield and lactose content in dairy cows. J Dairy Sci., 108(1):635–650.
4. Ajose DJ, Oluwarinde BO, Abolarinwa TO et al. (2022). Combating bovine mastitis in the dairy sector in an era of antimicrobial resistance: Ethno-veterinary medicinal option as a viable alternative approach. Front Vet Sci., 9:800322.
5. Severn MM, Horswill AR (2023). Staphylococcus epidermidis and its dual lifestyle in skin health and infection. Nat Rev Microbiol., 21(2):97–111.
6. Zheng Y, Hunt RL, Villaruz AE et al. (2022). Commensal Staphylococcus epidermidis contributes to skin barrier homeostasis by generating protective ceramides. Cell Host Microbe., 30(3):301-313.e9.
7. Burke Ó, Humphreys H, Fitzgerald-Hughes D (2024). The pathogenicity and virulence of the opportunistic pathogen Staphylococcus epidermidis. Virulence, 15(1):2359483.
8. Arciola CR, Campoccia D, Gamberini S, Cervellati M, Donati E, Montanaro L (2001). A rapid PCR method for the detection of slime producing strains of Staphylococcus epidermidis and Staphylococcus aureus. J Clin Pathol., 54(3):193–196.

9. Türkyılmaz S, Kaya O, Güler L (2006). Investigation of slime production and some virulence factors in staphylococci isolated from various animal clinical samples. Turk J Vet Anim Sci., 30(5):519–525.
10. Qi P, Gong F, Leng M, Wei Z (2025). Beneficial perspective on Staphylococcus epidermidis: a crucial species for skin homeostasis and pathogen defense. Front Immunol., 16:1674392.
11. Cheung GYC, Otto M (2025). Staphylococcus epidermidis key to understanding biofilms, commensalism, and more. J Bacteriol., 207(9):e00165-25.
12. Manandhar S, Singh A, Varma A, Pandey S, Shrivastava N (2018). Evaluation of methods to detect in vitro biofilm formation by staphylococcal clinical isolates. BMC Res Notes., 11: 714.
13. Delmas J, Chacornac JP, Robin F, Giammarinaro P, Talon R, Bonnet R (2008). Evaluation of the Vitek 2 system with a variety of Staphylococcus species. J Clin Microbiol., 46(1): 311–313.
14. Winn Jr W, Allen S, Janda W et al. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. Lippincott Williams & Wilkins, Baltimore.
15. Freeman DJ, Falkiner FR, Keane CT (1989). New method for detecting slime production by coagulase-negative staphylococci. J Clin Pathol., 42(8):872–874.
16. Newcombe RG (1998). Two-sided confidence intervals for the single proportion: comparison of seven methods. Stat Med., 17(8):857–872.
17. Kord M, Ardebili A, Jamalan M, Jahanbakhsh R, Behnampour N, Ghaemi EA (2018). Evaluation of biofilm formation and presence of ica genes in Staphylococcus epidermidis clinical isolates. Osong Public Health Res Perspect., 9(4):160–166.
18. França A, Gaio V, Lopes N, Melo LDR (2021). Virulence factors in coagulase-negative staphylococci. Pathogens, 10(2):170.
19. Le KY, Park MD, Otto M (2018). Immune evasion mechanisms of Staphylococcus epidermidis biofilm infection. Front Microbiol., 9:3593.
20. Patel JD, Colton E, Ebert M, Anderson JM (2012). Gene expression during S. epidermidis biofilm formation on biomaterials. J Biomed Mater Res A., 100(11):2863–2869.
21. Azeem K, Fatima S, Ali A, Ubaid A, Husain FM, Abid M (2025). Biochemistry of bacterial biofilm: insights into antibiotic resistance mechanisms and therapeutic intervention. Life, 15(1):49.
22. Mirzaei B, Faridifar P, Shahmoradi M et al. (2020). Genotypic and phenotypic analysis of biofilm formation in Staphylococcus epidermidis isolates from clinical specimens. BMC Res Notes., 13:114.
23. Ahmad S, Rahman H, Qasim M et al. (2022). Staphylococcus epidermidis pathogenesis: interplay of icaADBC operon and MSCRAMMs in biofilm formation of isolates from pediatric bacteremia in Peshawar, Pakistan. Medicina, 58(11):1510.
24. Juárez-Verdayes MA, Ramón-Peréz ML, Flores-Páez LA et al. (2013). Staphylococcus epidermidis with the icaA/icaD/IS256 genotype and protein or protein/extracellular DNA biofilm is frequent in ocular infections. J Med Microbiol., 62(10): 1579-1587.
25. Kumar S, Chandra N, Singh L, Hashmi MZ, Varma A (2019). Biofilms: The good and the bad. In: Biofilms in Human Diseases: Treatment and Control. S Sharma, N Chandra, L Singh, MZ Hashmi, A Varma (eds). Springer, Cham., 29-47.
26. Sharma S, Mohler J, Mahajan SD, Schwartz SA, Bruggemann L, Aalinkeel R (2023). Microbial biofilm: A review on formation, infection, antibiotic resistance, control measures, and innovative treatment. Microorganisms, 11(6):1614.
27. Shivaee A, Meskini M, Roodaki RRN, Kalani BS, Mirshekar M, Razavi S (2021). Evaluation of the effects of nano curcumin on the expression of genes involved in biofilm formation in Staphylococcus epidermidis. Gene Rep., 23:101026.
28. Zafer MM, Mohamed GA, Ibrahim SRM, Ghosh S, Bornman C, Elfaky MA (2024). Biofilm-mediated infections by multidrug-resistant microbes: a comprehensive exploration and forward perspectives. Arch Microbiol., 206:101.
29. Liu HY, Prentice EL, Webber MA (2024). Mechanisms of antimicrobial resistance in biofilms. NPJ Antimicrob Resist., 2:27.