Molecular Identification, Biofilm Formation Ability and Antimicrobial Resistance of Staphylococcus warneri Isolates Obtained from Mastitic Cows in the Aegean Region

Abstract

Staphylococcus warneri is a species belonging to the coagulase-negative staphylococci (CoNS) group and is commonly found as a commensal organism on the skin and mucosal surfaces of humans and animals. In recent years, the increasing isolation rates of CoNS from milk and dairy products have highlighted their significance in terms of both food safety and livestock production. This study aimed to confirm S. warneri isolates obtained from milk samples using molecular methods, to evaluate their biofilm-forming ability through the Congo Red Agar (CRA) method, and to determine their phenotypic antimicrobial resistance profiles. For this purpose, 650 milk samples were examined, the presence of S. warneri was investigated using phenotypic identification methods, and suspected isolates were confirmed by Polymerase Chain Reaction (PCR). The biofilm-forming ability of isolates identified as S. warneri was assessed on CRA medium. In addition, the susceptibility of the isolates to eight different antibiotics was determined by the Kirby-Bauer disk diffusion method according to CLSI standards. Among the 650 samples analyzed, 21 isolates were found to be positive for S. warneri, and all isolates demonstrated biofilm-forming capacity. According to the antibiotic susceptibility results, the isolates exhibited resistance to oxacillin (90.5%), trimethoprim–sulfamethoxazole (81.0%), penicillin (47.6%), tetracycline (38.1%), chloramphenicol (23.8%), erythromycin (14.3%), and doxycycline (14.3%), while all isolates were fully susceptible to gentamicin (100%). In conclusion, the presence of S. warneri, a CoNS species, in dairy cattle within the sampling region was confirmed; their biofilm-forming abilities were determined; and the most appropriate antibiotic options for treatment were evaluated phenotypically based on resistance profiles.

Keywords

• Antibiotic Resistance
• Biofilm
• Staphylococcus warneri

Ege Bölgesindeki Mastitisli Sığırlardan İzole Edilen Staphylococcus warneri Suşlarında Moleküler Tanımlama, Biyofilm Oluşturma Yeteneği ve Antimikrobiyal Direncin Değerlendirilmesi

Abstract

Staphylococcus warneri, koagulaz-negatif stafilokoklar (KNS) arasında yer alan ve insan ile hayvanların deri ve mukozalarında kommensal olarak bulunan bir türdür. Son yıllarda süt ve süt ürünlerinde KNS türlerinin artan oranlarda izole edilmesi, bu bakterilerin hem gıda güvenliği hem de hayvansal üretim açısından önemini artırmıştır. Bu çalışma, süt örneklerinden izole edilen Staphylococcus warneri suşlarının moleküler yöntemlerle doğrulanması, biyofilm oluşturma kapasitelerinin Congo Red Agar (CRA) yöntemiyle değerlendirilmesi ve antimikrobiyal direnç profillerinin fenotipik olarak ortaya konması amacıyla yürütülmüştür. Bu amaçla 650 süt örneği incelenmiş, fenotipik yöntemlerle S. warneri varlığı araştırılmış ve şüpheli izolatlar PCR ile doğrulanmıştır. S. warneri olarak tanımlanan izolatların biyofilm oluşturma yeteneği CRA besiyeri kullanılarak değerlendirilmiştir. Ayrıca izolatların sekiz farklı antibiyotiğe karşı duyarlılığı CLSI standartlarına göre Kirby-Bauer disk difüzyon yöntemi ile belirlenmiştir. Çalışmada 650 örnekten elde edilen 21 izolatın S. warneri yönünden pozitif olduğu ve tüm izolatların biyofilm oluşturma yeteneğine sahip olduğu tespit edilmiştir. Antibiyotik duyarlılık sonuçlarına göre izolatlarda %90.5 oksasilin, %81.0 trimetoprim-sülfametoksazol, %47.6 penisilin, %38.1 tetrasiklin, %23.8 kloramfenikol, %14.3 eritromisin ve doksisiklin direnci görülürken; tüm izolatların gentamisine karşı %100 duyarlı olduğu belirlenmiştir. Sonuç olarak, örnekleme bölgesindeki süt sığırlarında KNS türlerinden S. warneri varlığı ortaya konulmuş, biyofilm oluşturma kapasiteleri belirlenmiş ve tedavide kullanılabilecek en uygun antibiyotik seçenekleri fenotipik olarak değerlendirilmiştir.

Keywords

• Antibiyotik Direnci
• Biyofilm
• Staphylococcus warneri

References

1. Abdel Halim RM, Kassem NN, Mahmoud BS (2018). Detection of biofilm producing staphylococci among different clinical isolates and its relation to methicillin susceptibility. Open Access Maced J Med Sci, 6(8), 1335-1341.
2. Addis MF, Locatelli C, Penati M et al. (2024). Non-aureus staphylococci and mammaliicocci isolated from bovine milk in Italian dairy farms: A retrospective investigation. Vet Res Commun, 48(1), 547-554.
3. Al-Mousawi AH, Chessab RM, Alquraishy MK (2022). Inhibition of biofilm formation by methicillin-resistant Staphylococcus warneri using Citrus limon oil. Caspian J Environ Sci, 20(1), 197-201.
4. Alves E, Esteves AC, Correia A et al. (2015). Protein profiles of Escherichia coli and Staphylococcus warneri are altered by photosensitization with cationic porphyrins. Photochem Photobiol Sci, 14, 1169-1178.
5. Azimi T, Mirzadeh M, Sabour S et al. (2020). Coagulase-negative staphylococci (CoNS) meningitis: A narrative review of the literature from 2000 to 2020. New Microbes New Infect, 37, 100755.
6. Balows A, Hausler WJ Jr, Herrmann KL et al. (1991). Manual of Clinical Microbiology. V. Edition. American Society for Microbiology, Washington DC, USA.
7. Barigye R, Schaan L, Gibbs PS, Schamber E, Dyer NW (2007). Diagnostic evidence of Staphylococcus warneri as a possible cause of bovine abortion. J Vet Diagn Invest, 19, 694-696.
8. Clinical and Laboratory Standards Institute (2024). Performance Standards for Antimicrobial Susceptibility Testing. 34. Edition. Clinical and Laboratory Standards Institute, Malvern, USA.

9. De Buck J, Ha V, Naushad S et al. (2021). Non-aureus Staphylococci and bovine udder health: Current understanding and knowledge gaps. Front Vet Sci, 8, 658031.
10. de Oliveira RP, da Silva JG, Aragão BB et al. (2022). Diversity and emergence of multi-resistant Staphylococcus spp. isolated from subclinical mastitis in cows in the state of Piauí, Brazil. Braz J Microbiol, 53(4), 2215-2222.
11. Espino L, Bermudez R, Fidalgo LE et al. (2006). Meningoencephalitis associated with Staphylococcus warneri in a dog. J Small Anim Pract, 47, 598-602.
12. Freeman DJ, Falkiner FR, Keane CT (1989). New method for detecting slime production by coagulase-negative staphylococci. J Clin Pathol, 42(8), 872-874.
13. Garbacz K, Wierzbowska M, Kwapisz E et al. (2021). Distribution and antibiotic resistance of different Staphylococcus species identified by matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) isolated from the oral cavity. J Oral Microbiol, 13(1), 1983322.
14. Getahun YA, Abey SL, Beyene AM et al. (2024). Coagulase-negative staphylococci from bovine milk: Antibiogram profiles and virulence gene detection. BMC Microbiol, 24, 263.
15. Hamel J, Zhang Y, Wente N et al. (2020). Non-S. aureus staphylococci (NAS) in milk samples: Infection or contamination? Vet Microbiol, 242, 108594.
16. Hoque MN, Faisal GM, Das ZC et al. (2024). Genomic features and pathophysiological impact of a multidrug-resistant Staphylococcus warneri variant in murine mastitis. Microbes Infect, 26(3), 105285.
17. Hosseinzadeh S, Saei HD (2014). Staphylococcal species associated with bovine mastitis in the North West of Iran: Emerging of coagulase-negative staphylococci. Int J Vet Sci Med, 2(1), 27-34.
18. Kaiser TD, Pereira EM, Dos Santos KR et al. (2013). Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis. Diagn Microbiol Infect Dis, 75(3), 235-239.
19. Kim J, Hong J, Lim JA, Heu S, Roh E (2018). Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci. Arch Microbiol, 200(1), 73-83.
20. Kloos WE, Schleifer KH (1975). Isolation and characterization of staphylococci from human skin II. Descriptions of four new species: Staphylococcus warneri, Staphylococcus capitis, Staphylococcus hominis and Staphylococcus simulans. Int J Syst Bacteriol, 25, 62-79.
21. Kooken J, Fox K, Fox A et al. (2014). Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray). Mol Cell Probes, 28(1), 41-50.
22. Locatelli C, Gattolin S, Monistero V et al. (2023). Staphylococcus aureus coa gene sequence analysis can prevent misidentification of coagulase-negative strains and contribute to their control in dairy cow herds. Front Microbiol, 14, 1120305.
23. Mello PL, Riboli DFM, Martins LA et al. (2020). Staphylococcus spp. isolated from bovine subclinical mastitis in different regions of Brazil: Molecular typing and biofilm gene expression analysis by RT-qPCR. Antibiotics (Basel), 9(12), 888.
24. Musharrafieh R, Tacchi L, Trujeque J, LaPatra S, Salinas I (2014). Staphylococcus warneri, a resident skin commensal of rainbow trout (Oncorhynchus mykiss) with pathobiont characteristics. Vet Microbiol, 169, 80-88.
25. National Mastitis Council (2017). Laboratory handbook on bovine mastitis, III. Edition. Verona, WI: National Mastitis Council.
26. Nuhay C (2025). Investigation of Corynebacterium pseudotuberculosis and its antimicrobial susceptibility in subclinical mastitis cattle in the Aegean Region. J Anatol Env Anim Sci, 10(2), 202-207.
27. Phophi L, Petzer IM, Qekwana DN (2019). Antimicrobial resistance patterns and biofilm formation of coagulase-negative Staphylococcus species isolated from subclinical mastitis cow milk samples submitted to the Onderstepoort Milk Laboratory. BMC Vet Res, 15(1), 420.
28. Rasmussen TT, Kirkeby LP, Poulsen K, Reinholdt J, Kilian M (2000). Resident aerobic microbiota of the adult human nasal cavity. APMIS, 108, 663–675.
29. Raspanti CG, Bonetto CC, Vissio C et al. (2016). Prevalence and antibiotic susceptibility of coagulase-negative Staphylococcus species from bovine subclinical mastitis in dairy herds in the central region of Argentina. Rev Argent Microbiol, 48(1), 50-56.
30. Ravaioli S, De Donno A, Bottau G et al. (2024). The opportunistic pathogen Staphylococcus warneri: Virulence and antibiotic resistance, clinical features, association with orthopedic implants and other medical devices, and a glance at industrial applications. Antibiotics (Basel), 13(10), 972.
31. Regecová I, Výrostková J, Zigo F et al. (2022). Detection of resistant and enterotoxigenic strains of Staphylococcus warneri isolated from food of animal origin. Foods, 11, 1496.
32. Ryman VE, Kautz FM, Nickerson SC (2021). Case study: Misdiagnosis of nonhemolytic Staphylococcus aureus isolates from cases of bovine mastitis as coagulase-negative staphylococci. Animals (Basel), 11, 252.
33. Seng R, Kitti T, Thummeepak R et al. (2017). Biofilm formation of methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from community and hospital environments. PLoS One, 12(8), e0184172.
34. Sezener MG, Findik A, Erguden VE et al. (2019). Investigation of antibiotic resistance and some virulence genes in mastitis isolate Staphylococcus aureus strains. J Anatol Env Anim Sci, 4(2), 182-187.
35. Shrestha LB, Bhattarai NR, Khanal B (2017). Antibiotic resistance and biofilm formation among coagulase-negative staphylococci isolated from clinical samples at a tertiary care hospital of eastern Nepal. Antimicrob Resist Infect Control, 6, 89.
36. Sivaraman GK, Vijayan AA, Visnuvinayagam S et al. (2022). Incidence of multidrug-resistant coagulase-negative Staphylococci from seafood samples, Veraval, Gujarat. Indian J Anim Health, 61, 65-70.
37. Szczuka E, Krzymińska S, Kaznowski A (2016). Clonality, virulence and the occurrence of genes encoding antibiotic resistance among Staphylococcus warneri isolates from bloodstream infections. J Med Microbiol, 65(8), 828-836.
38. Taponen S, Simojoki H, Haveri M, Larsen HD, Pyörälä S (2006). Clinical characteristics and persistence of bovine mastitis caused by different species of coagulase-negative staphylococci identified with API or AFLP. Vet Microbiol, 115, 199-207.
39. Traversari J, van den Borne BHP, Dolder C et al. (2019). Non-aureus Staphylococci species in the teat canal and milk in four commercial Swiss dairy herds. Front Vet Sci, 6, 186.
40. Wang YT, Lin YT, Wan TW et al. (2019). Distribution of antibiotic resistance genes among Staphylococcus species isolated from ready-to-eat foods. J Food Drug Anal, 27(4), 841-848.
41. Yilmaz DK, Berik N (2024). Phenotypic and genotypic antibiotic resistance of Staphylococcus warneri and Staphylococcus pasteuri isolated from stuffed mussels. Aquat Sci Eng, 39, 172-178.
42. Yokoi KJ, Kuzuwa S, Iwasaki SI et al. (2016). Aureolysin of Staphylococcus warneri M accelerates its proteolytic cascade and participates in biofilm formation. Biosci Biotechnol Biochem, 80(6), 1238-1242.