Isolation, Antimicrobial Susceptibility Determination and Genotyping of Sphingomonas paucimobilis from Milk Samples of Cows with Mastitis

Abstract

Mastitis is one of the most economically significant diseases affecting dairy cattle worldwide, with its subclinical form causing greater production losses than the clinical form. Sphingomonas paucimobilis, a Gram-negative, yellow-pigmented, aerobic bacillus, has recently been recognized as an emerging environmental pathogen in cases of mastitis. This study aimed to isolate S. paucimobilis from milk samples of cows  with mastitis in the İzmir and Aydın regions, determine its antimicrobial susceptibility, and perform genotyping. A total of 100 milk samples were collected from cows with mastitis (50 clinical and 50 subclinical cases). Conventional bacteriological methods, VITEK® Compact 2, and PCR were used for identification. Eight (8%) S. Paucimobilis isolates were obtained, including six from subclinical mastitis samples and two from clinical mastitis samples. Antimicrobial susceptibility testing by the Kirby-Bauer disk diffusion method revealed high resistance rates to amoxicillin-clavulanic acid (87.5%), ampicillin-sulbactam (100%), and cefquinome (100%), while all isolates were susceptible to cefoperazone and gentamicin. Genotyping using the (GTG)₅-PCR method showed that all isolates had distinct patterns, with genetic similarity ranging from 32% to 98%. The findings indicate that S. paucimobilis should be considered among potential environmental mastitis pathogens and highlight the need for advanced diagnostic methods and effective antimicrobial stewardship to prevent resistance development.

Keywords

• Antibiotic Resistance
• Genotype
• Mastitis
• Sphingomonas paucimobilis.

Mastitisli İneklerin Süt Örneklerinden Sphingomonas paucimobilis'in İzole Edilmesi, Antimikrobiyal Duyarlılığının Belirlenmesi ve Genotiplendirilmesi

Abstract

Mastitis, dünya genelinde süt sığırlarını etkileyen en önemli ekonomik hastalıklardan biridir ve subklinik formu, klinik formuna göre daha büyük üretim kayıplarına yol açmaktadır. Gram-negatif, sarı pigmentli, aerob bir basillus olan Sphingomonas paucimobilis, son zamanlarda mastitis vakalarında ortaya çıkan çevresel bir patojen olarak tanımlanmıştır. Bu çalışmada, İzmir ve Aydın bölgelerindeki mastitisli ineklerden alınan süt örneklerinden S. paucimobilis izole edilmesi, antimikrobiyal duyarlılığının belirlenmesi ve genotiplendirme yapılması amaçlanmıştır. Sağlıklı ve mastitisli ineklerden toplam 100 süt örneği toplanmıştır. Tanımlama için konvansiyonel bakteriyolojik yöntemler, VITEK® Compact 2 sistemi ve PCR yöntemi kullanılmıştır. Sekiz (%8) S. paucimobilis izolatı elde edilmiştir. Kirby–Bauer disk difüzyon yöntemiyle yapılan antimikrobiyal duyarlılık testinde, amoksisilin–klavulanik aside (%87,5), ampisilin–sulbaktama (%100) ve sefkinoma (%100) karşı yüksek direnç oranları belirlenmiştir. Tüm izolatların sefopeperazon, gentamisin ve sefkinoma karşı duyarlı olduğu görülmüştür. (GTG)5-PCR yöntemiyle yapılan genotiplendirme sonucunda, tüm izolatların farklı tiplere sahip olduğu ve genetik benzerliğin %32 ile %98 arasında değiştiği tespit edilmiştir. Elde edilen bulgular, S. paucimobilis’in potansiyel çevresel mastitis etkenleri arasında değerlendirilmesi gerektiğini göstermekte olup, direnç gelişiminin önlenmesi amacıyla ileri tanı yöntemleri ve etkili antimikrobiyal yönetim stratejilerinin gerekliliğini ortaya koymaktadır.

Keywords

• Antibiyotik dirençliliği
• Genotiplendirme
• Mastitis
• Sphingomonas paucimobilis

References

1. Aghamohammadi, M., Haine, D., Kelton, D. F., Barkema, H. W. Hogeveen, H., & Dufour, S. (2018). Herd-level mastitis-associated costs on Canadian dairy farms. Front Vet Sci, 5, 100. https://doi.org/10.3389/fvets.2018.00100
2. Babacan, O. (2024). Methicillin and vancomycin resistance profiles of Staphylococcus aureus isolated from bovine mastitic milk samples. J Appl Biol Sci, 18(3), 331-341.
3. Bardhan, D. (2013). Estimates of economic losses due to clinical mastitis in organized dairy farms. Indian J Dairy Sci, 13(1), 8732. https://doi.org/10.21887/ijvsbt.v13i01.8732
4. Blowey, R., Edmondson, P. (2010). Milking machines and mastitis. In Mastitis control in dairy herds (2nd ed., pp. 60-94). CAB eBooks.
5. Bradley, A. J. (2002). Bovine mastitis: An evolving disease. Vet J, 164(2), 116-128. https://doi.org/10.1053/tvjl.2002.0724
6. Bray, D. R., Shearer, J. K. (1994). Milking machine and mastitis control handbook. University of Florida, Cooperative Extension Service.
7. Cengiz, M., Aydoğan, A., Çelik, O., Boynukara, B. (2014). Identificationof Sphingomonas paucimobilis isolated from bovine mastitis cases by PCR. Etlik Vet Mikrobiyol Derg, 25(1), 19-25.
8. Cengiz, S., Seyitoğlu, S., Kılıç Altun, S., Dinler, U. (2015). Detection of Sphingomonas paucimobilis infections in domestic animals by VITEK® Compaq 2 and polymerase chain reaction. Arch Med Vet, 47, 117-119. https://doi.org/10.4067/S0301-732X2015000100020

9. Clinical and Laboratory Standards Institute. (2006). Performance standards for antimicrobial susceptibility testing (Approved standard M100-S16). CLSI.
10. Das, D., Panda, S. K., Jena, B., Sahoo, A. K. (2018). Economic impact of subclinical and clinical mastitis in Odisha, India. Int J Curr Microbiol Appl Sci, 7(03), 3651-3654.
11. De Vliegher, S., Fox, L. K., Piepers, S., McDougall, S., Barkema, H. W. (2012). Mastitis in dairy heifers: Nature of the disease, potential impact, prevention, and control. J Dairy Sci, 95(3), 1025-1040. https://doi.org/10.3168/jds.2011-4542
12. Dego, O. K. (2020). Bovine mastitis: Risk factors, prevention, and control. Livest Res Int, 8(2), 56.
13. Halasa, T., Huijps, K., Østerås, O., Hogeveen, H. (2007). Economic effects of bovine mastitis and mastitis management: A review. Vet Q, 29(1), 18-31. https://doi.org/10.1080/01652176.2007.9695224
14. Hsueh, P. R., Teng, L. J., Yang, P. C., Chen, Y. C., Pan, H. L., & Luh, K. T. (1998). Nosocomial infections caused by Sphingomonas paucimobilis: Clinical features and microbiological characteristics. Clin Infect Dis, 26(3), 676-681. https://doi.org/10.1086/513939
15. Huang, Y. T., Zong, Z., Sun, W. H., Zhang, X. (2013). Antimicrobial susceptibility and molecular epidemiology of Sphingomonas paucimobilis. J Med Microbiol, 62, 1749-1754. https://doi.org/10.1099/jmm.0.057571-0
16. Izquierdo, A. C., Liera, J. G., Cervantes, R. E., Castro, J. I., Mancera, E. V., Crispin, R. H. (2017). Production of milk and bovine mastitis. J Adv Dairy Res, 5(2), 2.
17. Kenar, B., Ekin, İ. H., Sarımehmetoğlu, B., Güler, L. (2019). Isolation of Sphingomonas paucimobilis from bovine mastitis milk and determination of antimicrobial susceptibility. Kafkas Univ Vet Fak Derg, 25(1), 123-127.
18. Koskinen, M. T., Wellenberg, G. J., Sampimon, O. C., Holopainen, J., Rothkamp, A., Salmikivi, L., & Pyörälä, S. (2010). Field comparison of real-time PCR and bacterial culture for identification of bovine mastitis pathogens. J Dairy Sci, 93(12), 5707-5715. https://doi.org/10.3168/jds.2010-3132
19. Köstereli, S., Onuk, E. E. (2019). Yersinia ruckeri izolatlarının genotiplendirilmesinde PCR tabanlı DNA fingerprinting tekniklerinin karşılaştırmalı analizi. Acta Aquatica Turcica, 15(3), 262-271.
20. Kuang, Y., Tani, K., Synnott, A. J., Ohshima, K., Higuchi, T., Nagahata, H., & Tanji, Y. (2009). Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR-DGGE method. Biochem Eng J, 45(1), 76-81. https://doi.org/10.1016/j.bej.2009.02.007
21. Kuehn, J. S., Gorden, P. J., Munro, D., Rong, R., Dong, Q., Plummer, P. J., & Phillips, G. J. (2013). Bacterial community profiling of milk samples as a means to understand culture-negative bovine clinical mastitis. PLoS ONE, 8(4), e61959. https://doi.org/10.1371/journal.pone.0061959
22. Kurt, A., Eşki, F. (2021). Pathogen isolation and antibiogram analysis in dairy cows with clinical mastitis in Adana region, Turkey. Van Vet J, 32(2), 73-78. https://doi.org/10.36483/vanvetj.907535
23. Maragakis, L. L., Chaiwarith, R., Srinivasan, A., Torriani, F. J., Avdic, E., Lee, A., & Perl, T. M. (2009). Sphingomonas paucimobilis bloodstream infections associated with contaminated intravenous fentanyl. Emerg Infect Dis, 15(1), 12-18. https://doi.org/10.3201/eid1501.081062
24. Nuhay, Ç. (2025). Investigation of the presence and antimicrobial susceptibilities of Corynebacterium pseudotuberculosis in subclinical mastitis in cattle raised in the Aegean region. J Anatolian Environ Anim Sci, 10(2), 202-207. https://doi.org/10.35229/jaes.1629149
25. Nuhay, Ç., Deniz, D. A., Özavcı, V. (2025). Determination of antibiotic resistance profiles of Bacillus cereus from subclinical mastitis of cattle in İzmir province. J Adv VetBio Sci Tech, 10(2), 88-91. https://doi.org/10.31797/vetbio.1620730
26. Roque, K., Gyeong-Dong, L., Ji-Hoon, J., Kyung-Min, S., Eun-Seob, S., Ravi, G., Chang-Yul, K., & Hyoung-Ah, K. (2016). Epizootiological characteristics of viable bacteria and fungi in indoor air from porcine, chicken, or bovine husbandry confinement buildings. J Vet Sci, 17(4), 531-541. https://doi.org/10.4142/jvs.2016.17.4.531
27. Ruegg, P. L. (2017). A 100-year review: Mastitis detection, management, and prevention. J Dairy Sci, 100(12), 10381-10397. https://doi.org/10.3168/jds.2017-13023
28. Ryan, M. P., Adley, C. C. (2010). Sphingomonas paucimobilis: A persistent Gram-negative nosocomial infectious organism. J Hosp Infect, 75(3), 153-157. https://doi.org/10.1016/j.jhin.2010.03.007
29. Savaşan, S., Göksoy, E. O. (2018). Taze peynirlerden izole edilen Escherichia coli suşlarının genotiplendirilmesi. Etlik Vet Mikrobiyol Derg, 29(2), 127-135.
30. Seegers, H., Fourichon, C., Beaudeau, F. (2003). Production effects related to mastitis and mastitis economics in dairy cattle herds. Vet Res, 34(5), 475-491. https://doi.org/10.1051/vetres:2003027
31. Sinha, M. K., Thombare, N. N., Mondal, B. (2014). Subclinical mastitis in dairy animals: Incidence, economics, and predisposing factors. Scientifica, 2014, 265476. https://doi.org/10.1155/2014/265476
32. Tada, Y., Inoue, T. (2000). Use of oligotrophic bacteria for the biological monitoring of heavy metals. J Appl Microbiol, 88(1), 154-160. https://doi.org/10.1046/j.1365-2672.2000.00933.x
33. Tokajian, S., Bitew, M., Hashwa, F., Fares, M. (2008). Identification of Sphingomonas paucimobilis by molecular methods and antimicrobial susceptibility. J Med Microbiol, 57(11), 1340-1346. https://doi.org/10.1099/jmm.0.2008/002439-0
34. Yaman, Ş., Nuhay, Ç., Fındık, A., Çiftci, A. (2025). Genotyping of Corynebacterium pseudotuberculosis isolates using PCR-based DNA fingerprinting methods. J Microbiol Methods, 232, 107122. https://doi.org/10.1016/j.mimet.2025.107122