Antibiotic Resistance and Virulence Gene Profiles in Staphylococci Isolated from Cattle with Mastitis

Year 2021, Volume: 6 Issue: 3, 395 - 402, 28.09.2021

Mehmet Onur Gökdağ , Alper Çiftci

https://doi.org/10.35229/jaes.954156

Abstract

Staphylococcus spp. are the important bacterial agents of subclinical and clinical mastitis cases. This study was aimed to determine the vancomycin resistances, antibacterial resistance profiles, some virulence genes, and pheno- and genotyping of staphylococci from mastitis. For this aim, 121 staphylococcal isolates were analyzed. The identifications of isolates were confirmed with PCR for being Staphylococcus spp. and Staphylococcus aureus. The antibiotic resistance patterns were determined by Kirby bauer Disc Diffusion Tests and according to the resistance profiles, the isolates were antibiotyped. The vancomycin resistance genes were determined by PCR for investigating the vanA, vanH, vanR, vanS, vanZ, vanY and vanX genes. The vancomycin resistant isolates were genotyped with RAPD-PCR. The nuc gene was detected in 86 of 121 staphylococcal isolates examined and named as Staphylococcus aureus. The remaining 35 isolates were defined as Staphylococcus spp. S. aureus isolates were found to be resistant to penicillin G, amoxicillin/clavulanic acid, oxacillin, tetracycline, cefaperazone, teicoplanin, vancomycin and trimethoprim-sulfomethaxazole at 50% (43/86), 40.7% (35/86), 34.9% (30/86), 23.3% (20/86), 22.1% (19/86), 18.6% (16/86) 10.5% (9/86) and 8.1% (7/86) respectively. On the other hand, 4 (11.4%) of 35 Staphylococcus spp. isolates were resistant to penicillin G, 3 (8.6%) to amoxicillin/clavulanic acid, 2 (5.7%) to trimethoprim-sulfometaxazole, 1 (2.9%) to oxacillin, cefaperazone, teicoplanin, while all isolates were sensitive to vancomycin and tetracycline. Of the 9 S. aureus isolates that were phenotypically resistant to vancomycin, vanA gene was detected in 2 and vanR gene in 3 isolates. Multiple resistances to three or more antibiotics were determined in 42 of 86 S. aureus isolates. In addition, coa gene was detected in 61 (70.9%) of 86 S. aureus isolates. 10 different gene polymorphisms were detected in coa gene positive isolates. While the spa gene was determined in all S. aureus isolates, it was revealed that they had 4 spa gene polymorphisms. Nine different genotypes with a similarity between 51-75% were detected in the genotyping of vancomycin resistant 9 isolates. In conclusion, multiple antibiotic resistance rates in S. aureus isolates investigated were found to be important for mastitis treatment. The results obtained from this study show that milk and dairy products containing these factors pose a public health risk due to the determination of vancomycin resistance in mastitis-derived Staphylococcus strains.

Keywords

Antibiotic resistance, bovine, coa, mastitis, RAPD-PCR, spa, Staphylococcus

Supporting Institution

Scientific Research Projects Commission of Ondokuz Mayis University

Project Number

PYO.VET.1904.14.005

Mastitisli Sığırlardan İzole Edilen Stafilokoklarda Antibiyotik Direnci ve Virülens Gen Profilleri

Abstract

Staphylococcus spp. subklinik ve klinik mastitis olgularının önemli bakteriyel etkenleridir. Bu çalışmanın amacı, mastitis kaynaklı stafilokokların vankomisin dirençlerini, antibakteriyel direnç profillerini, bazı virülans genlerini ve feno- ve genotiplendirmesini belirlemektir. Bu amaçla 121 stafilokok izolatının analizi yapılmıştır. İzolatların Staphylococcus spp. ve Staphylococcus aureus olmak üzere identifikasyonları PCR ile ve antibiyotik direnç paternleri de Kirby Bauer Disk Difüzyon Testleri ile belirlendi. İzolatlar direnç profillerine göre antibiyotiplendirildi. VanA, vanH, vanR, vanS, vanZ, vanY ve vanX genlerinin PCR ile araştırılması sonucunda vankomisin direnç genleri belirlendi. Vankomisine dirençli izolatlar, RAPD-PCR ile genotiplendirildi. İncelenen 121 stafilokok izolatının 86'sında nuc geni saptandı ve Staphylococcus aureus olarak identifiye edildi. S. aureus izolatlarının penisilin G, amoksisilin/klavulanik asit, okzasilin, tetrasiklin, sefaperazon, teikoplanin, vankomisin ve trimetoprim-sulfometaksazole sırasıyla %50 (43/86), %40.7 (35/86), %34,9 (30/86), %23,3 (20/86), %22.1 (19/86), %18,6 (16/86) %10,5 (9/86) ve %8,1 (7/86) oranında dirençli bulundu. 35 adet Staphylococcus spp.'nin 4'ü (%11,4) penisilin G'ye, 3'ü (%8,6) amoksisilin/klavulanik aside, 2'si (%5,7) trimetoprim-sülfometaksazole, 1'i (%2,9) oksasilin, sefaperazon, teikoplanine direnç gösterirken, tüm izolatlar vankomisin ve tetrasikline duyarlıydı. Vankomisine fenotipik olarak dirençli olan 9 S. aureus izolatından 2'sinde vanA, 3'ünde vanR geni saptandı. 86 S. aureus izolatının 42'sinde üç veya daha fazla antibiyotiğe karşı çoklu direnç belirlendi. Ayrıca 86 S. aureus izolatının 61'inde (%70.9) coa geni tespit edildi. coa geni pozitif izolatlarda 10 farklı gen polimorfizmi tespit edildi. Tüm S. aureus izolatlarında spa geni belirlenirken, 4 spa gen polimorfizmi görüldü. Vankomisine dirençli 9 izolatın genotiplendirilmesinde %51-75 arasında benzerlik gösteren dokuz farklı genotip tespit edildi. Sonuç olarak, araştırılan S. aureus izolatlarında çoklu antibiyotik direnç oranları mastitis tedavisi için önemli bulunmuştur. Bu çalışmadan elde edilen sonuçlar, bu faktörleri içeren süt ve süt ürünlerinin mastitis kaynaklı Staphylococcus suşlarında vankomisin direncinin belirlenmesi nedeniyle halk sağlığı açısından risk oluşturduğunu göstermektedir.

Keywords

Antibiyotik direnci, coa, inek, mastitis, RAPD-PCR, spa, Staphylococcus

Project Number

PYO.VET.1904.14.005

References

• Antok, F.I., Mayrhofer, R., Marbach, H., Masengesho, J.C., Keinprecht, H., Nyirimbuga, V., Fischer, O., Lepuschitz, S., Ruppitsch, W., Ehling-Schulz, M., Feßler, A.T., Schwarz, S., Monecke, S., Ehricht, R., Grunert, T., Spergser, J. & Loncaric, I. (2020). Characterization of antibiotic and biocide resistance genes and virulence factors of Staphylococcus species associated with bovine mastitis in Rwanda. Antibiotics (Basel), 9, 1.
• Carter, P.E., Begbie, K., & Thomson, F.M. (2003). Coagulase gene variants associated with distinct populations of Staphylococcus aureus. Epidemiology and Infection, 130, 207–219.
• Centers for Disease Control and Prevention (CDCP). (1997). Staphylococcus aureus with reduced susceptibility to vancomycin-United States. MMWR, 46, 765–766.
• Ciftci, A., Findik, A., Onuk, E. E. & Savasan, S. (2009). Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis. Brazilian Journal of Microbiology, 40, 254−261.
• Clinical and Laboratory Standards Institute (CLSI). (2018). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, Approved Standard; CLSI Document VET08, 4th ed. Clinical and Laboratory Standards Institute, Wayne, PA, USA.
• DaSilva, E.R., Boechat, J.U.D. & DaSilva, N. (2006). Coagulase gene polymorphism of Staphylococcus aureus isolated from goat mastitis in Brazilian dairy herds. Letters in Applied Microbiology, 42, 30–34.
• de Jong, A., El Garch, F., Simjee, S., Moyaert, H., Rose, M., Youala, M. & Siegwart, E. (2018). Monitoring of antimicrobial susceptibility of udder pathogens recovered from cases of clinical mastitis in dairy cows across Europe: VetPath results. Veterinary Microbiology, 213, 73–81.
• Dezfulian, A., Aslani, M.M., Oskoui, M., Farrokh, P., Azimirad, M., Dabiri, H., Salehian, M.T. & Zali, MR. (2012). Identification and characterization of a high vancomycin-resistant Staphylococcus aureus harboring vanA gene cluster ısolated from diabetic foot ulcer. Iranian Journal of Basic Medical Science, 15, 803–806.
• Findik, A., Ica, T., Onuk, E.E., Percin, D., Kevenk, T.O. & Ciftci, A. (2011). Molecular typing and cdt genes prevalence of Campylobacter jejuni isolates from various sources. Tropical Animal Health and Production, 43, 711–719.
• Frenay, H.M.E., Theelen, J.P.G. & Schouls, LM. (1994). Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. Journal of Clinical Microbiology, 32, 846–847.
• Frenay, H.M.E., Bunschoten, A.E. & Schouls, L.M. (1996). Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. European Journal of Clinical Microbiology and Infectious Diseases, 15, 60–64.
• Goh SH, Byrne SK, Zhang JL, & Chow, AW. (1992). Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. Journal of Clinical Microbiology, 30, 1642–1645.
• Guimarães, F.F., Manzi, M.P., Joaquim, S.F., Richini-Pereira, V.B. & Langoni, H. (2017). Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd. Journal of Dairy Science, 100, 726–730.
• Hartstein, A.I. & Mulligan, M.E. (1986). Methicillin-resistant Staphylococcus aureus. In: Glen Mayhall C, ed. Hospital epidemiology and infection control. Maryland: Williams and Wilkins, 290–306.
• Haubert, L., Kroning, I.S., Iglesia, M.A., & da Silva, W.P. (2017). First report of the Staphylococcus aureus isolate from subclinical bovine mastitis in the South of Brazil harboring resistance gene dfrG and transposon family Tn916-1545. Microbial Pathogenesis, 113, 242–247.
• Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T. & Tenover, F.C. (1997). Methicillin- resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy, 40, 135–136.
• Hookey, J.V., Richardson, J.F. & Cookson, B.D. (1998). Molecular typing of Staphylococcus aureus based on PCR Restriction Fragment Length Polymorphism and DNA sequence analysis of the coagulase gene. Journal of Clinical Microbiology, 36, 1083–1089.
• Jagielski, T., Puacz, E., Lisowski, A., Siedlecki, P., Dudziak, W., Miedzobrodzki, J. & Krukowsk, H. (2014). Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland. Journal of Dairy Science, 97, 6122–6128.
• Jevons, M.P. (1961). Celbenin-resistant staphylococci. British Medical Journal, 1, 124–125.
• Kaatz, G., Seo, S.M., Dorman, N.J. & Lerner, S.A. (1990). Emergence of teicoplanin resistance during therapy of Staphylococcus aureus endocarditis. Journal of Infectious Diseases, 162, 103–108.
• Kalmus, P., Aasmäe, B., Kärssin, A., Orro, T. & Kask, K. (2011). Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia. Acta Veterinaria Scandinavica, 53, 4.
• Kapur, V., Sischo, W.M. & Greer, R.S. (1995). Molecular population genetic analysis of Staphylococcus aureus recovered from cows. Journal of Clinical Microbiology, 33, 376–380.
• Karahan, M. & Çetinkaya, B. (2007). Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey. The Veterinary Journal, 174, 428–431.
• Kot, B., Szweda, P., Frankowska-Maciejewska, A., Piechota, M. & Wolska, K. (2016). Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland. Journal of Dairy Research, 83, 228–235.
• Momtaz, H., Rahimi, E. & Tajbakhsh, E. (2010). Detection of some virulence factors in Staphylococcus aureus isolated from clinical and subclinical bovine mastitis in Iran. African Journal of Biotechnology, 25, 3753–3758.
• Monistero, V., Barberio, A., Biscarini, F., Cremonesi, P., Castiglioni, B., Graber, H.U., Bottini, E., Ceballos-Marquez, A., Kroemker, V., Petzer, I.M., Pollera, C., Santisteban, C., Dos Santos, M.V., Bronzo, V., Piccinini, R., Re, G., Cocchi, M. & Moroni, P. (2020). Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries. Journal of Dairy Science, 103, 3431–3446.
• Oliveira, C.J.B., Tiao, N., de Sousa, F.G.C., de Moura, J.F.P., Santos Filho, L. & Gebreyes, W.A. (2016). Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types. Zoonoses and Public Health, 63, 97–105.
• Pichette-Jolette, S., Millette, G., Demontier, E., Bran-Barrera, D., Cyrenne, M., Ster, C., Haine, D., Keefe, G., Malouin, F. & Roy, JP. (2019). Partial prediction of the duration and the clinical status of Staphylococcus aureus bovine intramammary infections based on the phenotypic and genotypic analysis of isolates. Veterinary Microbiology, 228, 188–195.
• Qu, Y., Zhao, H., Nobrega, D.B., Cobo, E.R., Han, B., Zhao, Z., Li, S., Li, M., Barkema, H.W. & Gao, J. (2019). Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis. Journal of Dairy Science, 102, 1571–1583.
• Sabat, A., Malachowa, N., Miedzobrodzki, J. & Hryniewicz, W. (2006). Comparison of PCR-based methods for typing Staphylococcus aureus isolates. Journal of Clinical Microbiology, 44, 3804–3807.
• Schabauer, A., Pinior, B., Gruber, C.M., Firth, C.L., Käsbohrer, A., Wagner, M., Rychli, K. & Obritzhauser, W. (2018). The relationship between clinical signs and microbiological species, spa type, and antimicrobial resistance in bovine mastitis cases in Austria. Veterinary Microbiology, 227, 52–60.
• Schmitz, F.J. & Jones, M.E. (1997). Antibiotics for treatment of infections caused by MRSA and elimination of MRSA carriage. What are the choices? International Journal of Antimicrobial Agents, 9, 1–19.
• Schwalbe, R., Stapleton, J.T. & Gilligan, P.H. (1987). Emergence of vancomycin resistance in coagulase negative staphylococci. New England Journal of Medicine, 316, 927–931.
• Schwarz, S., Feßler, A.T., Loncaric, I., Wu, C., Kadlec, K., Wang, Y. & Shen, J. (2018). Antimicrobial resistance among staphylococci of animal origin. Microbiol Spectrum, 6, ARBA-0010-2017.
• Sezener, M.G., Fındık, A., Erguden, V.E., Akgoz, S., Gulhan, T. & Ciftci, A. (2019). The determination of antibiotic resistances and some virulence genes of Staphylococcus aureus isolated from bovine mastitis. Journal of Anatolian Environmental and Animal Sciences, 4, 182–187.
• Shi, D., Hao, Y., Zhang, A., Wulan, B. & Fan, X. (2010). Antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis in China. Transboundary Emerging Diseases, 57, 221–224.
• Wang, D., Zhang, L., Zhou, X., He, Y., Yong, C., Shen, M., Szenci, O. & Han, B. (2016). Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China. Journal of Dairy Science, 99, 9560–9569.
• Xu, J., Tan, X., Zhang, X., Xia, X. & Sun, H. (2015). The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd. Microbial Pathogenesis, 88, 29–38.