This study investigated 960 Slovak and Czech spotted cattle from four different conventional (non-organic) dairy herds located in Eastern Slovakia and Czechia during early lactation (14-100 days after calving). Dairy cows were examined clinically; milk from fore-stripping of each udder quarter was subjected to sensory examination and assessed by the California mastitis test (CMT), and laboratory analyses of bacterial pathogens in milk, including virulence factors, were conducted. Positive CMT scores (1-3) for one or more quarters were detected in 271 (28.2%) of the examined animals. Out of 230 infected milk samples, representing 24.0% of all dairy cows, staphylococci (59.1% of positive findings) were the most commonly isolated organisms, followed by E. coli (11.3%), streptococci Str. uberis (9.1%) and Str. agalactiae (3.4%), and enterococci (6.1%). From 136 isolates of S. aureus (38 isolates) and non-aureus staphylococci (NAS; 98 isolates), virulence factors and their resistance to 14 antimicrobials were detected using the disk diffusion method, with PCR detection of the methicillin resistance gene, mecA. An increased incidence of clinical and chronic forms of mastitis has been reported in mastitic cows in which staphylococci, especially S. aureus and NAS (S. chromogenes, S. warneri, and S. xylosus), have been detected and compared to other isolated udder pathogens. From those species, S. aureus and isolates of NAS mentioned above showed multiple virulence factors that are more likely to hydrolyze DNA, hemolysis, produce gelatinase and biofilm, and have multi-drug resistance as compared to other less virulent staphylococci. Generally, the isolated staphylococci showed 77.2% resistance to one or more antimicrobials, in particular to aminoglycosides, β-lactams, macrolides, or cephalosporins. Isolates that showed the ability to form a biofilm were more resistant to more than one antimicrobial than isolates without biofilm production. Multi-drug resistance to three or more antimicrobial classes was recorded in 16 isolates (11.7%), and the presence of the mecA gene was also confirmed in two isolates of S. aureus and two species of NAS.

Department of Animal Origin Food and Gastronomic Sciences University of Veterinary Sciences Brno Palackého Tř 1946 1 CZ 61242 Brno Czech Republic

Department of Animal Physiology and Biostructure Wrocław University of Environmental and Life Sciences Norwida 31 50 375 Wroclaw Poland

Department of Biology and Physiology University of Veterinary Medicine and Pharmacy Komenského 73 04181 Košice Slovakia

Department of Food Hygiene Technology and Safety University of Veterinary Medicine and Pharmacy Komenskéhgo 73 04181 Košice Slovakia

Department of Nutrition and Animal Husbandry University of Veterinary Medicine and Pharmacy Komenského 73 04181 Košice Slovakia

Department of Physiology and Biochemistry University of Veterinary Medicine Budapest István Utca 2 H 1078 Budapest Hungary

Department of the Environment Veterinary Legislation and Economy University of Veterinary Medicine and Pharmacy Komenského 73 04181 Košice Slovakia

Zobrazit více v PubMed

Augère-Granier M.L. The EU Dairy Sector: Main Features, Challenges and Prospects. Briefing EU. 2018. [(accessed on 17 December 2018)]. p. 12. Available online: https://www.europarl.europa.eu/thinktank/en/document/EPRS_BRI(2018)630345.

Tančin V., Mikláš Š., Čobirka M., Uhrinčať M., Mačuhová L. Factors affecting raw milk quality of dairy cows under practical conditions. Potravin. Slovak J. Food Sci. 2020;14:744–749. doi: 10.5219/1336. DOI

Cobirka M., Tančin V., Slama P. Epidemiology and Classification of Mastitis. Animals. 2020;10:2212. doi: 10.3390/ani10122212. PubMed DOI PMC

Dufour S., Labrie J., Jacques M. The Mastitis Pathogens Culture Collection. Microbiol. Resour. Announc. 2019;8:e00133-19. doi: 10.1128/MRA.00133-19. PubMed DOI PMC

Zigo F., Vasil’ M., Ondrašovičová S., Výrostková J., Bujok J., Pecka-Kielb E. Maintaining Optimal Mammary Gland Health and Prevention of Mastitis. Front. Vet. Sci. 2021;8:607311. doi: 10.3389/fvets.2021.607311. PubMed DOI PMC

Zigo F., Farkašová Z., Lacková Z., Výrostková J., Regecová I., Vargová M., Sasáková N. Food Safety and Food Quality (Slovak) SPU; Nitra, Slovakia: 2021. Occurrence of some pathogenity factors in Staphylococci isolated from mastitic dairy cows; pp. 190–196. Proceedings of the Scientific Papers. DOI

Singha S., Koop G., Persson Y., Hossain D., Scanlon L., Derks M., Hoque M.A., Rahman M.M. Incidence, Etiology, and Risk Factors of Clinical Mastitis in Dairy Cows under Semi-Tropical Circumstances in Chattogram, Bangladesh. Animals. 2021;11:2255. doi: 10.3390/ani11082255. PubMed DOI PMC

Monistero V., Graber H.U., Pollera C., Cremonesi P., Castiglioni B., Bottini E., Ceballos-Marquez A., Lasso-Rojas L., Kroemker V., Wente N., et al. Staphylococcus aureus Isolates from Bovine Mastitis in Eight Countries: Genotypes, Detection of Genes Encoding Different Toxins and Other Virulence Genes. Toxins. 2018;10:247. doi: 10.3390/toxins10060247. PubMed DOI PMC

Condas L.A.Z., De Buck J., Nobrega D.B., Domonique A.C., Roy J.P., Greg P.K., De Vries T.J., Middleton J.R., Dufour S., Barkema H.W. Distribution of non-aureus staphylococci species in udder quarters with low and high somatic cell count, and clinical mastitis. J. Dairy Sci. 2017;100:5613–5627. doi: 10.3168/jds.2016-12479. PubMed DOI

El-Diasty M., Talaat H., Atwa S., Elbaz E., Eissa M. Occurrence of Coagulase-negative Staphylococcal mastitis in dairy cows. Mansoura Vet. Med. J. 2019;23:35–39. doi: 10.21608/mvmj.2019.23.207. DOI

Ferroni L., Lovito C., Scoccia E., Dalmonte G., Sargenti M., Pezzotti G., Maresca C., Forte C., Magistrali C.F. Antibiotic Consumption on Dairy and Beef Cattle Farms of Central Italy Based on Paper Registers. Antibiotics. 2020;9:273. doi: 10.3390/antibiotics9050273. PubMed DOI PMC

Holko I., Tančin V., Vršková M., Tvarožková K. Prevalence and antimicrobial susceptibility of udder pathogens isolated from dairy cows in Slovakia. J. Dairy Res. 2019;86:436–439. doi: 10.1017/S0022029919000694. PubMed DOI

Idriss S.E., Foltys V., Tančin V., Kirchnerová K., Zaujec K. Mastitis pathogens in milk of dairy cows in Slovakia. Slovak J. Anim. Sci. 2013;46:115–119.

Nascimento J.S., Fugundes P.C., Brito A.V., Dos Santos K.R., Bastos M.C. Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis. Vet. Microbiol. 2005;106:61–71. doi: 10.1016/j.vetmic.2004.10.014. PubMed DOI

De Buck J., Ha V., Naushad S., Nobrega D.B., Luby C., Middleton J.R., De Vliegher S., Barkema H.W. Non-aureus Staphylococci and Bovine Udder Health: Current Understanding and Knowledge Gaps. Front. Vet. Sci. 2021;8:658031. doi: 10.3389/fvets.2021.658031. PubMed DOI PMC

Fergestad M.E., Visscher A., De L’Abee-Lund T., Ngassam Tchamba C., Mainil J.G., Thiry D., De Vliegher S., Wasteson Y. Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples. J. Dairy Sci. 2021;104:10250–10267. doi: 10.3168/jds.2020-19988. PubMed DOI

Grinberg A., Hittman A., Leyland M., Rogers L., Le Quesne B. Epidemiological and molecular evidence of a monophyletic infection with Staphylococcus aureus causing a purulent dermatitis in a dairy farmer and multiple cases of mastitis in his cows. Epidemiol. Infect. 2004;132:507–513. doi: 10.1017/S0950268804002079. PubMed DOI PMC

Vasiľ M., Elečko J., Zigo F., Farkašová Z. Occurrence of some pathogenity factors in coagulase negative staphylococci isolated from mastitis milk in dairy cows. Potravin. Slovak J. Food Sci. 2012;6:60–63. doi: 10.5219/186. DOI

Regecová I., Výrostková J., Zigo F., Gregová G., Kováčová M. Detection of Antimicrobial Resistance of Bacteria Staphylococcus chromogenes Isolated from Sheep’s Milk and Cheese. Antibiotics. 2021;10:570. doi: 10.3390/antibiotics10050570. PubMed DOI PMC

Haveri M., Roslöf A., Pyörälä S. Virulence genes of bovine Staphylococcus aureus from persistent and nonpersistent intramammary infections with different clinical characteristics. J. Appl. Microbiol. 2007;103:993–1000. doi: 10.1111/j.1365-2672.2007.03356.x. PubMed DOI

National Research Council (NRC) Nutrient Requirements of Dairy Cattle: Seventh Revised Edition. The National Academies Press; Washington, DC, USA: 2001. p. 405. DOI

Tančin V. Somatic cell counts in milk of dairy cows under practical conditions. Slovak J. Anim. Sci. 2013;46:31–34. doi: 10.5219/767. DOI

Adkins P.R.F., Middleton J.R., Fox L.K., Pighetti G., Petersson-Wolfe C. Laboratory Handbook on Bovine Mastitis. National Mastitis Council; New Prague, MN, USA: 2017. p. 148.

Gregova G., Kmet V. Antibiotic resistance and virulence of Escherichia coli strains isolated from animal rendering plant. Sci. Rep. 2020;10:17108. doi: 10.1038/s41598-020-72851-5. PubMed DOI PMC

El-Aziz A.N.K., Ammar A.M., El Damaty H.M., Abd Elkader R.A., Saad H.A., El-Kazzaz W., Khalifa E. Environmental Streptococcus uberis Associated with Clinical Mastitis in Dairy Cows: Virulence Traits, Antimicrobial and Biocide Resistance, and Epidemiological Typing. Animals. 2021;11:1849. doi: 10.3390/ani11071849. PubMed DOI PMC

Hiko A. DNase-cross-Coagulase test and antimicrobial resistance test on Staphylococcus along beef abattoir line in Addis Ababa Ethiopia. Ethiop. Vet. J. 2019;23:90–110. doi: 10.4314/evj.v23i1.7. DOI

Vasil’ M., Farkasova Z., Elecko J., Illek J., Zigo F. Comparison of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis strains isolated from sheep milk using three diagnostic methods. Pol. J. Vet. Sci. 2017;20:795–801. doi: 10.1515/pjvs-2017-0100. PubMed DOI

Moraveji Z., Tabatabaei M., Shirzad Aski H., Khoshbakht R. Characterization of hemolysins of Staphylococcus strains isolated from human and bovine, southern Iran. Iran. J. Vet. Res. 2014;15:326–330. PubMed PMC

Performance Standards for Antimicrobial Disk Susceptibility Tests, Thirteenth Informational Supplement. Clinical and Laboratory Standards Institute; Wayne, PA, USA: 2018.

Performance Standards for Antimicrobial Susceptibility Testing, Thirty–First Informational Supplement. Clinical and Laboratory Standards Institute; Wayne, PA, USA: 2021.

Hein I., Jorgensen H.J., Loncarevic S., Wagner M. Quantification of Staphylococcus aureus in unpasteurised bovine and caprine milk by real-time PCR. Res. Microbiol. 2005;156:554–563. doi: 10.1016/j.resmic.2005.01.003. PubMed DOI

Poulsen A.B., Skov R., Pallesen L.V. Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit. J. Antimicrob. Chemother. 2003;51:419–421. doi: 10.1093/jac/dkg084. PubMed DOI

Nitz J., Wente N., Zhang Y., Klocke D., tho Seeth M., Krömker V. Dry Period or Early Lactation—Time of Onset and Associated Risk Factors for Intramammary Infections in Dairy Cows. Pathogens. 2021;10:224. doi: 10.3390/pathogens10020224. PubMed DOI PMC

Persson Waller K., Bengtsson B., Lindberg A., Nyman A., Ericsson Unnerstad H. Incidence of mastitis and bacterial findings at clinical mastitis in Swedish primiparous cows-influence of breed and stage of lactation. Vet. Microbiol. 2009;134:89–94. doi: 10.1016/j.vetmic.2008.09.004. PubMed DOI

Silva A.C., Laven R., Benites N.R. Risk Factors Associated with Mastitis in Smallholder Dairy Farms in Southeast Brazil. Animals. 2021;11:2089. doi: 10.3390/ani11072089. PubMed DOI PMC

Rahman M., Bhuiyan M., Kamal M., Shamsuddin M. Prevalence and risk factors of mastitis in dairy cows. Bangladesh Vet. 2010;26:54–60. doi: 10.3329/bvet.v26i2.4951. DOI

Wenz J.R., Barrington G.M., Garry F.B. Bacteremia associated with naturally occuring acute coliform mastitis in dairy cows. J. Am. Vet. Med. Assoc. 2001;219:976–981. doi: 10.2460/javma.2001.219.976. PubMed DOI

Pyörälä S., Taponen S. Coagulase-negative staphylococci—Emerging mastitis pathogens. Vet. Microbiol. 2009;134:3–8. doi: 10.1016/j.vetmic.2008.09.015. PubMed DOI

Zigo F., Elecko J., Vasil M., Ondrasovicova S., Farkasova Z., Malova J., Takac L., Zigova M., Bujok J., Pecka-Kielb E., et al. The occurrence of mastitis and its effect on the milk malondialdehyde concentrations and blood enzymatic antioxidants in dairy cows. Vet. Med. 2019;64:423–432. doi: 10.17221/67/2019-VETMED. DOI

Supre K., Haesebrouck F., Zadoks R.N., Vaneechoutte M., Piepers S., De Vliegher S. Some coagulase-negative Staphylococcus species affect udder health more than others. J. Dairy Sci. 2011;94:2329–2340. doi: 10.3168/jds.2010-3741. PubMed DOI

Hosseinzadeh S., Saei H.D. Staphylococcal species associated with bovine mastitis in the North West of Iran: Emerging of coagulase-negative staphylococci. Int. J. Vet. Sci. Med. 2014;2:27–34. doi: 10.1016/j.ijvsm.2014.02.001. DOI

Fredheim E.G.A., Klingenberg C., Rodhe H., Frankenberger S., Gaustad P., Flaegstad T., Sollid J.E. Biofilm formation by Staphylococcus haemolyticus. J. Clin. Microbiol. 2009;47:1172–1180. doi: 10.1128/JCM.01891-08. PubMed DOI PMC

Pérez V.K.C., Da Costa G.M., Sá Guimarães A., Heinemann M.B., Lage A.P., Dorneles E.M.S. Relationship between virulence factors and antimicrobial resistance in Staphylococcus aureus from bovine mastitis. J. Glob. Antimicrob. Res. 2020;22:792–802. doi: 10.1016/j.jgar.2020.06.010. PubMed DOI

Melchior M.B., Van Osch M.H.J., Lam T.J.G.M., Vernooij J.C.M., Gaastra W., Fink-Gremmels J. Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: Evidence for association between genetic makeup and biofilm susceptibility. J. Dairy Sci. 2011;94:5926–5937. doi: 10.3168/jds.2011-4243. PubMed DOI

Khazandi M., Al-Farha A.A.B., Coombs G.W., O’Dea M., Pang S., Trott D.J., Aviles R.R., Hemmatzadeh F., Venter H., Ogunniyi A.D., et al. Genomic characterization of coagulase-negative staphylococci including methicillin-resistant Staphylococcus sciuri causing bovine mastitis. Vet. Microbiol. 2021;219:17–22. doi: 10.1016/j.vetmic.2018.04.004. PubMed DOI

Vyleťelova M., Vlkova H., Manga I. Occurrence and Characteristics of Methicillin Resistant Staphylococcus aureus and Methicillin Resistant Coagulase-negative Staphylococci in Raw Milk Manufacturing. Czech. J. Food Sci. 2011;29:11–16. doi: 10.17221/4443-CJFS. DOI

Bogdanovičová K., Skočková A., Šťástková Z., Karpíšková R. Occurrence and antimicrobial resistance of Staphylococcus aureus in bulk tank milk and milk filters. Potravin. Slovak J. Food Sci. 2014;8:97–101. doi: 10.5219/363. DOI

Seixas R., Santos J.P., Bexiga R., Vilela C.L., Oliveira M. Short communication: Antimicrobial resistance and virulence characterization of methicillin-resistant staphylococci isolates from bovine mastitis cases in Portugal. J. Dairy Sci. 2014;97:340–344. doi: 10.3168/jds.2013-7130. PubMed DOI

Corti S., Sicher D., Regli W., Stephan R. Current data on antibiotic resistance of the most important bovine mastitis pathogens in Switzerland. Schweiz Arch. Tierheilkd. 2003;145:571–575. doi: 10.1024/0036-7281.145.12.571. PubMed DOI

Vinodkumar K., Neetha N., Ashok S., Suchithra S., Justin D., Radhik S. Genotypic and phenotypic β-lactam resistance and presence of PVL gene in Staphylococci from dry bovine udder. PLoS ONE. 2017;12:e0187277. doi: 10.1371/journal.pone.0187277. PubMed DOI PMC

Bardiau M.K., Yamazaki J.N., Duprez B., Taminiau J., Mainil G., Ote I. Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk of bovine mastitis. Lett. Appl. Microbiol. 2013;57:181–186. doi: 10.1111/lam.12099. PubMed DOI

Vanderhaeghen W., Cerpentier T., Adriaensen C., Vicca J., Hermans K., Butaye P. Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows. Vet. Microbiol. 2010;144:166–171. doi: 10.1016/j.vetmic.2009.12.044. PubMed DOI

EU . European Union’s Farm to Fork Strategy—For a Fair, Healthy and Environmentally-Friendly Food System. EU; Brussels, Belgium: 2020. [(accessed on 17 June 2020)]. p. 13. Available online: https://ec.europa.eu/food/sites/food/files/safety/docs/f2f_action-plan_2020_strategy-info_en.pdf.