Bovine mastitis is the most widespread and important disease of the mammary gland worldwide. The health of the mammary gland is critical for dairy industry because the disease has negative impact on milk yield and its quality, animal welfare, farm economy and food safety. Mastitis is mainly caused by the pathogens which based on their habit, interaction with teat and pathogenicity could be classified into contagious, opportunistic and environmental ones (Cerioli et al., Reference Cerioli, Moliva and Reinoso2024). Important approach to treat mastitis is the application of antibiotic therapy (Duse et al., Reference Duse, Persson-Waller and Pedersen2021). Despite the fact that antibiotics are still the most common treatment for mastitis, concerns about the rise of antibiotic-resistant bacteria are increasing (Persson et al., Reference Persson, Nyman and Gronlund-Andersson2011; Kerro Dego and Vidlun, Reference Kerro Dego and Vidlun2024). The European Community Action Plan has described surveillance systems including monitoring of antimicrobial resistance in animal medicine as one of the key actions (European Commission (EC), 2011). Based on our previous study by Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019), where the most common pathogens identified were Coagulase-negative staphylococci (CNS), Escherichia (E.) coli, Streptococcus (Str.) uberis, Staphylocossus (S.) aureus and Str. agalactiae, we decided to continue monitoring of these pathogens and their sensitivity to antibiotics.
Material and methods
Sampling
The milk samples were taken by veterinarians from dairy cows suspected mastitis in dairy farms based on high somatic cell count or California mastitis test or visible abnormalities in milk. The samples came from 52 samplings in 2017 (47 farms), from 32 samplings in 2018 (29 farms), from 31 samplings in 2019 (29 farms), from 44 samplings in 2020 (41 farms), from 40 samplings in 2021 (35 farms), from 33 samplings in 2022 (31 farms) and form 38 samplings in 2023 (35 farms). A total of 2236 quarter udder milk samples were collected (Council, Reference Council1999).
Bacterial culture
The milk samples (10 μl) were incubated aerobically on blood agar plates (MkB Test a.s. Rosina. SR) at 37°C for 24 h. Grown bacterial colonies were initially identified by their morphology, haemolysis, activities of catalase, aesculin hydrolysis, Gram staining, cell morphology and of cytochrome oxidase C (Bactident Oxidase. Merck) were tested. Presumptive S. aureus was verified with the clumping factor test (DiaMondiaL Staph Plus Kit. Germany). Aesculin-positive streptococci were subcultivated on modified Rambach agar to identify Str. uberis or Enterococcus sp. (Watts et al., Reference Watts, Salmon and Yancey1993). Aesculin-negative streptococci were identified by Lancefield serotyping (DiaMondiaL Strept Kit. Germany). Streptococci of group B were referred to as Str. agalactiae and those from group C as Str. dysgalactiae. Gram-positive and catalase-positive small colonies were identified as coryneform bacteria. Bacillus sp. are Gram-positive, catalase-positive large colonies on blood agar. All grown colonies were identified using MALDI-TOF MS (Bruker Daltonics. Bremen. Germany) (Tvarožková et al., Reference Tvarožková, Vašíček, Uhrinčať, Mačuhová, Hleba and Tančin2021). Milk samples was considered positive if at least one colony-forming unit (CFU) of contagious pathogens (S. aureus, Str. agalactiae) was detected. For other pathogens positive classification required at least five CFU. Sample was classified as contaminated and removed from data analysis if three and more different colony types were detected on blood agar.
Antibiotic susceptibility testing
S. aureus, CNS, Str. agalactiae, Str. uberis and E. coli were subjected to susceptibility testing to 19 antibiotic agents (in online Supplementary tables) by the standard disk diffusion procedure (CLSI, 2018). The diameters of the inhibition zones were evaluated (susceptible intermediate, resistant) according to EUCAST and CLSI breakpoints.
Results
Of the total number of milk samples analysed from suspected cows on mastitis, only 4.74% bacteriological negative samples were recorded. The total number of identified isolates during the recording period was 2486. The most frequent pathogens (CNS 36.89%, E. coli 24.26%, Str. uberis 16.21%, S. aureus 8.41% and Str. agalactiae 2.86%) accounted for up to 88.62% of the identified isolates. In Table 1, there are presented the occurrence of monitored pathogens in study years.
The number of isolates in milk of dairy cows during 2017–2023 (n – number isolates, % of occurrence from all identified isolates)
Detailed data on the resistance of the monitored to selected antibiotics are presented in Tables S1–S5 (online Supplementary). The highest resistance of CNS isolates was observed to lincomycin (100%) and also high resistance showed isolates to streptomycin from 88.7% to 100% and neomycin from 60.9% to 97.6%. The low resistance showed CNS isolates to amoxicillin/clavulanic acid from 0.0% to 11.9% in tested years (Table S1). The highest resistance of E. coli was recorded to streptomycin (100%), neomycin (100%), cloxacillin from 87.5% to 100%, lincomycin (100%) and lincomycin/neomycin (100%) in monitored years (Table S2). The highest resistance of Str. uberis was detected to streptomycin from 93.9% to 100% and neomycin from 92.0% to 100%. No resistance showed Str. uberis isolates to amoxicillin/clavulanic acid in tested years (Table S3). S. aureus isolates showed the highest resistance to streptomycin and neomycin from 90% to 100% in monitored years, while we detected the lowest resistance of isolates to amoxicillin/clavulanic acid from 0% to 12.5% (Table S4). The tested Str. agalactiae isolates showed 100% resistance to streptomycin, neomycin and tetracycline during our study in 2017–2023. The most effective antibiotic (100% susceptibility) was amoxicillin/clavulanic acid (Table S5).
Discussion
The most frequently pathogens in our research were CNS. Similarly, Idriss et al. (Reference Idriss, Foltys, Tančin, Kirchnerová and Zaujec2013) and Zigo et al. (Reference Zigo, Farkašová, Výrostková, Regecová, Ondrašovičová, Vargová, Sasáková, Pecka-Kielb, Bursová and Kiss2022) found that the most frequently identified pathogens in milk samples from dairy cows were CNS (17.95%, 42.30%, respectively). The second common pathogen was E. coli in our study. E. coli was also the second common pathogen (26.56%) in study by Sweeney et al. (Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024). Str. uberis was the third most abundant pathogen identified in our research. The prevalence of Str. uberis was 20.02% in cows with clinical and subclinical mastitis (Sweeney et al., Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024). Also in the study of Langhorne et al. (Reference Langhorne, Horsman, Wood, Clark, Price, Henning, Grewar, Wood, Ranjbar, McGowan and Gibson2024), Str. uberis was the most common pathogen (16.80%).
The occurrence of both contagious pathogens S. aureus and Str. agalactiae was relatively low in our study. The incidence of S. aureus in other study realized in Slovakia was 12.50% (Holko et al., Reference Holko, Tančin, Vršková and Tvarožková2019) and 16.50% (Zigo et al., Reference Zigo, Farkašová, Výrostková, Regecová, Ondrašovičová, Vargová, Sasáková, Pecka-Kielb, Bursová and Kiss2022). Persson et al. (Reference Persson, Nyman and Gronlund-Andersson2011) and recent study by Sweeney et al. (Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024) recorded S. aureus as the most common pathogen (19.00%, 29.33%). The prevalence of Str. agalactiae in another studies conducted in Slovakia was also low at 5.80% (Holko et al., Reference Holko, Tančin, Vršková and Tvarožková2019) or at 3.40% (Zigo et al., Reference Zigo, Farkašová, Výrostková, Regecová, Ondrašovičová, Vargová, Sasáková, Pecka-Kielb, Bursová and Kiss2022).
The most important part of our study are results describing antibiotic resistance of mastitis pathogens. We found the highest resistance of CNS isolates to streptomycin, neomycin and lincomycin, but neomycin and lincomycin were not tested throughout the whole study period. Amoxycillin/clavulanic acid and rifaximin were found to be the most effective antibiotics. Under Slovakian dairy farms, Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019) reported the highest resistance CNS to streptomycin (36.4%) and neomycin (20.9%), but their values are clearly lower compared to our results. Also in evaluated susceptibility of CNS Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019) found more effective antibiotics: amoxicillin/clavulanic acid, amoxicillin, ceftiofur, cefquinome, tetracycline and rifaximin compared to our results. Brito and Costa (Reference Brito and Costa2024) detected lower resistance of CNS isolates to lincomycin (52.3%), streptomycin (60.0%) and higher resistance isolates to penicillin (80.0%) and ampicillin (75.4%) compared to our results.
E. coli isolates had the highest occurrence of multi-antibiotics resistance. E. coli was found to be highly resistant to almost all antibiotics tested, with the exception of marbofloxacin, which was resistant only in 2017, but otherwise showed the lowest resistance among the antibiotics tested. Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019) detected the highest resistance of E. coli to rifaximin (58.4%), streptomycin (35.1%) and neomycin (24.7%) which are lower compared to our findings and they reported 100% susceptibility E. coli isolates to ceftiofur and cefquinome.
Sweeney et al. (Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024) observed low resistance of E. coli isolates to ceftiofur (2.8%) and cefoperazone (3.4%) and ampicillin (9.2%), in contrast to our results since we found higher resistance of the isolates to the mentioned antibiotics.
Str. uberis proved to be the most resistant to streptomycin and marbofloxacin. Neomycin and novobiocin also showed high resistance but were not tested during all years of the study. Resistance to amoxicillin/clavulanic acid, penicillin and ceftiofur was shown to be lowest in the Str. uberis isolates tested. Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019) reported resistance Str. uberis isolates to cloxacillin (42.1%), streptomycin (78.9%) and neomycin (74.0%). Different results they found were in 100% susceptibility isolates to lincomycin in contrast to our findings. Str. uberis isolates showed the lowest resistance to ampicillin (0%) and ceftiofur (0.2%) in study of Sweeney et al. (Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024). We observed also similar low resistance isolates of Str. uberis to ceftiofur but higher resistance isolates to ampicillin compared with Sweeney et al. (Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024).
In our study, S. aureus showed the highest antibiotic resistance to streptomycin and neomycin, and the lowest resistance to amoxicillin/clavulanic acid and rifaximin. Zigo et al. (Reference Zigo, Farkašová, Výrostková, Regecová, Ondrašovičová, Vargová, Sasáková, Pecka-Kielb, Bursová and Kiss2022) observed that amoxicillin/clavulanic acid (100%), rifaximin (94.7%) and tetracycline (94.7%) were the most effective antibiotics and the highest resistance of S. aureus they detected to streptomycin (29.0%) and neomycin (29.0%). The highest susceptibility (100%) of S. aureus was observed to amoxicillin, amoxicillin/clavulanic acid, ceftiofur, cefquinome and rifaximin in study by Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019). The highest resistance of S. aureus isolates to streptomycin (44.6%) and neomycin (21.5%) was reported in the same study. However, Shehab et al. (Reference Shehab, Elkenany and Younis2023) reported high resistance of S. aureus to ampicillin (100%), amoxicillin/clavulanic acid (98.1%). Brito and Costa (Reference Brito and Costa2024) found out higher resistance of S. aureus to penicillin (94.3%) and ampicillin (88.6%) and on the other hand they detected lower resistance isolates to streptomycin (22.9%). The low resistance of S. aureus isolates Sweeney et al. (Reference Sweeney, Gunnett, Kumar, Lunt, Moulin, Barrett, Gurjar, Doré, Pedraza, Bade and Machin2024) detected to ceftiofur (0.1%) and cefoperazone (1.2%) which was lower compared with our results.
Str. agalactiae isolates showed resistance to streptomycin, marbofloxacin and kanamycin. The most effective antibiotic during our study was amoxicillin/clavulanic acid. The high resistance of Str. agalactiae was observed by Holko et al. (Reference Holko, Tančin, Vršková and Tvarožková2019) in their study to streptomycin (70.0%) and neomycin (80.0%).
Conclusion
In dairy cows with suspected mastitis, the most common mastitis pathogens were CNS, followed by E. coli, Str. uberis, S. aureus and Str. agalactiae. Identified pathogens showed the lowest resistance to amoxicillin/clavulanic acid. S. aureus isolates showed the lowest resistance to amoxicillin/clavulanic acid and rifaximin. Str. agalactiae isolates showed the highest susceptibility to amoxicillin/clavulanic acid. E. coli isolates showed the lowest resistance to marbofloxacin. Identification of mastitis pathogens is necessary not only for the application of subsequent therapy but also for the application of appropriate anti-mastitis programmes. Decisions on the use of antibiotics should be based on resistance tests to prevent the spread of resistant strains in the dairy cow population.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029925101258
Acknowledgements
The publication was supported by the project APVV-18-0121: The effect of animal and environmental factors on milk production and udder health in dairy cows in Slovakia.
