The irrational use of antimicrobials in animal husbandry has led to livestock becoming a reservoir of antibiotic-resistant bacteria. Resistant strains of microorganisms pose a threat not only to animal health, but also to human health as they can also enter the human body with products of animal origin (meat, eggs, and dairy products). There is now a pressing need to develop a unified strategy for the rational use of antimicrobials, which includes monitoring of microbial susceptibility, rotation of veterinary medicinal products and use of alternative methods that allow reducing their use. Important measures also include a transition to extensive farming systems, reducing animal stress, and maintaining high hygiene standards. Scientists worldwide emphasize the global nature of the antimicrobial resistance (AMR) problem and the importance of international cooperation in solving it [1][2][3][4]. Foreign authors stress the need for coordinated global, regional, and national strategies, based on the “One World, One Health” approach, to reduce the use of antimicrobials and find alternatives [5][6][7]. The World Health Organization and the World Organization for Animal Health have developed lists of critically important antibiotics for human medicine and veterinary medicine in order to limit their irrational use [3].

Russian scientists have experimentally established that the repeated use of the same antibiotics in treatment and prevention protocols both in cattle and in poultry leads to the AMR development in pathogenic microflora. This reduces efficacy of veterinary medicinal products, negatively impacts productivity, and increases risks to animal health [8][9][10].

Experience from leading international medical researchers indicates that the periodic rotation of antibiotics can help reduce the risk of AMR development. Rotation of veterinary medicinal products can significantly increase the susceptibility of antibiotic-resistant bacterial strains. Modified treatment protocols, routinely applied in practice, can yield positive results even after several years. The authors have also conducted multi-center studies to confirm these findings and to optimize both the frequency and rotation options of antibiotics [11][12].

Experts agree that an effective countermeasure against AMR necessitates an integrated approach, combining optimized antibiotic therapy, stringent infection control, innovative methods (such as rapid resistance diagnostics), and AMR monitoring to achieve maximum effect [13][14].

Modern Russian publications also take into account the ecological status of the territories of the Russian Federation when developing measures to control AMR. Authors discuss enhanced monitoring of radionuclides and heavy metals in feed, as well as antibiotic resistance on farms in industrial zones, alongside the development of adaptive livestock farming technologies to reduce animal stress in polluted areas [15]. Researchers emphasize the need for widespread application of alternative methods, such as vaccination, probiotics, phytobiotics, bacteriophages, bacteriocins, rotation of antibiotics, and controlled application of these alternative methods in industrial livestock and poultry farming [1][16][17]. However, despite promising results from using these methods, most of them require additional research, particularly within the context of specific agricultural farms [17][18][19][20].

Research aimed at identifying antibiotic resistance in bacterial pathogens is highly relevant due to the complex AMR situation in animal husbandry, which poses a serious threat to both animal and human health through the food chain. The irrational use of antimicrobials has led to the emergence and spread of resistant microbial strains, significantly reducing treatment efficacy and necessitating new approaches to managing infectious diseases in livestock. In Sverdlovsk Oblast, a region with developed livestock sectors, the AMR problem is particularly significant, underscoring the need for localized monitoring and development of tailored recommendations for specific farms.

The novelty of this study is twofold. First, it provides a comprehensive analysis of the dynamics of the microbial landscape and the resistance profiles of mastitis pathogens on the operational farm in Sverdlovsk Oblast. Second, it develops and validates a practical algorithm for rotating antimicrobials, based on regular molecular genetic monitoring, which has proven effective in a commercial herd.

This study aimed to identify the primary bacterial pathogens responsible for mastitis in cows on the farm in Sverdlovsk Oblast and assess their resistance to antimicrobials. The findings provide a basis for implementing an antimicrobial rotation strategy and delivering tailored farm-specific recommendations.

This research was conducted as part of the Russian Ministry of Science and Higher Education’s state assignment “Development of Methodological Approaches for Monitoring, Controlling, and Containing Antibiotic Resistance of Opportunistic Microorganisms in Animal Husbandry” (No. 0532-2021-0004). The work was carried out across several departments of the Ural Federal Agrarian Scientific Research Centre, Ural Branch of the Russian Academy of Sciences: the Department of Genomic Research and Animal Selection, the Laboratory of Microbiological and Molecular Genetic Research Methods, and the Laboratory of Biological Technologies within the Department of Veterinary Laboratory Diagnostics and its testing facility.

The study involved monitoring circulation of pathogenic and opportunistic microorganisms, determining their susceptibility to standard antibiotics and the antimicrobials/disinfectants in use, identifying resistance genes, and developing recommendations for rotating antimicrobials used in treatment of bovine mastitis. This was implemented and evaluated over a three-year period (2022, 2023, 2024) on the dairy farm located in Sverdlovsk Oblast.

Sampling was conducted as follows: in 2022, 10 samples of mammary-gland secretion were collected from cows with clinical mastitis; in 2023, 3 composite samples were collected from 15 cows with subclinical mastitis on the same farm; and in 2024, 16 samples were collected.

Microbiological tests were performed in accordance with the “Methodological Guidelines for the Bacteriological Examination of Milk and Udder Secretions from Cows” (No. 115-69, approved by the Main Veterinary Directorate of the USSR Ministry of Agriculture on December 30, 1983)1.

The following nutrient media were used in this study: “Columbia Blood Agar Base” (Bio-Rad Laboratories, Inc., France), defibrinated sheep blood (EKOlab, Russia), dry nutrient medium for accumulation of Salmonella (magnesium medium), bismuth sulfite agar, Ploskirev’s agar, GRM nutrient agar for microorganism cultivation (State Research Center for Applied Microbiology and Biotechnology, Russia), Sabouraud Dextrose agar with 2% glucose and chloramphenicol, Mueller – Hinton agar (SIFIN diagnostics GmbH, Germany), and trypticase soy broth with 20% glyce­rol (Condalab, Spain).

Grown colonies were identified using MALDI-ToF mass spectrometry (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) on a Vitek® MS device (bioMérieux, France). For this purpose, bacterial biomass was applied to a target slide spot, then covered by 1 μL of matrix (α-cyano-3-hydroxycinnamic acid), and air-dried at room temperature, and its ribosomal mass spectra were read with a special device and were compared with ones from the database using the MYLA® software (bioMérieux, France).

Antibiotic susceptibility was determined by a disk-diffusion test on Mueller – Hinton agar (Bio-Rad Laboratories, Inc., France) following European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard guidelines and disks impregnated with preparations of a specific concentration (Bio-Rad Laboratories, Inc., France). Antibiotic susceptibility patterns were read by an ADAGIO automatic analyzer (Bio-Rad Laboratories, Inc., France). Interpretation of susceptibility categories was performed following EUCAST criteria: Clinical breakpoints-bacteria (v 10.0).

The antibiotic disks used in the study included: amoxicillin / clavulanic acid, gentamicin, oxytetracycline, tigecycline, levofloxacin, norfloxacin, cefepime, cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, ciprofloxacin, and ceftiofur (Bio-Rad Laboratories, Inc., France). Microbiological tests also included determining susceptibility to combined antibacterials used on the farm for treating bovine mastitis (2023–2024), which contained antibiotics from the following classes: cephalosporins, aminoglycosides, tetracyclines, and polypeptide antibiotics.

The isolated microbial cultures were frozen at –20 °C in tubes containing trypticase soy broth with 20% glycerol as a cryoprotectant.

Real-time polymerase chain reaction was performed using the Diatom™ DNA Prep 200 kit (Laboratory Isogene, Russia) for DNA extraction from biological materials and the “COMPLEX RESISTOM ESKAPE-V” reagent kit (Lytech, Russia) – for detecting pathogen DNA and antibiotic resistance genes. Amplification was performed in real time using a QuantStudio 5 system (Thermo Fisher Scientific Inc., USA).

Based on the laboratory findings, tailored recommendations for antibacterial therapy of bovine mammary gland diseases were developed. Anti­biotic selection followed established methodological guidelines [21], ensuring a scientifically grounded approach to rotation of antibiotics, and complied with Order No.  771 of the Ministry of Agriculture of the Russian Federation (November 18, 2021)2 on restrictions governing use of antimicrobials in veterinary medicine.

The obtained data were processed using Microsoft Excel software (Microsoft Office Pro 19).

In 2022, microbiological tests using MALDI-ToF mass spectrometry of the collected biological materials (10 samples of mammary gland secretion from cows) revealed the following bacterial isolates: Streptococcus spp. (present in 70.6% of samples), Streptococcus agalactiae (23.5%), Staphylococcus aureus (35.3%), and Escherichia coli (52.9%).

Data on the antibiotic resistance and the presence of AMR genes in these bacterial pathogens are presented in Table 1.

When determining antibiotic resistance by a disco diffusion test, it was found that all identified E. coli isolates were resistant to several groups of antimicrobials (aminoglycosides, penicillins, tetracyclines) and sensitive to cefoxitin (cephalosporin of the II generation), ciprofloxacin (fluoroquinolone of the II generation). S. aureus, isolated from all 10 samples, was resistant to ciprofloxacin (fluoroquinolone of the II generation), vancomycin (a glycopeptide antibio­tic), tetracyclines and was sensitive to chloramphenicol, cefoxitin, and in single cases it was sensitive to tobramycin (aminoglycoside) and linezolid (oxazolidinone). Streptococci isolates were susceptible to all tested antimicrobials. Notably, isolates of S. aureus and E. coli also exhibited resistance to chlorhexidine- and iodine-based disinfectants used for pre- and post-milking udder hygiene.

Key resistance genes were detected by qPCR: blaDHA, blaCTX-M, blaOXA-10 genes (conferring resistance to β-lactams – cephalosporins and protec­ted penicillins) were detected in 5% of E. coli isolate; ErmB gene (responsible for resistance to macrolides, lincosamides, and streptogramins) was found in 4% of Streptococcus spp. isolates; MecA gene (regulating resistance to cephalosporins of the II generation) was identified in one S. aureus isolate.

Based on these findings, the following evidence-based recommendations were developed to enhance therapeutic efficacy and curb the further spread of antibiotic resistance. Priority antimicrobials for mastitis treatment were recommended: cefazolin, ceftiofur, cefquinome (representing cephalosporins of the I, III, and IV generation, respectively), and ciprofloxacin (fluoroquinolone of the II generation). Previously used multi-component medicinal products containing tetracyclines, aminoglycosides, macrolides, and cephalosporins of the II generation were recommended for removal from treatment protocols. It was recommended to use antimicrobials of penicillin group with caution. With regard to hygiene and monitoring it was recommended to implement regular disinfection control of milking equipment and conduct a semi-annual (every 6 months) AMR monitoring program of detected pathogens.

In 2023, microbiological tests performed by the disk diffusion test on 3 pooled samples of mammary gland secretion obtained from 15 cows with subclinical mastitis revealed that single E. coli and S. aureus isolated from the biological material by MALDI-ToF mass spectrometry, possessed resistance to ciprofloxacin. Other bacterial isolates (S. aureus, Escherichia, Enterobacter, Streptococcus spp., Enterococcus faecalis/faecium) were susceptible to all tested antimicrobials. It should be noted that the E. coli and S. aureus isolates exhibited susceptibility to chlorhexidine- and iodine-based agents used for udder disinfection before and after milking. Using the qPCR method, the blaVIM and blaNDM genes, responsible for resistance to carbapenems, were detected in a single Pseudomonas aeruginosa isolate. The other bacterial isolates exhibited no genetic mutations, indicating rational use of antibacterials on the farm during the study period and the future possibility of using a broader spectrum of antimicrobials in the treatment of inflammatory diseases of the mammary gland in cows, taking into account their identification of the phenotypic antibiotic susceptibility.

Throughout 2022–2023, it was established that the detected isolates were resistant to the agents used for treatment after milking. So, the use of combinations of disinfectants with different mechanisms of action was recommended to optimize hygienic measures during milking. A product based on a polyvinylpyrrolidone-iodine complex was proposed as the disinfectant of choice for post-milking teat treatment.

Microbiological tests conducted on the same farm in 2024 showed the predominance of E. coli and Staphylococcus spp. (100% of samples) in 16 samples of mammary gland secretions collected from cows with mastitis; in contrast, K. pneumoniae (30%), Enterobacter spp. (20%) and E. faecalis/faecium (10%) were less frequently detected.

Data on antibiotic resistance and the presence of AMR genes in bacterial pathogens isolated from cow mammary gland secretions in 2024 are presented in Table 2.

The disk diffusion test revealed that all E. coli, S. aureus, and Staphylococcus spp. isolates exhibited resistance to cephalosporins and carbapenems. The blaOXA-10 genes, conferring resistance to cephalosporins, were detected in 30% of E. coli isolates by qPCR, and in single cases the blaCTX-M genes were detected. The blaKPC and blaOXA-48-like genes responsible for carbapenem resistance were identified in 50% of K. pneumoniae isolates. The MecA gene, conferring β-lactam resistance, was confirmed in 50% of Staphylococcus spp. isolates. 30% of the Enterobacter spp. isolates harbored resistance genes (blaGes, blaDHA) that confer resistance to carbapenems protected by penicillins and cephalosporins. E. faecalis/faecium carrying the VanB gene, associa­ted with glycopeptide (vancomycin) resistance, were detected in single cases. Thus, microbial cultures isolated in 2024 from bovine mammary gland secretions exhibited 8 distinct AMR genes. The findings demonstrate a high prevalence of multi-drug resistance in the bacterial flora of mammary secretions, including resistance to reserve antibiotics.

All isolates detected in 2024 demonstrated susceptibility to the post-milking teat disinfectant containing polyvinylpyrrolidone-iodine complex that was recommended in 2023.

Based on the research findings, the following recommendations were provided to the farm: revision of mastitis treatment protocols with mandatory susceptibility testing of identified pathogens, enhanced biosafety measures (equipment disinfection, animal quarantine), implementation of regular antibiotic resistance monitoring. The recommendations emphasized that critically important antibiotics (cephalosporins and fluoroquinolones of the III and IV generation) should be strictly restricted to use as a last-line therapy in exceptional cases only, to preserve their efficacy.

Microbiological tests and MALDI-ToF mass spectrometry identified the following dominant bacterial pathogens in bovine mammary gland secretions: in 2022 – Streptococcus spp. (70.6%), S. agalactiae (23.5%), S. aureus (35.3%), and E. coli (52.9%) isolates; in 2023 – antimicrobial-susceptible S. aureus, Escherichia coli, Enterobacter spp., Streptococcus spp., E. faecalis/faecium, and P. aeruginosa isolates, and in single cases – ciprofloxacin-resistant E. coli and ciprofloxacin-resistant S. aureus; in 2024 – E. coli and Staphylococcus spp. were detected in 100% of samples, alongside newly emerging pathogens: K. pneumoniae (30%), Enterobacter spp. (20%), and E. faecalis/faecium (10%).

In 2022, E. coli exhibited resistance to aminoglycosides, penicillins, tetracyclines, with 5% of isolates carrying several resistance genes blaDHA, blaCTX-M and blaOXA-10 conferring resistance to cephalosporins and protected penicillins; S. aureus demonstrated resistance to fluoroquinolones, vancomycin, tetracyclines, and the MecA gene resistant to cephalosporins of the II generation was identified in a single isolate; 4% of Streptococcus spp. group bacteria had the resistance gene to macrolides, lincosamides, streptogramins. In 2023, no AMR genes were detected in the tested isolates, except for one P. aeruginosa isolate, which carried the carbapenem resistance genes blaVIM and blaNDM. In 2024, blaOXA-10 genes were identified in 30% of E. coli isolates, while blaCTX-M genes, conferring resistance to cephalosporins, were identified in a single isolate. In 50% of K. pneumoniae isolates blaKPC/OXA-48-like carbapenem resistance genes were identified, while the MecA gene conferring β-lactam resistance was detected in Staphylococcus spp.; the blaGes/DHA resistance genes to carbapenems, protected penicillins, and cephalosporins were detected in 30% of Enterobacter spp. A few single isolates of E. faecalis/faecium that harbored the VanB gene, which confers resistance to glycopeptides, were reported.

In 2022 it has been established that multicomponent veterinary medicinal products based on tetracyclines, aminoglycosides, macrolides and cephalosporins of the II generation should be excluded from the treatment protocols used in the bovine mastitis treatment. As an alternative, the use of cefazolin, ceftiofur, cefquinome (cephalosporins of the I, III, and IV generation) and ciprofloxacin (fluoroquinolone of the II generation) was recommended. The implementation of an antibiotic rotation system based on monitoring made it possible to temporarily reduce resistance levels in 2023. However, the subsequent return to previous treatment protocols in 2024 provoked a sharp increase in multi-drug resistance among bacterial mastitis pathogens. The obtained results confirm the need for continuous monitoring of antibiotic resistance, strict adherence to recommendations for the rotation of antimicrobials, and the integration of molecular genetic methods into the veterinary control system as a tool for tracking the occurrence of AMR genes in bacteria.

In 2022–2023, an increase in resistance of bacterial isolates to the disinfectants used on the dairy farm was identified. A veterinary medicinal product based on a polyvinylpyrrolidone-iodine complex was proposed as the disinfectant of choice for post-milking teat treatment. Control studies in 2024 confirmed the effectiveness of this measure: no resistance to the disinfectant was detected, justifying its continued use at the farm.

The results of the work are of practical importance for the veterinary service of the farm and can be used in the development of regional programs for AMR control in animal husbandry.

Contribution of the authors: Bezborodova N. A. – administration, study design, material sampling, laboratory studies, paper preparation and editing; Isakova M. N. – study design, material sampling, paper preparation and editing; Sokolova O. V. – material sampling, paper editing; Zubareva V. D. – material sampling, laboratory studies, literature searches, paper editing; Yusupova Ch. R. – literature searches, paper editing; Vasilyeva A. N. – literature searches, paper editing.

Вклад авторов: Безбородова Н. А. – администрирование, дизайн исследования, отбор проб, лабораторные исследования, подготовка и редактирование текста; Исакова М. Н. – дизайн исследования, отбор проб, подготовка и редактирование текста; Соколова О. В. – отбор проб, редактирование текста; Зубарева В. Д. – отбор проб, лабораторные исследования, работа с литературой, редактирование текста; Юсупова Ч. Р. – работа с литературой, редактирование текста; Васильева А. Н. – работа с литературой, редактирование текста.

1. https://base.garant.ru/72125912/?ysclid=mguhhtg7xh175440448 (in Russ.) 2. https://fsvps.gov.ru/files/prikaz-minselhoza-rossii-ot-18-nojabrja-2021-2/?ysclid=mgqesh36jf335708795 (in Russ.)