Bulletin "Veterinary biotechnology"

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Veterynarna biotehnologija – Veterinary biotechnology, 2018, 32(1), 107-115 [in Ukrainian]. https://doi.org/10.31073/vet_biotech32(1)-12

KASYANCHUK V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., BERHYLEVYCH O., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Sumy State University

LOTSKIN I., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., 

Sumy National Agrarian University

DERIABIN O., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

State Scientific Control Institute of Biotechnology and strains (DNKIBSHM)

DETERMINATION OF THE RESISTANCE GENE TO METHICILLIN IN ISOLATES S. AUREUS, COLLECTED FROM MILK OF COWS

Introduction. S. aureus is one of the most important microorganisms that must be controlled in the production of raw milk because it can cause dangerous diseases in humans. Antibiotic resistant S. aureus – MRSA is a particular danger to human health. It is believed that the main source of MRSA is the agrarian sector, therefore it is important to continuously monitor these bacteria in products and the environment of livestock farms, including milk for preventive measures. Among the existing methods for determining MRSA, PCR is considered to be the most effective method for identifying specific antibiotic resistance genes.

The aim of the present study was to determine MRSA in raw milk of cows by detecting the mecA gene by PCR method.

Materials and methods. The studies were conducted during 2016–2017. 36 samples of raw milk from farms of the Sumy region were investigated. Classical bacteriological methods were used to identify bacteria S. aureus. Coagulase-positive isolates of S. aureus were tested for susceptibility to 10 antibiotics: penicillin, amoxicillin, oxacillin, cefazolin, gentamicin, tetracycline, vancomycin, lincomycin, erythromycin, rifampicin. A DNA-sorbent kit (Ukraine) was used to extract DNA. The reaction was carried out on a thermocycler "T1" (Biometra, Germany). A reaction mixture was prepared with the physical separation of PCR components. To prepare the "lower" reaction mixture, the primers MecA147-F 5'-GTGAAGATATACCAAGTGATT-3 'and MecA147-R5'-ATGCGCTATAGATTGAAAGGAT-3 were mixed. Thermoprofile of the reaction: 95°C for 4 min (1 cycle), followed by 35 cycles of 95°C, 30 s, 50°C for 30 seconds, 72°C for 30 seconds, 72°C for 7 minutes (1 cycle). Analysis of the amplification products was carried out in 1.5% agarose gel, and the determination of the DNA by the Gel DocXR Plus system (BioRad Laboratories, USA).

Results of research and discussion. 12 coagulase-positive isolates S. aureus (33.3%) were identified by bacteriological method. Three isolates from 12 were assigned to MRSA (25%). The bacteriological method using antibiotic disks revealed that MRSA isolates identified were highly resistant to six antibiotics: penicillin, amoxicillin, oxacillincephazolin, tetracycline, erythromycin from 10 subjects, and these isolates were sensitive to gentamicin, vancomycin, lincomycin, rifampicin. Three isolates, that have been selected from microbiological studies as potential MRSA have been tested for the presence of the mecA gene in the PCR with the MecA147-F and MecA147-R primers. For comparison, isolated isolates from other types of animals and humans were investigated. The PCR method confirmed the presence of the mecA gene in the three S. aureus isolates isolated from milk samples, as well as in 19.4% of cases, the presence of this gene in other S. aureus isolates was confirmed from humans and isolated S. aureus isolates from pigs, cattle and fish. This confirms the possibility of interspecific cross-contamination of MRSA.

Conclusion and prospects for further research. The information obtained during this study is useful for understanding the prevalence of S. aureus and its antibiotic sensitivity in dairy farms and may be useful for local and national monitoring and for developing specific programs for monitoring MRSA isolates in the milk production chain. In addition, the study of antibiotic resistance among isolates of S. aureus in each farm is very important, especially for the successful treatment of staphylococcal animal infections in order to prevent cross-contamination by foodstuffs. The prospect of further research is the study of the spread of MRSA in the food chain livestock production and the improvement of hygienic and sanitary practices in the agrarian sector.

Keywords: antibiotic resistance, milk of cows, S. aureus, MRSA, PCR, mecA gene.

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