Phenotypic resistance to antibiotics of Staphylococcus aureus strains isolated from cow milk

The emergence of multiresistant strains has sharply escalated in recent decades due to the wide use of antimicrobials. The infections caused by Staphylococcus aureus are most common in highly productive dairy animals. The aim of our work was to assess the phenotypic resistance of S. aureus strains isolated from cow milk to the main antibacterial drugs used to treat various forms of mastitis in the Russian Federation, as well as to determine their minimum inhibitory concentrations (MIC, MIC50 and MIC90). From January to December 2018 milk samples were taken from cows of black-motley Holstein breed at the farms of the Central region of the Russian Federation. A total of 314 milk samples were examined and 447 potential staphylococci were isolated. All isolates were evaluated by conventional phenotypic methods. Only 103 isolates met all the identification criteria and were used for further analysis. The disk diffusion method (DDM) was used to test the susceptibility of the isolated strains to the following antibiotics: penicillin (PEN, 10 U), oxacillin (OX, 10 μg), gentamicin (GN, 10 μg), erythromycin (ER, 15 μg), and lincomycin (LN, 15 mcg), rifampicin (RF, 5 mcg), ciprofloxacin (CP, 5 mcg), vancomycin (VA, 30 μg), fusidine (FZ, 10 μg) (Pharmacotherapy Research Center, Russia; Mueller Hinton Agar, HiMedia Laboratories Pvt. Ltd., India). A method of double (Log2) serial dilutions of antibiotics (penicillin, erythromycin, gentamicin, and ciprofloxacin) in TSB medium (from 64 to 0.125 mg/l) with 5×105 CFU/ml inoculum was used to determine the minimum inhibitory concentrations (MIC, MIC50, MIC90) and LD50. The incubation was carried out at 37 °C for 20 hours (220 rpm). The bacterial growth in TSB medium with antibiotics was evaluated by plating on Baird Parker Agar medium (HiMedia Laboratories Pvt. Ltd., India). The interpretation of the results was carried out according to the recommendation of The European Committee on Antimicrobial Susceptibility Testing (EUCAST). The highest degree of resistance was observed to erythromycin (82.5 %) and fusidine (75.7 %). Seven of 103 strains tested were susceptible to all studied antibiotics, 96 isolates were resistant to at least one of them. When assessing multiple resistance (MAR), 65 (63.1 %) strains were resistant to four or more antibiotics. The predominant phenotypes for the isolates were ER + LN + FZ and ER + CP (58.3 % and 47.6 %, respectively). A total of 31 strains of the 47 isolates DDM-resistant to penicillin grew at ≥ 64 mg/l penicillin, while in the concentration range of ≤ 1.0 mg/l, the growth was observed in 33.0 % of the strains. Fifteen of the 85 erythromycin-resistant isolates showed growth at MIC ≥ 64 mg/l; only 7 strains out of 18 DDM-susceptible ones were in the MIC ≤ 0.5 mg/l range. A total of 29 (28.2 %) of the 103 studied strains were DDM-resistant to gentamicin, 72 (60.2 %) showed growth at MIC ≥ 0.5 mg/l, and 5 strains at MIC ≥ 64.0 mg/l. Only 2 of the 49 DDM-resistant strains grew at MIC ≥ 64 mg/l. It is important to point out that the MIC50 was not in the susceptible range for any of the studied antibiotics, and the MIC90 showed the susceptible range for penicillins, aminoglycosides, and fluoroquinolones (> 0.5, > 0.25, and > 0.25 mg/l, respectively). In our study the high phenotypic resistance indices of S. aureus isolates from cow milk emphasizes the importance of routine screening of S. aureus isolates for inducible phenotype resistance.