Antibiotic resistance, pathogenicity and virulence genes of Staphylococcus aureus and Escherichia coli, isolated from the reproductive tract and mammary gland of cattle (Bos taurus) during inflammation

Mastitis and metritis are the main diseases that cause significant economic damage to dairy cattle breeding, which is associated with a decrease in fertility and productivity, premature culling of highly productive cows, as well as the costs of diagnostics and treatment of affected animals. In this work, we have for the first time carried out genotyping of Staphylococcus aureus and Escherichia coli isolates obtained from cattle in the Sverdlovsk region for the genes of antibiotic resistance, pathogenicity, and virulence. The presence of isolates with a combination of genes determining resistance to antibiotics and the simultaneous manifestation of pathogenic and virulent properties was found. It was revealed that a high frequency of fimA and fimH virulence genes may be important for E. coli colonization of epithelial cells of the mammary gland and reproductive tract of cows. The aim of our research was to study the genetic profile of S. aureus and E. coli isolates for genes responsible for antibacterial resistance to drugs of the fluoroquinolone group, second-generation cephalosporins, macrolides, aminoglycosides, amphenicols, tetracyclines, oxazolidinones, glycopeptides, as well as for the pathogenicity and virulence genes of S. aureus (sea , seb , sec , seg , tst , luks-PV , lukED , fnbA , fnbB , cna) and E. coli (K99 , fimA , fimH , F41 , Sta , STΙa , STΙb , stx1 , stx2). E. coli (n = 26) and S. aureus (n = 16) isolates from mammary gland secretion and cervicovaginal swabs of 29 Holstein cows with signs of inflammatory process in the reproductive tract and mammary gland were studied (2021,. Ready-made nutrient media were used for the microbiological study: Endo medium (State Research Center of Applied Microbiology and Biotechnology), salt broth, yolk-salt agar, elective salt medium (mannitol-salt agar) (OOO BioMedia, Russia), chromogenic medium for E. coli , chromogenic agar for uropathogenic bacteria (Condalab, Spain), Russell double sugar agar (HiMedia Laboratories Private Limited, India). DNA was isolated using the Ribosorb test system for bacterial DNA isolation (OOO InterLabservice, Russia). The sensitivity of isolates to antimicrobial drugs was determined by the disk diffusion method. Discs with antibiotics of known concentration of cefoxitin (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), tobramycin (10 μg), tigecycline (15 μg), linezolid (30 μg) (OOO NITsF, Russia) were used. Testing for resistance to linezolid by the disk diffusion method was performed only for S. aureus in accordance with EUCAST Clinical Breakpoint Tables V.12.0. Detection of antibiotic resistance genetic markers mecA , blaDHA , blaOXA-48 , blaСTX-M was carried out in Real-time PCR using commercial kits (NPF Litech, Russia) on a QuantStudio 5 analyzer (Thermo Fisher Scientific, USA). To determine the antibiotic resistance genes mecA , tetM , vanA , blaFOX , blaEBC , blaACC , blaDHA , blaCITM , blaMOX , blaIMP , the pathogenicity genes of E. coli (stx1 , stx2), as well as the virulence genes of E. coli (K99 , fimA , fimH , F41 , Sta , STIa , STIb) and S. aureus (sea , seb , sec , seg , tst , luks-PV , lukED , fnbA , fnbB , cna), specific oligonucleotide primers synthesized by OOO DNA-Synthesis (Russia) were used. By the PCR results, iIn 12.5 % of S. aureus isolates with the antibiotic resistance phenotype, the lukED genes encoding leukocidins were detected. The f nbA / B genes regulating fibronectin-binding proteins were detected in S. aureus : fnbA in 18.75 % of isolates, fnbB in 12.5 % (mastitis milk samples), fnbB in 6.25 % (endometritis swab samples). The studied S. aureus isolates showed combinations of antibiotic resistance genes with virulence and pathogenicity genes. The fnbA / fnbB / ermB combination was characteristic of 6.25 % isolates from the reproductive tract of cows. The lukED / fnbB / fnbA / ermB combination was found in 12.5 % isolates from mammary gland secretion. The ermB / ermC / fnbA combination was established in 6.25 % isolates from mammary gland secretion. The fimA gene was detected in 100 % of E. coli isolates with the antibiotic resistance phenotype, while the fimH gene was also detected in 88.46 % of cases; both genes encode adhesion factors. The stx2 gene was detected in one E. coli isolate from cervicovaginal swabs of a cow with endometritis. The most common combination was the fimA / fimH encoding adhesion factors and regulating biofilm formation; it was found in 92.31 % isolates. The fimA adhesin gene combined with the blaDHA antibiotic resistance gene were found in one E. coli isolate from mammary gland secretion. The fimA / fimH / blaDHA / blaOXA combination was detected in one E. coli isolate from mammary gland secretion of a cow with mastitis.