Comparison between Some Phenotypic and Genotypic Methods for Assessment of Antimicrobial Resistance Trend of Bovine Mastitis Staphylococcus aureus Isolates from Bulgaria

Abstract

The aim of this study was to assess the resistance of bovine mastitis S. aureus isolates from farms in Bulgaria to different classes of chemotherapeutic drugs by comparison of some phenotypic and genotypic methods by means of Cohen's kappa statistics. The study comprised 546 milk samples from subclinical and clinical mastitis at 14 farms from 9 districts in the country. A total of 92 Staphylococcus aureus strains were isolated from tested samples and identified by nuc PCR. The results demonstrated high levels of resistance to sulfadimethoxine (87%), followed by resistance to penicillin (33.7%), erythromycin (13%), streptomycin (8.7%), tetracycline (6.5%) and gentamicin (1.1%). The comparison of both phenotypic tests with respect to 9 antimicrobials revealed strong agreement with kappa coefficient 0.836. An almost complete agreement was evidenced between phenotypic resistance to penicillin and blaZ gene presence, to methicillin with mecA gene, to tetracycline with tet genes, but the agreement between erythromycin resistance and erm genes presence was moderate. This study was the first to demonstrate discrepancy between the behaviour to cefoxitin in the disk diffusion test and oxacillin in the MIC test for an isolate shown to carry the mecA gene in the subsequent genetic analysis. Considering the detected discrepancies for some of isolates, an integral evaluation through phenotypic and molecular methods for monitoring of antimicrobial resistance of Staphylococcus aureus is recommended.

Keywords: Staphylococcus aureus; antimicrobial resistance; cows; mastitis; resistance genes.

Figure 1

PCR identification of S. aureus isolates on the basis of nuc gene. M–100 bp molecular marker (BrightMax 100–1000 bp, Canvax, Spain); 1–positive control S. aureus ATCC 25923; 2-8–tested isolates; 9-negative control (water).

Figure 2

Percentage distribution of S. aureus isolates from bovine mastitis, resistant to tested chemotherapeutic drugs in the Bauer-Kirby disk diffusion test. Legend: P-penicillin, A-ampicillin, E-erythromycin, S-streptomycin, T-tetracycline, Eft-ceftiofur, G–gentamicin. * Statistically significantly higher resistance against penicillin in comparison to the other antibiotics in the investigated farms.

Figure 3

PCR demonstrating mecA and blaZ genes in bovine mastitis S. aureus isolates from Bulgaria. M–DNA ladder 100 bp (BrightMax 100–1000 bp, Canvax, Spain), 1–MRSA DSM 29134 mecA positive control, 2–mecA positive isolate, 3–negative control of first reaction mix; 4–MRSA DSM 29134 blaZ positive control, 5-8–blaZ positive isolates, 9–negative control of second reaction mix.

Figure 4

PCR demonstrating ermC and tetK genes in bovine mastitis S. aureus isolates from Bulgaria. M–DNA ladder 100 bp, 1–4–ermC positive isolates, 5-negative control of first reaction mix; 6–8–tetK positive isolates, 9-negative control of second reaction mix.

Figure 5

Multiplex PCR demonstrating the presence of blaZ/tetK and blaZ/ermC genes in some S. aureus isolates. 4, 9–negative controls of first and second reaction mix.