Mechanisms Of Erythromycin Resistance In Staphylococcus Aureus Isolates From Cows Mastitis

A total of 26 isolates of Staphylococcus aureus from cow clinical and subclinical mastitis in China were investigated for their resistance to erythromycin and the resistance mechanisms. Kirby-Bauer (K-B) diffusion method and macrodilution were used to test the resistance phenotype of S.auruus to erythromycin. The inducible clindamycin resistance was checked by D-test and the responsibility for efflux of the antibiotic was measured by the active efflux inhibitiors. The erythromycin resistance genes ermA, ermB, ermC, msrA, and mefA were detected by PCR. Six isolates with resistance genes but without resistance phenotype were selected for further analysis of mutation of their Erm genes with the promotor region and the 23S-rDNAs, meanwhile, five isolates with resistance phenotype and resistance genes were randomly selected as control.20 isolates showed resistance to erythromycin by Kirby-Bauer (K-B) diffusion and macrodilution among 26 isolates. The rate of erythromycin resistance was 76.9% and MICs value of erythromycin was between 64μg/mL and 2560μg/mL. 15 isolates were inducible resistant (D-test positive) and 5 isolates were constitutive resistant (D-test negative) among resistance isolates. The active efflux inhibitiors have no effect on MIC value. All erythromycin resistant strains carried ermB and ermC gene. Among the six erythromycin-sensitive isolates, all had ermB, and two had both ermB and ermC genes. None of the isolates harboured the ermA, msrA or mefA gene. ErmBs and their promoters share sequence similarity with the transposon Tn551 (GenBank: Y13600.1) between 99% to 100%, and ermCs and their promoters show sequence similarity with S. aureus plasmid pWBG751 (GenBank: GQ900393.1) from 99% to 100%. The promoter region of ermB in a sensitive isolate mutate from A to T at 23 base site, and the promoter region of ermC mutate from A to G at 46 base site in a resistant isolate, the other point mutations occur on Erm structure gene. The sequence homology of 23S-rDNA genes shows sequence similarity with S. aureus Newman's 23S-rDNA(AP009351.1)from 99% to 100%. Both sensitive strain SH-11 and SH-12 had the same mutation at the A989G, and the A to G mutation frequency is higher than the other mutation frequency.The results show that the rate of erythromycin resistance in S. aureus isolates from cow mastitis is as high as 76.9%, meanwhile, the erythromycin-resistance is determined by both ermB and ermC gene. These data confirm that high-level resistance to erythromycin in Staphylococcus aureus was mainly due to erythromycin ribosome methylation. Resistance genes ermB and ermC were firstly detected in sensitive S. aureus isolates from cow mastitis.