| Bovine mastitis is the most cost disease of dairy farm. Staphylococcus aureus is one of the main pathogens of the disease. It is reported that 20%-50% milk samples were positive for S. aureus. Currently, antibiotics are only effective method for treatment and control of bovine mastitis. However, antibiotics and multiple-drug resistance (MDR) S. aureus isolates have been developing resistance due to adaptive evolution and excessive usage of antibiotics, which resulted in great challenge in clinical treatment.The objective of present study is to investigate the status of drug resistance and epidemic of drug resistance genes in Chinese S. aureus isolates for the effective drug treatment of mastitis, and also to find the major epidemic strains and genomic features of bovine MRSA for acknowledging the new trend and mechanism of drug-resistant S. aureus.A total of 219 S. aureus strains were isolated from 1069 bovine subclinical mastitis cases, which identified from 2103 samples collected from Beijing, Shanxi, Shandong Inner Mongolia, Zhejiang and Xinjiang provinces of China between 2010 and 2013. All strains were detected for antimicrobial susceptibility to ten usually used antibiotics in clinical veterinary by disc diffusion test and out of 81 strains were carried out for detection of resistance genes including six genes classified into three categories by multiplex PCR assay. The profiles revealed that more than 70% strains showed resistance to erythromycin, clindamycin, penicillin, cotrimoxazole, doxycycline and tetracycline. And among 60% MDR strains showed common resistant to more than five categories of antibiotics. But there were significant differences in sensitivity to different provinces and regions. Meanwhile, more than 90% strains were positive for the resistance gene, and ermC, aac(6’)-/aph(2"), aph(3’)-Ⅲ, tetM and tetK genes were main epidemiological. Additionally, the mecA-negative MRSA strains were identified.The 81 strains were also carried out for molecular typing by multilocus sequence typing (MLST) and SCCmec typing was carried out in bovine MRSA by multiplex PCR. The MLST profiles depicted that these strains mainly belonged to CC5 and CC398 lineage, especially within ST97 and ST398. The six bovine MRSA strains belonged to CC5 lineage, and out of three strains were ST97-SCCmec Ⅳ, others were ST965-SCCmec Ⅳ, ST6-SCCmec Ⅳ and ST9-SCCmec NT. Meanwhile, base on MLST data, the evolutionary relationship between human MRSA and bovine MRSA was evaluated by eBURST v3 software. The analysis displayed that the generation of human MRSA and bovine MRSA occurred independently and emerge of bovine MRSA depended on migration of SCCmec to MSSA which belong to different CCs, while, the strains do not spread widely by rapid diffusion.Furthermore, two bovine MDR MRSA strains’ genome (Z35 and Z43) were achieved by de novo method base on Hiseq 2000 and PacBio SMRT data and the SCCmec composition was checked. The features showed that Z35 was ST9-SCCmec ⅩⅡ, which carried ccrC2 complex; Z43 was ST97-SCCmec IV, the genome sequence was homologous with S. aureus mu50 and the longer J2 region (about 15 Kb) was found in its SCCmec Ⅳ sequence which carried drug resistance genes.The research suggests that the strains, which isolated from bovine subclinical mastitis cases collected from six provinces of China, mainly belong to CC5 and CC398 lineage, especially within ST97 and ST398, and ermC, aac(6’)-/aph(2"), aph(3’)-Ⅲ and tetM genes were main epidemiological in the area; The generation of bovine MRSA depends on migration of SCCmec to MSSA and the strains do not spread widely by rapid diffusion; the genome sequence of bovine ST9-MRSA-SCCmec XII and ST97-MRSA-SCCmec IV shows SCCmec composition carries ccrC2 complex and J2 region which imported resistance genes, respectively. |
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