Antimicrobial susceptibility testing The MIC values of all cfr-positive original Staphylococcus isolates and transformants were determined by the broth microdilution method, according to the recommendations specified in CLSI documents M100-S22 . The results were interpreted according to Eucast breakpoints ( http://www.eucast.org/clinical_breakpoints/).
Isolates with an MIC of ≥16 mg/L were tentatively considered to be florfenicol-resistant . The reference strain S. aureus ATCC 29213 was used for quality control. Cloning and sequencing selleck chemical of the regions flanking cfr The regions flanking cfr in the transformant obtained from the isolate TLKJC2 were determined by PCR mapping. The plasmid DNA of the isolate TLD18 was extracted and digested with EcoRI. The digested fragments were cloned into the pUC18 vector, and the recombinant plasmid (designated as pUC18-cfr) was introduced into Escherichia coli DH5α with subsequent selection for the transformant (designated as E. coli DH5α- pUC18-cfr) on media supplemented with 10 mg/L florfenicol. The approximately 5.7-kb segment in pUC18-cfr,
including cfr and its flanking regions, was sequenced by primer walking. The DNA sequences were compared to those deposited in GenBank using the BLAST program ( http://www.ncbi.nlm.nih.gov/BLAST). Barasertib Nucleotide sequence accession number The nucleotide sequences of cfr-containing fragments of plasmids pHNLKJC2 and pHNTLD18 have been deposited in the GenBank under the accession numbers KF751701 and KF751702, respectively. Acknowledgements This work was supported in part by grants from National Key Basic Research Program of China (No. 2013CB127200), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13063) and the fund for Training of PhD Students from the Ministry of Education of China (201044041100). References 1. Bozdogan B, Appelbaum PC: Oxazolidinones: activity, mode of action, and mechanism of resistance. Int J Antimicrob Ro 61-8048 purchase Agents 2004, 23:113–119.PubMedCrossRef 2. Shaw KJ, Barbachyn MR: The oxazolidinones: past, present,
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