Purified spa PCR products were sequenced, and spa types were assigned by using the spa database website (http://www.ridom.de/spaserver). Multilocus sequence typing (MLST) MLST of MRSA isolates was conducted through amplification of internal fragments of seven housekeeping genes of S.aureus as described previously [10]. Following purification and sequencing of these genes, allele quantification and sequence typing were assigned using a well-characterized online database (http:// saureus.mlst.net/). Results Antimicrobial
susceptibility patterns Antimicrobial susceptibility testing by the disc diffusion method revealed that all RIF-R S.aureus isolates were MRSA and were resistant to β-lactam, ciprofloxacin, erythromycin, levofloxacin, gentamycin and tetracycline. Of the S.aureus isolates, 88.6% were resistant selleck products to clindamycin. Isolates also Selleck AZD6244 displayed low levels of resistance to sulfamethoxazole (9.1%), quinupristin (2.3%). There were no vancomycin-resistant
S.aureus isolates in our study. Distribution of mutations associated with rifampicin resistance Among the 88 RIF-R MRSA isolates, 83 isolates showed high-level rifampicin resistance (MIC ≥8 mg/L) and 5 isolates showed low-level rifampicin resistance (MICs 2 to 4 mg/L) [3, 11]. Four amino acid substitutions were found in 88 RIF-R isolates. Results are shown in Table 1. Mutation at 481His/Asn was the most common and found in 95.5% of RIF-R isolates. Mutation 466Leu/Ser was found in 87.5% of isolates. The remaining mutations included 477Ala/Asp (6.8%) and 486Ser/Leu (4.5%). Five low-level resistant isolates had only one mutation, while 83 high-level resistant isolates had two or more mutations. The single mutation 481His/Asn and 486Ser/Leu were conferring low-level rifampicin resistance. Two mutations, 481His/Asn+466Leu/Ser, were the most common multiple mutations found in 92.8% (77/83) of samples. The remaining multiple mutated clones consisted of 481His/Asn+477Ala/Asp (6.0%, 5/83)
and 481His/Asn+466Leu/Ser+477Ala/Asp http://www.selleck.co.jp/products/Rapamycin.html (1.2% and 1/83, respectively). Table 1 The characteristics of the rifampicin-resistant S. aureus isolates studied MRSA rpoB mutations Number of isolates Mutation frequency % Rifampicin MIC Resistance pattern Nucleotide mutation Amino acid substitution MIC(mg/L) Number of isolates TCA/TTA 486Leu/Ser 4 4.5% 4 4 CIP+E+GEN+TET(3) CIP+E+GEN+TET+CC (1) CAT/AAT 481His/Asn 1 1.1% 4 1 CIP+E+GEN+TET(1) CAT/AAT+TTA/TCA 481His/Asn+466Leu/Ser 45 87.5% 32 45 CIP+E+GEN+TET(7) CIP+E+GEN+TET+CC (35) CIP+E+GEN+TET+CC+SXT(2) CIP+E+GEN+TET+CC+SXT+QD(1) CAT/AAT+TTA/TCA 481His/Asn+466Leu/Ser 14 64 14 CIP+E+GEN+TET+CC (12) CIP+E+GEN+TET+CC +SXT(2) CAT/AAT+TTA/TCA 481His/Asn+466Leu/Ser 11 128 11 CIP+E+GEN+TET+CC (8) CIP+E+GEN+TET+CC +SXT(3) CAT/AAT+TTA/TCA 481His/Asn+466Leu/Ser 7 256 7 CIP+E+GEN+TET+CC +SXT(7) CAT/AAT+GCT/GAT 481His/Asn+477Ala/Asp 5 5.7% 64 5 CIP+E+GEN+TET+CC (5) CAT/AAT+TTA/TCA+GCT/GAT 481His/Asn+466Leu/Ser+477Ala/Asp 1 1.