Study On Drug Resistance Characteristic Of Staphylococcus Aureus In Changsha And Cloning And Prokaryotic Expressionof FemB Gene

Objective (1) To investigate the characteristic of drug resistance and current situation of methicillin-resistance and vancomycin-resistance in clinical isolates of staphylococcus aureus in Changsha. (2) To construct prokaryotic expression vector of femB gene (Factors essential for the expression of methicillin resistance gene), and express it in E. coli and establish foundation for further investigations of methicillin-resistance in staphylococcus aureus.Methods (1) Totally 279 non-duplicate clinical isolates of staphylococcus aureus were collected during November 2009 to November 2010 from 11 hospitals in Changsha and then identified by Vitek-2 system. K-B disk method was used to test drug sensitivity of 24 commonly used antibiotics, chromogenic cephalosporin spot test was applied to detectβ-lactamase and D-test were used to check inducible resistance of erythromycin to clindamycin. (2) MRSA was screened by oxacillin and cefoxitin disk diffusion methods. Agar dilution method was used to determine the minimal inhibitory concentrations (MICs) of oxacillin, cefoxitin and vancomycin to isolates of staphylococcus aureus. (3) Genomic DNA were extracted from staphylococcus aureus and used as templete for PCR amplification of femB gene fragment using special PCR primers. Recombinant plasmids pGEX-4T-1-femB were constructed and transformed into E.coli BL21. The expressed product was identified by SDS-PAGE and Western blot.Results (1) Of the 279 S. aureus isolates, The sensitive rates to 9 of the 24 antibiotics tested were higher than 50%. All the isolates were susceptible to tecoplanin, vancomycin and linezolid, and the sensitive rate to Nitrofurantoin, chlormycetin and trimethoprim-sulfamethoxazole was 97.1%,93.5% and 87.1% respectively. The resistant rates to 11 of the 24 antibiotics tested were higher than 50%, resistant rates to penicillin and ampicillin were the highest (both 97.1%).Among 279 strains, MRSA accounted for 54.5%, which were high sensitive to vancomycin, tecoplanin and linezolid. The resistant rates of MRSA to 16 antibiotics were higher than MSSA. (2) Of the 279 strains, the positive rate ofβ-lactamase was 89.6%(250/279). Of the 30 isolates resistant to erythromycin but susceptible or intermediate to clindamycin, 22(73.3%)showed a positive result of D-test. (3) The MIC range of OXA and FOX on staphylococcus aureus was 0.125->256μg/ml and 2～>256μg/ml respectively. The MIC50 and MIC90 of OXA and FOX was 128μg/ml and 256μg/ml,64μg/ml and 256μg/ml respectively. The MIC range of vancomycin on staphylococcus aureus was 1～2μg/ml, both the MIC50 and MIC90 of vancomycin were 2μg/ml. (4) Verified by PCR, double-enzyme digested assessment and sequencing, recombinant plamid pGEX-4T-1-femB was successfully constructed. SDS-PAGE and Western blot analysis confirmed the recombinant plasmid pGEX-4T-1-femB expressed a 49KD target protein in E.coli BL21.Conclusions (1) Clinical isolates of staphylococcus aureus in Changsha are multiple resistant to commonly used antimicrobial agents, positive rate ofβ-lactamase and rate of inducible resistance of erythromycin to clindamycin are high. (2) Clinical isolates of staphylococcus aureus in Changsha have a high isolated rate of MRSA, and are highly resistant to methicillin. All the strains are sensitive to vancomycin, but special attention should be paied to the incresed MICs which nearly reached intermediate level. (3) Recombinant plasmid pGEX-4T-1-femB is successfully constructed and high-effective expressed in E. Coli.