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Study on Drug Resistance Characteristic of Staphylococcus Aureus in Changsha and Cloning and Prokaryotic Expressionof FemB Gene

Author: WuWenJun
Tutor: ZouMingXiang
School: Central South University
Course: Clinical Laboratory Science
Keywords: Staphylococcus aureus Resistance Drug sensitivity test femB gene Prokaryotic expression
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 38
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Abstract


The purpose of (1) to understand Changsha clinical isolates of Staphylococcus aureus (S. aureus) drug resistance to commonly used antibiotics, the level of resistance of Staphylococcus aureus to methicillin and vancomycin. (2) Construction of Staphylococcus aureus methicillin-resistant auxiliary gene femB of prokaryotic expression vector and expressed in E. coli, and to lay the foundation for further study of Staphylococcus aureus methicillin-resistance mechanisms. (1) collected in Changsha Hospital in November 2009 -2010 11 279 non-repetitive clinical isolates of Staphylococcus aureus application Vitek-2 automated microbial analysis system to identify 11 KB assay of S. aureus to 24 kinds of drug sensitivity of the chromogenic cephalosporin assay of beta-lactam enzyme D test for detection of inducible clindamycin resistance. (2) the application of cefoxitin and oxacillin disk diffusion screening for methicillin-resistant Staphylococcus aureus (MRSA); agar dilution method detection cefoxitin, oxacillin and vancomycin minimum inhibitory concentration ( MIC). (3) Extraction of S. aureus genomic DNA, and femB gene as a template PCR amplification to construct the recombinant plasmid pGEX-4T-1-femB expression and the recombinant plasmid was introduced into E. coli BL21. SDS-PAGE and Western blot analysis to verify the expressed protein. Results (1) 279 S. aureus isolates 24 drug resistance analysis showed that the sensitivity rate gt; 50% of the drug 9, was most sensitive to vancomycin, teicoplanin and linezolid amine were 100%, followed by nitrofurantoin (97.1%), chloramphenicol (93.5%) and cotrimoxazole (87.1%). Resistance rates gt; 50% of the 11 antimicrobial agents, including penicillin G and ampicillin resistance rates are 97.1%. MRSA isolation rate was 54.5%, in addition to vancomycin, teicoplanin, linezolid, nitrofurantoin a result are highly sensitive to 16 commonly used antibiotics, MRSA resistance rates were significantly higher than MSSA. (2) 279 golden GPB, p-within-lactamase positive and 250 (89.6%) were resistant to erythromycin and clindamycin-sensitive, 30 strains of the intermediary, D test was positive in 22 (73.3% ). (3) oxacillin (OXA) and cefoxitin (FOX) MIC range were O.125μg/ml - gt; 256μg/ml and 2μg/ml - GT; 256μg/ml, the oxacillin The MIC50 and MIC90 for 128μg/ml and 256μg/ml, cefoxitin MIC50 and MIC90 were 64μg/ml and 256μg/ml. Vancomycin MIC range of 1 to 2μg/ml MIC50 and MIC90 are 2μg/ml. (4) by PCR, double enzyme digestion and sequencing, the constructed recombinant plasmid pGEX-4T-1-femB; recombinant plasmid was transformed into E. coli BL21 induced by IPTG, SDS-PAGE and Western blot analysis confirmed the expression of the 49kDa purposes proteins. Conclusions (1) The positive rate of Changsha clinical isolates of Staphylococcus aureus p-lactamase enzyme, inducible clindamycin resistance rate is high, and were multidrug-resistant characteristics. (2) Changsha clinical isolates of Staphylococcus aureus MRSA isolation not only high, but also showed a high level of resistance to methicillin; All strains were sensitive to vancomycin MIC close to the intermediary level, attention should be paid. (3) successfully constructed a recombinant plasmid pGEX-4T-1-femB and expression in E. coli.

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