Study On AMEs And Fluoroquinolones Resistance Genes Of Staphylococcus Aureus

Objective: Staphylococcus aureus(SA), an aerobic, non-fermentative, Gram-negative rod, resides in the nature generally, like various source of water、soil、animal and human body; It is also found in hospital environments such as dialysis apparatus,centr-A/VP tester、artificial respiration device and ventilation pipe. SA plays an important role as an opportunistic nosocomial pathogen in recent years,its detection rates in hospitalized patients were increased and it can cause a broad spectrum of nosocomial infectious, e.g: pneumonia, meningitis, endocarditis, cellulitis, urinary tract and wound infection, bacteremia and sepsis. The majority of SA clinical isolates are intrinsically resistant to many antimicrobials, which caused great difficulties for clinical treatment. Therefore, research on the Methicillin-resistant Staphylococcus aureus( MRSA) and the dissemination of resistance genes of SA have great clinical significance.To clone and express 2 drug resistance genes adenylyltransferase, NORA of SA, provide the material for further study about crystal structure of adenylyltransferase and NORA protein, lay foundation to understand the characteristics and function of expression products above, and lay foundation to monitor and control nosocomial infections caused by SA from the molecular level.Methods: Used the chromosome DNA which extracted from SA clinical isolated as template, amplified the adenylyltransferase, NORA genes by PCR, the PCR products were ligated with p GEX-4T-1 vector, and transformed to E.coli DH5α respectively. Extracted recombinant plasmid were identified by double enzyme digestion and sequenced. Recombinant protein were expressed in E.coli BL21 under the induction of IPTG. The expression of the recombinant adenylyltransferase, NORA were analyzed by SDS-PAGE and confirmed by western blot. Antibiotic resistance of recombinant p GEX-4T-1-adenylyltransferase were analyzed. The prevalence of adenylyltransferase, NORA in SA clinical isolated were identified by PCR method.Results: Used the chromosome DNA as template, amplified production were about 783 bpand 1167bp and successfully cloned into T vector. Sequence analysis showed that adenylyltransferase contained an open reading frame of 793 bp, which encoded a 29.9k Da protein; NORA contained an open reading frame of 1173 bp, which encoded a 44.9k Da protein; Recombinant p GEX-4T-1-adenylyltransferase/NORA plasmids were constructed. The recombinant protein was expressed as a fusion protein with a GST tag in E.coli BL21 induced by IPTG. Two major protein bands corresponding to the expected recombinant GST- fusion proteins(55k Da, 69 k Da respectively) were indentified by SDS-PAGE and western blot. Recombinant p GEX-4T-1-NORA/BL21 has no effects on levofloxacin and gatifloxacin, but a reduction in the susceptibility with ciprofloxacin. The adenylyltransferase gene was identified in 9(40%) of the 22 SA isolates; The NORA were identified in 10(45%) isolates.Conclusion:SA adenylyltransferase,NORA were coloned and expressed in p GEX-4T-1 plasmid, which made a good foundation for understanding the characteristics and function of above. The prevalence of adenylyltransferase, NORA gene in SA clinical isolation were 40%, 45% respectively.