Study On The Mechanisms Of Resistance To Fluoroquinolones In Clinical Isolates Of Enterococci

Enterococci are common opportunistic pathogens and. currently ascendant nosocomial pathogens. Enterococci have become the second most common organisms recovered from nosocomial urinary tract and wound infection and the third most common cause of nosocomial bacteriemia in the United States. They are intrinsic resistant to β-lactam, low concentrations of aminoglycosides, clindamycin, and w and acquired resistant to fluoroquinolones, tetracycline, Chloramphenicol erythromycin and glycopeptides. As a result, Enterococci have emerged as one of the leading clinical challenges.The main mechanism of Enterococci resistant to fluoroquinolones include: (1). The target alterations caused by parC or gyrA mutantion (2). Reducing the plasma concentration of antibiotic by the multiresistant efflux pump. In this study, the Enterococcus faecalis and Enterococcus faecium were selected as the representative species to investigate the role of the efflux pump and the target mutation in Enterococci resistant to fluoroquinolones.Part Ⅰ: Antibacterial Activity of Clinical Commonly usedAntibiotics and Newer Fluoroquinolones to Clinical Isolated Enterococcus faecalis and Enterococcus faeciumObjective: To investigate the in vitro activity of commonly used antibiotic in clinical and new fluoroquuinolones (levofloxacin, gatifloxacin, and moxifloxacin) to Enterococcus faecalis and Enterococcus faeciumMethod: Clinical isolated strains of Enterococcus faecalis and Enterococcus faecium from 8 hospital in Tianjin during 2001-2002 were collected. The MIC of these bacteria were performed by disk diffusion and standard agar dilution. The data was analyzed by WHONET5 software offered by WHO.Results: 108 Enterococci were mainly collected from wards of internal medicine (41.7%), surgery (23.1%), and intensive care units (20.4%). More than half of the patients is elder than 60y (53.7%). In 108 clinical isolated stains, 66 is Enterococcus faecalis and 35 strains is Enterococcus faecium. The ratio of Enterococcus faecalis resistant to the Ampicillin is 28.8%, Enterococcus faecium resistant to Chloramphenicol is 22.9%. To other commonly used antibiotic in clinical the resistant rate is 30.3%~98.5% in Enterococcus faecalis, 68.6%~100% in Enterococcus faecium. The ratio of Enterococcus faecium resistant to new fluoquinolones, levofloxacin, gatifloxacin and moxifloxacin, is 28.8%,16.7%,12.1% respectively. To Enterococcus faecium the ratio is 88.6%-. 91.4% and 91.4%. No vancomycin resistant strains founded.Conclusion: The clinical isolated strain of Enterococci, include Enterococcus faecalis and Enterococcus faecium in this survey were resistant to the clinical commonly used antibiotic in different degree. In vitro activity of new fluoroquinolones to Enterococcus faecalis has been strengthened, especially in Gatifloxain and Moxifloxacin. This provide more selection in treatment of infection caused by Enterococcus faecalis. But there still need more study to find out effective agents in treating Enterococcus faecium infection. Vancomycin is still an effective means in treating the Enterococci infection .Part II Study on the Mechanisms of Resistance to Fluoroquinolones in Clinical Isolates of EnterococciObjective: To explore the mechanisms of resistance to fluoroquinilones involved target alterations and active efflux in clinical isolates of Enterococci.Methods : MICs of norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin in clinical isolates Enterococcus faecalis and Enterococcus faecium, were determined by an agar dilution method in the presence or absence of reserpine. Fragments of emeA were amplified by PCR and sequenced. The features of emeA of Enterococcus faecalis W79 and W62 were analyzed by bioinformatics. The expression level of emeA were detected by RT-PCR. The fragments of parC and gyrA QRDRs were amplified by PCR and sequenced. MICs of berberine in enterococci were determined by an agar dilution method in the presence or absence of reserpine or CCCP.Results: In the presence of reserpine, the rates of strains with MIC decreased of norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, moxifloxacin were respectively 100%(66/66),81.8%( 54/66),48.5%(32/66),6.0%(4/66),6.0% (4/66) and 4.5% (3/66) in Enterococcus faecalis, and were 100% (35/35) , 82.9% (29/35) ,20.7% (6/35) ,2.9% (1/35) ,0% (0/35) and 5.7% (2/35) in Enterococcus faecium respectively. The Fragments of emeA were amplified in all the 66 strains of Enterococcus faecalis The emeA sequence of W79 was submitted to GenBank and the accession NO. was DQ004851. Expression of emeA was increased in the Enterococcus faecalis strains with susceptibilities...