Study On The Susceptibility And Resistance Mechanism Of Enterococci To Fluoroquinolones

Objective: To detect the distribution of clinical isolates of enterococci in clinical specimens and determine the susceptibility of fluoroquinolones against enterococci for the guidance of rational use of antibiotics and effective surveillance of nosocomial infections. To understand the resistance mechanism of enterococci to FQs by studying the correlation of gene mutations of type II topoisomerase with the resistance to FQs and exploring whether active efflux system exsist in enterococci or not. Method: The in vitro activities of 6 fluoroquinolones against 78 clinical isolates of enterococci were compared by the agar dilution mothod. Clinical isolates showing different resistance to cipfloxacin were selected. Polymerase chain reaction(PCR) was used to amplify the quinolones resistance-determining region(QRDR) of gene gyrA, gyrB, parC and parE, and amplicons were sequenced. The nucleotide sequences of resistant isolates were compared with the reference strain's counterparts, and analysised the correlation of gene mutation with the resistance to FQs. The accumulation of ciprofloxacin in enterococci and the influence of reserpine were measured by a fluorescence method.Results: Among 78 clinical isolates, there were 61(78.2%)strains of E.faecalis, 15(19.2%)of E. faecium, 2(2.6%)of other enterococcus species.The enterococcus isolates rates from urine, secretion with pus, sputum, and others were 61.6%. 25.6%, 7.7%, and 5.1% respectively.In decreasing order, the relative activities (MIC₅₀S, MIC90S) of 6 fluoroquinolones against enterococci were as follows: tosufloxacin (MIC₅₀,0.5mg/L;MIC9o, 2mg/L) >gatifloxacin (MIC50, 1mg/L; MIC90, 4mg/L), sparfloxacin (MIC50, 1mg/L; MIC90, 4mg/L) >levofloxacin (MIC50, 2mg/L; MIC90, 16mg/L )>ofloxacin (MIC₅₀, 4mg/L; MIC90, 64mg/L), cipfloxacin (MIC50, 4mg/L; MIC90, 64mg/L).Among 6 fluoroquinolones, tosufloxacin was the most active while ofloxacin and ciprofloxacin were the least active. According to the criteria of MICs≥4mg/L to cipfloxacin, 11 cipfloxacin-resistant E.faecalis isolates,8 cipfloxacin -resistant E. faecium isolates were selected from78 isolates.Type II topoisomerase genes were amplified by PCR ,the products were sequenced and analyzed. Results found that a G to T or T to G change at 98nt or 99nt of gyrA QRDR of E.faecalis led to the substitution of Ser83 by isoleucine or arginine.while Ser83(AGT) change to arginine(AGG) among E.faecium strains. A A to G change at llOnt of gyrA QRDR of E.faecalis and a G to A change at 109nt of gyrA QRDR of E.faecium led to the substitution of Glu87 by glycine or lysine. A G to T or T to G change at 96 or 97nt of parC QRDR of E.faecalis led to the substitution of Ser80 by isoleucine or arginine,while Ser80(AGC) change to isoleucine(ATC) among E.faecium . A A to C change at 108nt of parC QRDR of E.faecalis led to the substitution of Glu84 by alanine. The strains' MIC of single mutation occurred in parC was between the strains without mutation and with both gyrA and parC mutations. No amino acid change observed in gyrB and parE.The quantity of ciprofloxacin accummulation was detected by Fluorescence assay .Results showed that the first 5 min was key to ciprofloxacin accummulation,and then accummulation was limited.The quantity of ciprofloxacin accummulation of resistant strains was less than that of susceptible strains, reserpine had different influence on clinical isolates of enterococci. Reserpine could dramatically increased the accumulation of ciprofloxacin, espesically in ciprofloxacin-resistant strains. Conclusion: 1. Among enterococcal species, E.faecalis is the most frequently,followed by E.faecium ,other enterococcal species are unusual.2. New fluoroquinolones such as tosufloxacin, gatifloxacin and sparfloxacin are more active against enterococci than the older fluoroquinolones; Enterococci has varying degree of resistance to commonly used fluoroquinolones.3. The mutations of 83 and 87 condon of gyrA and the mutations of 80 and84 condon of parC genes are related to fluroquinolone resistance,and the mutations of 80 condon of parC genes are more important. The low-level cipfloxacin-resistance strains are detected a single mutation in parC gene but high level cipfloxacin-resistance are associated with simultaneous alterations in both GyrA and ParC. Maybe topoisomerase IV is the primary target of cipfloxacin in enterococci.