Mutant Prevention Concentration And Molecular Resistant Mechanism Of Fluoroquinolones Against Acinetobacter Baumannii

Objective(1)To establish a method to determine mutant prevention concentrateion (MPC) in vitro. The potential for restricting the selection of resistant mutants of four fluoroquinolones (ciprofloxacin, pazaufloxacin, levofloxacin, gatifloxacin) with different structures against Acinetobacter baumannii was compared. (2) The effect of drug concentration, drug structure on the resistant gene was discussed and the process of Acinetobacter baumannii from sensitivity to resistance was studied. (3) The rationality of clinical use of fluoroquinolones dicussed according to the MSW and the PK/PD theory.Method(1)The agar dilution method was carried out on Mueller-Hinton agar containing fluoroquinolones according to CLSI guidelines to determine minimal inhibition concentration(MIC) of ciprofloxacin, pazaufloxacin, levofloxacin and gatifloxacin (CPLX, PZLX, LVLX, GTLX)against ATCC19606. Mutant prevention concentration was determined by the agar dilution according to MIC determining except that ATCC19606 were enriched in broth and the bacterial concentrations were adjusted to 1010 colony form units per milliliter. The fraction of bacteria recovered curve was traced by colony counting method.The MSW,MPI and the mutant frequency of four fluoroquinolones against ATCC19606 and the mutations. (2)The two quinolone resistance-determining regions of target genes, gyrA and parC of mutants selected in the MSW were obtained by PCR method and sequenced by DNA sequencing and BLAST.(3) The MPC and MPI ( MPC/MIC ) of four fluoroquinolones (ciprofloxacin, pazaufloxacin, levofloxacin, gatifloxacin) against 34 ciprofloxacin sensitive clinical strains of Acinetobacter baumannii. The potential for restricting the selection of resistant mutants of four fluoroquinolones against Acinetobacter baumannii was compared.Results(1) The MIC of CPLX,PZLX,LVLX and GTLX against ATCC19606 was 1,1,1,0.5μg/ml. The MIC99 of four fluoroquinolones against ATCC19606 order was generally CPLX>PZLX>LVLX>GTLX; The MPC order was generally CPLX>PZLX>LVLX>GTLX. The MPI order was generally CPLX>PZLX> LVLX>GTLX .The bacteria recovered curve showed that the MSW of GTLX was narrower than that of CPLX, LVLX and PZLX. The mutant frequency of ATCC19606 GTLX was the smallest, LVLX and PZLX were the smaller and CPLX was the largest. In low concentration ,the potential for restricting the selection of resistant mutants of GTLX was stronger than that of LVLX,PZLX and CPLX ,but it was not obviously in hight concentration . (2)The 37 one-step mutants were seleted by CPLX,LVLX,PZLX and GTLX .The one-step mutants were resistant to CPLX and PZLX ,but to LVLX and GTLX were still sensitive or middle-resistant . The QRDRs of ParC and GyrA were sequenced with the 37 one-step mutation Acinetobacter baumannii.Two of 37(5.4%) had mutations both in GyrA and ParC .Twenty-one of 37 (51.4%)mutants had mutations in GyrA and six of 37 (16.2%) isolates had mutations in ParC.(3) The MPCs for 90% of the isolates(MPCpr90) of CPLX,PZLX, LVLX and GTLX for 34 ciprofloxacin-susceptible Acinetobacter baumannii clinical isolates were 8,16,2and 1μg/ml,and the MPCpr90/MIC90 order was generally CPLX>PZLX> LVLX>GTLX. The MPC/MIC of the clinical isolates were 8-16 times to CPLX and PZLX,and 4 times to LVLX and GTLX.ConclusionThe potential for restricting the selection of resistant mutants of GTLX was stronger than that of LVLX, PZLX and CPLX,but it was not obviously in hight concentration. It means that enough dose LVLX, PZLX and CPLX can restrict the resistant mutation,but we must pay more attention to the toxic reation caused by the high blood concentration.The mutants seleted by LVLX and PZLX had a wider MSW than ATCC19606 and a new mutant could be easier to be seleted. CPLX,PZLX,LVLXand GTLX concentrations have strong effect on the recovery of resistant colonies and selection of resistant alleles.Combined with pharmacokinetic parameters, the potential for restricting the selection of resistant mutants of GTLX was stronger than that of LVLX, PZLX and CPLX,but it cann't restrict the next-step selective enrichment of resistant mutants among ciprofloxacin-or levofloxacin-resistant Acinetobacter baumannii. DNA gyrase and topoismerase are the primary target of fluoroquinolone against Acinetobacter baumannii to be seleted.The mutation at position gyrASer81→Leu was the most frequent. When the resistant colonies was in low level resistant condition, the mutation at positionIV parC Ala→Asp. When the resistant colonies was in severely resistant condition, mutations occurrence both in gyrA and parC .