| Quinolones has a wide antimicrobial spectrum, especiallyagainst Gram-Negative bacillus. Quinolones kill bacteria byinhibiting the activity of gyrase and type IV topoisomerase. Becauseof widespread usage of antibiotic, many bacteria obtained the abilityof resistance to antibiotics including ciprofloxacin. The increasedincidence of drug resistant P. aeruginosa obtained in recent yearsmay be due to selective pressure caused by drug abuse. The mechanisms of drug resistance had been widely study in lastdecade, and the mechanisms to various antibiotics are known asfollows: (1). alteration of target sites; (2). existence of degradingenzymes; (3). modification of antibiotics; (4). reduced permeability;(5). existence of efflux pump. As all quinolones act in the same way,resistance to one member of the class will also confer decreasedsusceptibility to all members of the family. In the present study, we in vitro mimicked the progress of P.aeruginosa ciprofloxacin resistance occurred in human body andpresented molecular genetic analysis of the resistance. During theserial stimulation of drug in gradient concentrations formed bydiffusion of drug on MH agar medium (although the exactconcentrations of drug in MH agar were not known), the bacteria candevelop the ability of drug resistance in vitro as in vivo. The earliestmutation in QRDRs of gyrA was detected by agar disk diffusion testscombined with PCR-SSCP method. As compared with DNAsequencing, the results of PCR-SSCP showed the combinationmethod could precisely provide genetic information about the one ormore mutations in gene fragments and may be used to detect earlymutations in QRDRs during quinolone resistant process ofpathogenic bacteria. As the concentration of the gel was regarded as the mostimportant factor which affects the resolution of single strand DNAmigration in SSCP analysis. We used different concentration ofPAGE to test which concentration can provide the most informationabout mutations. The conformations with hyper-spatial-impedimentcan be separated by lower concentration of PAGE (5% 6.5%), andthe conformations with hypo-spatial-impediment can be separated byhigher concentration of PAGE (6.5% 8%). This hinted that the bestconcentration of PAGE (6.5% in this study) used for SSCP couldgive the maximum resolution of all single strand DNA shift bands ofamplified fragments and may be different for detection of differentfragments of DNA. Only one mutation (Thr83Ile) in P. aeruginosa of bothATCC27853 and clinical isolate was observed, this suggests that themutation may be the most important to quinolones resistance. Theearliest time of gene mutations in process of P. aeruginosainteracting with ciprofloxacin was once the diameters of zone ofcomplete inhibition less than 21mm, the gene mutations of gyrA canbe detected by both SSCP and sequencing. In practice, the rangewhose diameters of zone sizes of 20mm and of 16mm wereregarded as "intermediate" category, which includes isolates withantimicrobial agent MICs that approach usually attainable blood andtissue levels and for which responses rate may be lower than forsusceptible isolates, and implies clinical applicability in body siteswhere the drugs are physiologically concentrated or when a highdosage of a drug can be used. However, from the study, once the P.aeruginosa entered "intermediate" category, at least one base inQRDRs of gyrA mutated and could lead to drug resistance. From the results of serially subcultures without ciprofloxacinwe observed once the gene mutations occurred, the mutated QRDRscould not be repaired and the phenotype of mutant would be resistantto quinolones. This suggests that the first reason of drug resistancewas the gene mutation after the drug interacted with its targets, DNAgyrase or type IV topoisomerase. From the results, the whole process of ciprofloxacin resistancecould be consist of two stages: susceptibl...
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