Study To Plasmid-mediated Genotypes Of CTX-M-14and CTX-M-15in Multiple Drug-resistant Escherichia Coli

Abstract objective: To explore the genotypes and drug-resistance ofCTX-M-14and CTX-M-15in plasmid-mediated drug resistance Escherichiacoli(E.coli) from the Affiliated Hospital of Lu Zhou medical college, Andinvestigate the plasmid curing effect by EB in E.coli. Methods:1Genotypesof CTX-M-14and CTX-M-15in E.coli.1.1The Kerby-Bauer disk diffusion:25strains selected were multi-drug resistance strains,15strains of dual-drugresistance strains,16strains of simple-drug resistance strains, which71strainsof E.coli from the Affiliated Hospital of Lu Zhou from July2007to July2008were isolated.1.2PCR technique: The plasmid DNA was refined according tospecification. CTX-M-14, CTX-M-15genes were amplified by PCR technique.Gene primer sequence of CTX-M-14, P1:5′-G A A A G A G A G T G C A A CG G A T G-3′, P2:5′-A T T G G A A A G G G T T C A T C A C C-3′; Geneprimer sequence of CTX-M-15, P1:5′-A A A A A T C A C T G C G C C A GT T C-3′, P2:5′-A G C T T A T T C A T C G C C A C G T T-3′.The volumeof PCR reaction was26μl(5.5μl of ddH2O,12.5μl of2×TaqPCR MasterMix,6μl of refined DNA template,1μl of each primer. Amplification was running asfollows: Initial denaturation at94℃for3min, denaturation at94℃for30s,annealing at55℃for30s, extension at72℃for60s, amount of30cycles,and a final step of72℃for5min. PCR products were visualized in theultraviolet light after2%agarose gels electrophoresis, and photographed withgelatin camera, then sent to ShangHai Biotechnology co. for gene sequencing. 1.3The delivering of strains carrying CTX-M-14, CTX-M-15positive andnegative in E. coli: Drug-resistance of strains to7antibiotics was comparedbetween the positive and negative of E.coli CTX-M-14, CTX-M-15.2Plasmidcuring in multiple drug-resistant E.coli:21strains of E.coli carryingCTX-M-14from multi-drug resistance group were selected. PCR techniquewas used to amplify the plasmid carrying CTX-M-14gene in the E.coli, whichgrew in LB broth with30μg/ml of EB for64hours and120hours respectively.Drug-resistance of E.coli to antibiotics was compared between two groups ofbefore and after plasmid curing. Results:1. Genotypes of CTX-M-14andCTX-M-15in E.coli:1.1Drug-resisitance rate of IMP was0, AMK12.5%,CAZ55.4%, LEV60.7%, GEN62.5%, AZT71.4%, CTX83.9%respectivelyin56drug-resistance strains.1.2The plasmids with genotypes CTX-M-14,CTX-M-15among56drug-resistance strains had44(78.6%),28(50%)respectively, and96.0%,48.0%respectively among25multiple drugresistance strains,53.3%,60.0%respectively among15dual-drug resistancestrains,75.0%,43.8%respectively among16simple-drug resistance strains.Chi-square test shew that it has statistical significant(P＜0.05) the differencebetween the two groups of multiple and dual drug-resistance CTX-M-14positive strains. The difference between the two groups of multiple anddual-plus-simple drug-resistance CTX-M-14positive strains shew statisticalsignificant(P＜0.05).1.3The difference between the two groups of CTX-M-14positive and negative to7antibiotics resistance did not show statisticalsignificant(P＞0.05).The difference between the two groups of CTX-M-15 positive and negative to AZT,CTX resistance shew statistical significant(P＜0.05). The difference between the two groups of CTX-M-15positive andnegative to IMP,GEN,A,LEV,CAZ resistance did not show statisticalsignificant(P＞0.05).The difference between strains with CTX-M-14,CTX-M-15gene to CTX and CAZ resistance shew statistical significant(P＜0.05).2The results of plasmid curing in multiple drug resistance E.coli:2.121strains of the E.coli were still detected carrying CTX-M-14when theygrew in LB broth with30μg/ml of EB for64hours; one of21strains was notdetected carrying CTX-M-14when they grew in LB broth with30μg/ml of EBfor120hours.2.2It shew statistical significant(P＜0.05) the differencebetween strains to Streptomycin resistance before and after plasmid curingexperiments for120hours. It did not show statistical significant(P＞0.05) thedifference between strains to susceptibility of FOX, CIP, PRL/TZB, PRL, CFT,CTX, CAZ before and after plasmid curing experiments for120hours.Conclusion:1. The strains of plasmid carrying CTX-M-14and CTX-M-15inthe drug-resistance E.coli are common.2. multiple drug-resistance CTX-M-14positive strains is more than other strains in E.coli.3. The drug-resistancerate of CTX is higher than that of CAZ in the CTX-M-14, CTX-M-15positiveE.coli.4. The effect may need more further studies about plasmid curing inE.coli by using30μg/ml EB.