| Cephalosporin antibiotics were usually used to treat infectious diseases which were caused by bacterias.The existence of the extended spectrum ?-lactamases(ESBLs)conferred the bacteria resistant to antibiotic.The ESBLs drug resistance genes were located in the plasmid,which could transfer horizontally between different bacterias,causing great disturbance to the treatment of infectious disease.At present,the distribution of the ESBLs of CTX-M had been reported,while it was not clear that the transfer regularity of CTX-M genes between bacterias and the mechanism of ESBLs strains resistance to cephalosporins.In the study,we aimed to describe the distribution of drug-resistance in Escherichia coli(short for E.coli)which were isolated from hospital,analyze the type of CTX-M-carrying plasmids which were obtained from clinical and Gen Bank E.coli and explore the role of CTX-M in the resistance to cephalosporin.There was important public health significance for elucidating the mechanism of ESBLs strains resistance to cephalosporins and controlling the epidemic of ESBLs-positive strains.Objective 1 To describe the distribution of drug-resistant phenotype,resistance genes and class I integron in the E.coli which were isolated from hospital and analyze the molecular distribution of ESBLs-positive clinical E.coli.2 To analyze the plasmid types containing the gene of CTX-M-15 and CTX-M-55 and to master how the plasmid was transferred.3 To explore the effect of cephalosporin on CTX-M-55 gene.Methods 1 Identification of ESBLs-positive strains and susceptibility test were performed by Kirby-Bauer slip diffusion,PCR was performed to amplify the genes.2 CGE web server was used to collect the information of MLST,the types of plasmids and drug-resistant genes,and the e BURST software was used to draw the dendrogram of MLST results.3 The electroporation was used to transfer the drug resistance plasmid which carried the gene of CTX-M-55 into the DH5?.The micro-broth dilution method was used to test the minimum inhibitory concentration of the transformants of cefotaxime and ceftazidime.The transformants were induced by the sub-inhibitory concentration of cefotaxime and ceftazidime,and the MIC of the induced strains were detected after induction.The RT-PCR was implemented to compare the expression level of CTX-M-55 gene between the experimental group and the control group.Results 1 The distribution of drug resistance was analyzed in E.coli of clinical isolates.The positive rate of ESBLs in 181 strains of E.coli was 65.75%.In all E.coli,the antibiotics resistance rate of AMP,CZO,CXM,CRO,CEP,CAZ,CTX and FEP were 97.79%,86.74%,79.56%,75.69%,78.45%,51.93%,77.35% and 53.04%,respectively.In terms of cephalosporin antibiotics,the resistance rates of ESBLs-positive strains were higher than those in negativestrains and there was a statistical difference(P< 0.05).2 The drug-resistant genes was analyzed in clinical E.coli.Among all the ESBLs-positive strains,the gene of CTX-M existed in 118 strains,and the positive rates of TEM,OXA-1,aac(6')-Ib-cr,qnrs,and SHV were 52.94%(63/119),24.37%(29/119),29.41%(35/119),5.88%(7/119),3.36%(4/119),respectively.In the only one type of CTX-M-carrying isolates,the CTX-M-14(32/108)played a leading role,followed by CTX-M-15(30/108),CTX-M-55(17/108)and CTX-M-27(17/108),and the main combination of CTX-M types were CTX-M-55+CTX-M-1(n=4)and CTX-M-55+CTX-M-27(n=4).3 The class I integron was detected in ESBLs-positive E.coli.Among all the CTX-M-positive strains,94 isolates carried class I integron.Among the int I1+CTX-M-positive E.coli,73 isolates were tested positive for the presence of variable regions,while 21 of the isolates could not be amplified.Furthermore,the predominant gene cassette was dfr A17-aad A5(60/73)in the variable regions positive isolates.In the CTX-M-14-carrying strains,the amounts and kinds of the gene cassette were the most.4 The distribution of drug-resistant genes and MLST types were described in CTX-M-15-carrying E.coli which were collected from Gen Bank.All the 232 strains were clustered into 37 sequence types(STs),and among them ST131(35.24%,82/232)was the dominant type.The number of the drug resistance genes ranged from 1 to 22,of which ST131 and ST617 harbored the maximum genes.The positive rate of genes of OXA-1,aac(6')-Ib-cr,aac(3)-II,aad A,mph(A)in ST131 were higher than other STs.What's more,there were statistic differences about distribution of those genes between ST131 isolates and other STs(P<0.05).5 The subtypes of the plasmids were analyzed.The incompatibility plasmids were nine types in 232 E.coli of Gen Bank,of which Inc F plasmids existed in 221 isolates.The Inc F subtypes of F36:A4:B1 and F31:A4:B1 existed in ST131 and many STs which belonged to one clonal complex CC10(ST10,ST44,ST744,ST167,ST617,ST1284,ST3835).During analyzing the types of plasmids,thirteen CTX-M-55-positive plasmids could be divided into four types,namely Inc F(5/13),Inc I1(4/13),Inc N(2/13)and Inc H1(2/13).Further typing of the Inc F plasimids revealed two subgroups: F18:A-:B1(n=2)and F33:A-:B-(n=3).Inc I1 plasmids were classified into two STs,with Inc I1/ST167(n=2)and Inc I1/ST167(n=1),while one plasmid could not be typed.The plasmid of F33:A-:B-could exist in the strains of ST10 and ST617 which had close phylogenetic relationship and Inc I1 plasmids were found consisted in closely related STs(ST10 and ST167).6 The expression of CTX-M-55 was detected in transformed strain.Under the subinhibitory concentration of the cefotaxime,the expression of CTX-M-55 in the transformed strain was increased in experimental group compared with the control group and the difference was statistically significant(P< 0.001).Conclusions 1 The drug resistance of cephalosporin antibiotics was very serious in clinical E.coli.2 The class I integron was widely distributed in ESBLs-positive E.coli which were isolated from the clinic,and the variable regions presented diversely.3 Among the E.coli,the same p MLST subtype Inc F plasmid could transfer to the isolates which had the intimate relationship.4 The Cefotaxime could enhance the expression of CTX-M-55 in transformed strain. |
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