Resistance And Associated Resistance Mechanism Of Escherichia Coli Producing Plasmid-mediated AmpC β-lactamases

Objective:To investigate the production and the resistance of plasmid-mediated AmpCβ-lactamase-producing Escherichia coli and analyze their associated resistance mechanism,and guide rational use of antimicrobial agents.To study the biochemical properties of the novel AmpC-Type plasmid-mediatedβ-lactamase.Materials and Methods:Totally 402 clinical isolates of Escherichia coli were collected from 35 hospitals of Anhui province in 2005.M-H agar dilution method was used to determine MICs of 12 antimicrobial agents against wild-type isolates.The AmpC-producing isolates were choosed by cefoxitin,and identified by the three-dimensional test.The plasmid-mediated AmpCβ-lactamases were detected by Multiplex PCR.Extended-spectrumβ-lactamases(ESBLs) gene,inserted gene cassettes of class 1 integrons and active efflux system acrAB-tolC gene were amplified by PCR.The encoding genes of the novel AmpC-type plasmid-mediatedβ-lactamase were amplified by PCR.The purified PCR products were ligated with pUC-118 vectors, expressed in Escherichia coli JM109,and sequenced by Sanger's dideoxy chain termination composition method.Then blastn program was used to ascertain the genotype at GenBank.After digestion by EcoRI and BamHI,the whole ORF amplicon was linked into the vector Escherichia coli pHSG398 by T₄DNA lingase.And then,the recombinant plasmid was introduced into the component cell E.coli JM109,which transformed by CaCl₂ method,and the transformant was selected on M-H agar plate supplemented with 50μg/ml of chloromycetin and 60μg/ml of ampicillin.Agar dilution method was used to determine MICs against wild-type isolates,its transconjugants and transformants.The crude enzyme was extracted from transcojugant by sonication method,pI values were determined using polyacrylamide gel by isoelectric focusing.The enzymes were used for subsequentβ-lactamase assays,and checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Coomassie Blue staining. Southern blot was used to reveal that the gene encoding the novel enzyme was on a plasmid.Results:Among the 402 isolates collected,28(7.0%) produced plasmid-mediated AmpCβ-lactamases.The resistant rates of AmpCβ-lactamase-producing strains against most kinds of antibiotics were higher than the unproducing strains.They were all sensitive to imipenem and meropenem.Among the 28 strains,23 were multidrug-resistant,20 were determined as ESBLs gene, 21 as class 1 integrons sequence,and 20 as acrAB-tolC gene.Among the 28 isolates collected,genotypes of AmpCβ-lactamases were CIT(11), EBC(7),DHA(7),ACC(2) and MOX(1),respectively.DNA sequence analysis conformed that 1 CIT and 1 EBC were novel ampC gene(GenBank accession is EF054895 and EF417572,respectively).One of novel enzyme had been designated as MIR-4 by G.A.Jacoby.A clinical strain of the novel plasmid-mediated AmpCβ-lactamase-producing and its transconjugant had the same resistance spectrum,which exhibited the same high resistant rate to ampicillin,cefuroxime,cefotaxime,ceftriaxone,and cefoxicin. However,compared with wild-type strains,the transconjugant had decreased resistant ability to cefuroxime,ceftazidime,eefepime,aztreonam,gentamicin,and ciprofloxacin. This novel enzyme with apparent pI of approximately 8.2 was identified,transferred by conjugation and associated with a plasmid.Southern blot revealed that the gene encoding MIR-like was on a 54-kb plasmid.Kinetic study of this enzyme suggested that it effectively hydrolyzed broad-spectrumβ-lactams.Affinity of thisβ-lactamase for ceftazidime was quite low and resulted in the lowest hydrolytic efficiency for substrates with measurable rates of hydrolysis.Conclusion:The resistant rates of AmpCβ-lactamase-producing E.coli against most kinds of antibiotics reach a high level.The AmpC producers are multidrug-resistant and have several resistance mechanisms.Class 1 integrons and production of AmpC and ESBLs are the main resistance mechanisms.Carbopenems,amikacin and fourth cephalosporins should be chosen in clinical empirical medication.CIT,EBC and DHA were the mainly epidemic genotypes of plasmid-mediated AmpCβ-lactamase in our area.Meanwhile,two novels of subtypes were found on the basis of the AmpC-type enzymes,and multiply resistance mechanisms evolved in those isolates. Therefore,it is necessary to strengthen surveillance of antimicrobial resistance in local areas and exchange data between different areas.The rational use of antimicrobial agents may improve the situation.There is extremely important epidemiology significance in this work to prevent dissemination of resistant genes.