Studies On Plasmid-mediated AmpC β-lactamase And Class Ⅰ Integrons In Isolates Of Escherichia Coli In Anhui Province

Objective:To investigate the production and the resistance of plasmid-mediated AmpCβ-lactamase-producing Escherichia coli and guide rational use of antimicrobial agents. To study the isoelectric point and the enzymatic hydrolyzing kinetics of the novel enzyme.To investigate the genotypes and epidemiology of class I integron in enzyme carrier, Escherichia coli isolates, and the gene cassettes in class I integron.Materials and Methods:Totally 355 strains of Escherichia coli were collected on 2007 from several hospitals of Anhui Province. All isolates were identified with the Microscan WalkAway-40 identification System, and excluded the repeated isolates from the same patients.The AmpC-producing isolates were identified by the three-dimensional test, the three-dimensional test positive strains were subsequent analysised by multiple PCR. The conjugation experiment was performed in AmpC-producing isolates, the susceptibility to antibiotics of AmpC-producing isolates of Escherichia coli were studied by agar dilution method. The entire encoding genes of the AmpC-type plasmid-mediatedβ-lactamase were amplified by PCR. The susceptibility to antibiotics of AmpC-producing isolates of Escherichia coli were studied by agar dilution method.The purified PCR products were ligated with pMD-18T 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. The whole ORF amplicon was linked into the vector Escherichia coli pHSG398. And then,the recombinant plasmid was introduced into the component cell E.coli JM109, and the transformant was selected on M-H agar plate supplemented with antibiotics. Agar dilution method was used to determine MICs against wild-type isolates its transconjugant and transformants.The crude enzyme was extracted from transformants by sonication method,pI values were determined using polyacrylamide gel by isoelectric focusing. We desired southern blot hydridization to demonstrate the novel gene was in plasmid. Kinetic parameters forβ-lactamas were detected by spectrophotometrically. Lastly, the AmpC-producing isolates of Escherichia coli were followed by PCR analysis with primers specific for classⅠintegron.The blaint1-positive strains were subjected to PCR analysis of gene cassettes.ClassⅠintegron gene cassettes were detected using PCR with primers previously described to amplify gene cassettes inserted between the 5' and 3' conserved sequences.Results:21 strains were confirmed as AmpC-producing isolates by the three-dimensional test. The percentage of AmpCs producing isolates in all isolates is 5.9%(21/355), and the number of plasmid-mediated AmpCs producing isolates in all isolates detected by conjugation is 16. Then BLAST program was used to ascertain the genotypes of the sequencing of PCR products at GenBank Sequence and Blastn results indicated that positive results for CIT group were 9 strains; positive results for DHA group were 6 isolates; for EBC group were only 3 strains. One single strain harboured both EBC and DHA gene. A novel EBC gene was confirmed by DNA sequence analysis (GenBank accession is FJ237369). All wild-type isolates exhibited the highest resistant rate to mostβ-lactams and were susceptible to cefepime, imipenem, meropenem. The clinical strain of the novel plasmid-mediated AmpCβ-lactamase-producing and its transformants had the same resistance spectrum, which exhibited the same high resistant rate to ampicillin, piperacillin, ceftazidime and cefoxitin. However, compared with wild-type strains, the transconjugants had decreased resistant ability to aztreonam, gentamicin, and ciprofloxacin.The novel AmpC beta-lactamase is mediated by the plasmid and its pI is 8.6, and the enzyme was not inhibited by clavulanic acid. Kinetic study of this enzyme suggested that it effectively hydrolyzed broad-spectrumβ-lactams. Affinity of thisβ-lactamase for cefoxitin was quite high and resulted in the high hydrolytic efficiency for substrates with measurable rates of hydrolysis. The spread of classⅠintegron and gene cassettes among AmpC-producing Escherichia coli was studied.The incidence of classⅠintegron was 16 strains of all. DNA sequence analysis demonstrated that the inserted gene cassettes with strong homology to the dhfrA17, aadA5, aadA3 and aadB genes, which confers resistance to trimethoprim and aminoglycoside antibiotics. blaACT-5 resistance gene sequences were not amplified in gene cassettes of the integron.Conclusion:It suggested that carbopenems and the fourth generation cephalosporins could be chosen in clinical empirical medication. Plasmid-mediated ampC genes were found in Escherichia Coli strains isolated from Anhui Province and CIT and DHA were the mainly epidemic genotypes in 2007. It is the first report that a novel plasmid-encoded class Cβ-Lactamase called ACT-5 in the world. GenBank accession number FJ237369. ClassⅠintegron test was positive in most of the AmpCβ-lactamase-producing islates. Gene cassettes confer resistance to trimethoprim and aminoglycoside antibiotics. Therefore, it is necessary to strengthen surveillance of antimicrobial resistance in local 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.