Resistance And Associated Resistance Mechanism Of Klebsiella Pneumoniae Producing CTX-M β-lactamases And Characterization Of The Novel CTX-M-Type β-lactamase

Objective:To investigate the production and the resistance of CTX-Mβ-lactamase-producing Klebsiella pneumoniae and analyze their associated resistance mechanism, and guide rational use of antimicrobial agents.To investigate the genotypes and epidemiology of CTX-M enzyme carrier, K. pneumoniae isolates, in several hospitals of Anhui Province. To study the biochemical properties of the novel CTX-M-Typeβ-lactamase.Materials and Methods:Totally105 strains of ESBLs-producing Klebsiella pneumoniae were collected on 2006 from several hospitals of Anhui Province.Adopting PCR methods, universal primers of blaCTX-M were used for CTX-M+ isolates screening; Conjugative transfer method was used between CTX-M+ isolates and E.coli C600 to transfer resistance plasmid.The entire encoding genes of the CTX-M-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 EcoRΙand BamHΙ, 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 transformants.The crude enzyme was extracted from transformants 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). ERIC-PCR method was carried to analyze the homology of novel CTX-M-producing isolates. Lastly, kinetic parameters forβ-lactamas were detected by spectrophotometricallyResults:Positive amplification results were observed for 52 K.pneumonia isolates which expressing ESBLs phenotype.. The percentage of CTX-M-producing isolates in all isolates and ESBLs positive strains were 23.4% and 49.5%, respectively. Sequence and blastn results indicated that positive results for CTX-M-1 group were comprised of 12 blaCTX-M-22-carried strains, 2 blaCTX-M-15- carried strains and 1 blaCTX-M-3- carried strain; positive results for CTX-M-9 group were comprised of 35 blaCTX-M-14-producing isolates; CTX-M-2 .CTX-M-8 group were not detected . All wild-type isolates exhibited the highest resistant rate to mostβ-lactama and were susceptible to piperacillin/tazobactam,amikacin,and ceftazidime, respectively.Two clinical strains of the novel plasmid-mediated CTX-Mβ-lactamase-producing and its transformants had the same resistance spectrum, which exhibited the same high resistant rate to ampicillin, cefuroxime, cefotaxime, and cefurxime. However, compared with wild-type strains, the transconjugant had decreased resistant ability to cefuroxime, ceftazidime, aztreonam, gentamicin, and ciprofloxacin.This novel enzyme with apparent pI of approximately 8.1, 8.4 was identified, transferred by conjugation and associated with a plasmid. The two new lactamases molecular weight were approximately 28Kda. The analysis of ERIC-PCR indicated that Two isolates, displayed a different pattern.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 CTX-Mβ-lactamase-producing K.pneumonia against most kinds of antibiotics reach a high level. CTX-M-14, CTX-M-22 were the mainly epidemic genotypes of plasmid-mediated CTX-Mβ-lactamase in our area. Meanwhile, two novels of subtypes were found on the basis of the CTX-M -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.