Analysis Of The Resistance And Molecular Biology Property Of CTX-M-Type ESBLs-Producing Strains Of Anhui Province

Objective:To study the resistance of the CTX-M-producing Escherichia coli strains against 18 antibiotics, and offer the resistance data to clinical therapy. For the rational use of antimicrobial agents to provide a basis and is conducive to the prevention of producing CTX-M-type extended-spectrumβ-lactamase prevalence of Escherichia coli.To study the biochemical properties of the novel CTX-M enzyme and the resistance of the novel enzyme producing strains. Research from the hospital in Anhui Province at all levels if a new collection of strains resistant gene and protein molecular structure caused by changes in locus of change, with a view to discover new enzyme active site, and found a new gene cloning and expression, to study its biological characteristics, specifically its function, in order to design new drugs based on targets, thereby reducing mortality, reducing medical costs.Materials and Methods:Isolates:Totally 416 strains of ESBLs-producing Escherichia coli were collected between 2006 in 41 hospitals of Anhui Province.Method:Adopting PCR methods, universal primers of blaCTX-M were used for CTX-M+ isolates screening; according to the universal primers PCR results, two pairs of entire coding gene primers were designed, and to convenient the consequence experiments, endonucleotide cleavage site of EcoRΙand BamHΙwere added at 5'terminal of the upstream and downstream of the two groups of primers. The encoding genes of the CTX-Mβ-lactamase were amplified by PCR. The purified PCR products were ligated with pUC-118 vectors, expressed in Escherichia coli JM109, single-strand conformation polymorphism was used for choosen the mutant possible. The sequence was determined by direct sequencing of PCR products, carried out by the dideoxy chain termination procedure of Sanger on an automatic sequencer. 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 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 cefotaxime (2mg/L).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.Results:Positive amplification results were observed for 217 E.coli isolates which expressing CTX-M-M type of ESBLs phenotype. The percentage of CTX-M-producing isolates ESBLs positive strains was 52.2%. All wild-type isolates exhibited the highest resistant rate to piperacillin,cefuroxime and cefatrioxime, and all strains were susceptible to imipenan. The resistance of the transconjugants has decreased, and the susceptibility to ciprofloxacin and levofloxacin have obviously enhanced.53 and 164 strains were confirmed positive for CTX-M-1 group and CTX-M-9 group entire coding gene primers, respectively. Of 19 strains were positive for the both group primers. SSCP data showed that one stain of CTX-M-1 is the possible mutant, and four stains of CTX-M-9 were the possible mutants.Sequence and blastn results indicated that one in five the stains which choosen were carried novel CTX-M type gene (GenBank accession is EU545409. The novel enzyme had been designated as CTX-M-87 by G. A. Jacoby.Compared to the wild-type isolate, the correlative transformant elevated the susceptible to all antimicrobial agents, but still exhibited a moderate resistant to ampicillin,cefotaxime, cefuroxime and ceftriaxone.This novel enzyme with apparent pI of approximately 9.1 was identified, transferred by conjugation and associated with a plasmid. Kinetic study of this enzyme suggested that it effectively hydrolyzed broad-spectrumβ-lactams. Affinity of thisβ-lactamase for ceftazidime and aztreonam were quite low and resulted in the lowest hydrolytic efficiency for substrates with measurable rates of hydrolysis.