| ObjectiveTo study the resistance of the SHV-producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) strains against 15 antimicrobial agents, and offer the resistance data to clinical therapy.To investigate the genotypes and epidemiology of SHV enzyme carrier, E. coli and K. pneumoniae isolates, in several hospitals of Hefei, Anhui Province.To study the biochemical properties of the novel SHV enzyme and the resistance of the novel enzyme producing strains.Materials and MethodsIsolatesTotally 98 strains of ESBLs-producing E. coli and K. pneumoniae from 299 clinical strains were collected between 1999 and 2000 from four hospitals in Hefei.MethodsAdopting PCR methods, universal primers of blaSHV were used for SHV+ isolates screening; matting method was used between SHV+ isolates and E. coli C600 to transfer resistance plasmid; agar dilution method was used to determine MICs of 15 antimicrobial agents against wild-type isolates and the transconjugants.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 SHV-typeβ-1actamases produced by 3 transconjugant were amplified by PCR. The purified PCR products were ligated with pGEM-Teasy vectors, expressed in E. coli DH5a, and sequenced by Sanger's dideoxy chain termination composition method, on an ABI377 automatic sequencer (Invotrigen, Shanghai, China). Then BLAST 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 T4 DNA 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 Mueller-Hinton (M-H) agar plate supplemented with 50μg/mL of chloromycetin and 60μg/mL of ampicillin. Agar dilution method was used to determine MICs of 13 antimicrobial agents against transformants.The crude enzyme was extracted from transcojugant by sonication method,pI values were determined using polyacrylamide gel by isoelectric focusing.Pulsed-field gel electrophoresis method was carried to analyze the homology of novel SHV-producing isolates.ResultsPositive amplification results were observed for 24 strains of K. pneumonia and 5 strains of E. coli which expressing ESBLs phenotype. The percentage of SHV-producing isolates in all isolates and ESBLs positive strains were 9.7% and 29.6%, respectively. 21 strains of K. pneumonia and 5 strains of E. coli were successfully conjugated to the recipient cell. All wild-type isolates exhibited the highest resistant rate to ampicillin, piperacillin, cefuroxime and ceftriaxone, and all strains were susceptible to imipenan and meropenem. The susceptibility of the transconjugants has increased, and the susceptibility to ciprofloxacin and levofloxacin have obviously enhanced.Sequence and BLAST results indicated that positive results for SHV-type were comprised of 8 blaSHV-1-carried strains, 5 blaSHV-11-carried strains, 12 blaSHV-12-carried strains, 1 blaSHV-18-carried strains, 1 blaSHV-28-carried strains, and 3 novel blaSHV-harboured which amino acid substitution occurred at two positions (Leu35Gln, Met129Val), in comparison with the parent enzyme, SHV-1(Accession No. X98098).Compared to the wild-type isolates, the correlative transformant elevated the susceptible to all antimicrobial agents, but still exhibited a moderate resistant to ampicillin,piperacillin,cefuroxime and ceftriaxone.The results of isoelectric focusing indicated that there were totally four positive bands at the position of pI5.4, pI7.6, pI8.0.The analysis of pulsed-field gel electrophoresis (PFGE) indicated that three isolates displayed different patterns, and the remainders had no relationship.ConclusionSHV enzyme-producing strains were screened in all the four hospitals. All the wild-type isolates and transconjugants were exhibited high resistance to ceftriaxone and cefotaxime, but the resistance of the transconjugants to antimicrobial agents decreased, compared to the wild-type isolates.BlaSHV-12 was the mainly epidemic genotypes in our area. Meanwhile, three novels of subtypes were found on the basis of the SHV-type enzymes, and had no evident difference between the novel enzymes and parent enzymes in resistance. It was the first report of SHV-89 (DQ193536) typeβ-lactamase produced from China in the world. There were no genetic relationships among those novel enzymes carriers.
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