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Objective To investigate the prevalence of 16S rRNA methylases gene in imipenem-resistant Acinetobacter baumannii isolates from China. Methods A total of 342 imipenem-resistant A.baumannii isolates were collected between December 2004 and December 2005, from 25 hospitals of China. Agar dilution was used to determinate the minimal inhibitory concentration (MIC) of these isolates. The homology of these isolates was analyzed by pulse-field gel electrophoresis (PFGE). Several 16S rRNA methylase genes and carbapenemase genes were detected by PCR-based assays and PCR products were sequenced. Results The rates of resistance to ampicillin-sulbactam, cefoperazone-sulbactam, tobramycin, and minocycline were 68.0%, 54.2%, 87.4%, and 75.9%, respectively. The rate of resistance to polymyxin E was 10.8%, the lowest among the tested agents. The rates of resistance to all other tested antimicrobial agents were more than 90%. The A.baumannii isolates belonged to 29 distinct clones. Among them, 6 clones were dominant, consisting of 303 isolates in total. All isolates contained the blaOXA-51-1ike gene (blaOXA-66) and 322 isolates contained the blaOXA-23-1ike gene. PCR with the ISAbal-OXA-23-like primers generated a PCR product in 314 isolates, and PCR with the ISAbal-OXA-51-1ike primers generated a PCR product in 13 strains. 221 armA-positive isolates were identified. Conclusion Most of the imipenem-resistant A.baumannii contained blaOXA-23, with ISAbal upstream of the gene. 16S rRNA methylase gene armA was widely distributed in these isolates. The results suggested that the spread of clones played an important role in the outbreak of imipenem-resistant A. baumannii in China.
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Objective To characterize the antibiotic resistance,homology and carbapenemase genotypes of imipenem resistant Acinetobac1ter baumannii isolated from our hospital,and analyze the clonal relatedness of the test strains.Methods Ninety five strains of imipenem resistant A.baumannii were isolated from August 2003 to December 2004 in the First Affiliated Hospital, College of Medicine,Zhejiang University.The MICs of 16 antimicrobial agents against these strains were determined by agar dilution and E-test method.The homology of these isolates was analyzed by pulse-field gel electrophoresis(PFGE).The coding gene of carbapenemases was amplified.PCR products were purified,cloned and sequenced.Plasmid DNA was extracted and purified.Conjugation and Southern blot were performed to locate the position of oxa 23 gene.Results The resistance rates to ampicillin-sulbactam and cefoperazone sulhactam were 67.9% and 30.2%.Polymyxin E had the lowest resistance rate of 17%. The resistance rate to other antimicrobial agents was higher than 90%.The 95 strains,isolated from 10 clinical units,were classified into 6 clones.Clones A and B were predominant clones.All strains produced carbapenemases which were confirmed as OXA 23 by PCR and sequencing analysis.No plasmid was extracted and conjugation was not successful.Southern bolt showed that oxa-23 gene was located on Apal-digested chromosomal segments about 220 kb and 200 kb in Clones A and B,re spectively.Conclusions OXA 23-producing A.baumannii has become one of the most important multi-resistant pathogens in our hospital.Clones A and B have widely spread in our hospital.Oxa-23 gene is located on chromosomal DNA.
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Objective To investigate the resistant mechanism of imipenem-resistant K. pneumoniae.Methods The minimal inhibitive concentrations (MICs) of the antimicrobial agents were determined by Etest.Isoelectric focusing electrophoresis (IEF),plasmid extraction,conjugation, transformation,PCR amplification,cloning and sequencing were carried out for analyzing the encoding gene of ?-1actamases.Results Three kinds of ?-1actamases were detected with pIs of 7.2,6.7,and 5.4.in a clinical strain of K.pneumoniae.These ?-1actamases were TEM-I (pI,5.4),SHV-12 (pI,8.2) and KPC-2 ( pI,6.7 ) confirmed by sequencing of the PCR products.Only one band of ?-1actamase with pI 6.7 was displayed in the transformant.A 1500 bp segment,which contained the KPC-2 gene confirmed by nucleotide sequence analysis,was cloned from a 60 000 bp plasmid of the transformant.Conclusion The strain of K.pneumoniae resistant to imipenem produces a plasmid-mediated carbapenemase KPC-2 which belongs to Bush group 2f,class A ?-1actamase.