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Objective To investigate the situation of Pseudomonas aeruginosa strains carrying metallo-β-lactamases and inte-grases in the Second Affiliated Hospital of Harbin Medical University.Methods The phenotype of metallo-β-lactamases were detected by modified Hodge test,double-disc synergy and combination paper method,respectvily.PCR method was used to detecte metallo-β-lactamases genotypes and the integrationⅠ,Ⅱ and Ⅲ.The PCR products of the whole length bla-IMP gene were purified,sequenced and analyzed by Blast.Results Among 62 Pseudomonas aeruginosa strains,metallo-β-lactamases phenotype of 3 by modified hodge test,4 by double-disc synergy test,4 by combination paper method and were all positive 4 of IMP-1 type metallo-β-lactamases of Pseudomonasaeruginosa strains (10%,4/40)were positives by PCR.Inte-grase Ⅰ of 16(40 %,16/40)strains were positives by PCR method in IMP-resistant Pseudomonasaeruginosa,and integraseⅠ of 22.73 %(5/22)were positives in IMP-sensitive Pseudomonasaeruginosa.No other metallo-β-lactamases,integraseⅡandⅢ were detected in this study.Conclusion IMP-type metallo-β-lactamase existed in IMP-resistant Pseudomonasaerugi-nosa isolates of the Second Affiliated Hospital of Harbin Medical University.Most strains carried integraseⅠ,and other re-sistance mechanisms may be associated with multi-drug resistance,so it is important to prevent the Pseudomonasaeruginosa strains which carried metallo-β-lactamases and integrons widely spread in the hospital.
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Objective To construct mutant strains of Klebsiella pneumoniae with ampG gene dele-tion by homologous recombination and to evaluate the role of ampG gene in inducing the expression of AmpC enzyme.Methods Polymerase chain reaction ( PCR) was used to amplify the upstream and downstream fragments of ampG gene.The gene splicing by overlap extension PCR ( SOE-PCR) technique was used to construct the fusion fragment , which was then ligated into the temperature sensitive suicide vector pKO 3-km after enzyme digestion as pKO3-km-ΔampG.To achieve allelic exchange , the plasmid pKO3-km-ΔampG was introduced into Kp1 and Kp NTUH-K2044 strains by electroporation .The mutant strains of Klebsiella pneu-moniae with ampG gene deletion were screened out .The plasmid pACYC184-ampCR was introduced into the Kp NTUH-K2044 wild-type strain and its mutant strain with ampG gene deletion to make them harbor the gene encoding AmpC enzyme .The disk diffusion method was used to evaluate the effects of ampG gene on the expression of AmpC enzyme in Klebsiella pneumoniae strains with cefoxitin as the inducer .Results The recombinant plasmid pKO3-km-ΔampG was constructed successfully .The mutant strains of Klebsiella pneu-moniae with ampG gene deletion were constructed as verified by PCR and DNA sequencing .Compared with the Kp1 wild type strain, no AmpC enzyme was produced by the ampG gene knock-out Kp1 strain.The Kp NTUH-K2044 strain could produce AmpC enzyme , while the mutant strain of Kp NTUH-K2044 with ampG gene deletion could not after introduced the pACYC 184-ampCR plasmid .Conclusion The mutant strain of Klebsiella pneumoniae with ampG gene deletion was successfully constructed .The Klebsiella pneumonia strain without the ampG gene could not produce the AmpC enzyme .
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OBJECTIVE To investigate the occurrence and the genotype character of AmpC ?-lactamases-producing Escherichia coli isolated from the Second Hospital affiliated to Harbin Medical University.METHODS K-B disk diffusion test and FOX(≤17mm) test were used as initial screen tests to detect the clinical isolates;AmpC ?-lactamases were confirmed by three-dimentional extract tests;multiple-PCR was used for detecting plasmid-mediated AmpC gene and their genotypes were determined by DNA sequencing;the regulator genes of high producing AmpC isolates of E.coli were cloned to pUCm vectors and sequenced,the difference among them was analyzed by blast method.RESULTS Among total 586 E.coli clinical isolates,10 isolates(1.70%) resisted to cefoxitin;three-dimensional extract tests were positive for all 10 isolates;4 isolates of E.coli were plasmid-mediated DHA-1 type AmpC ?-lactamases by using multiplex PCR and DNA sequencing;6 isolates of chromosomal high level AmpC ?-lactamases-producing E.coli were sequenced and contrasted to that of E.coli K12,showed that they were gene polymorphism.CONCLUSIONS E.coli clinical isolates producing high level AmpC ?-lactamases not only acquire plasmid-mediated DHA-1 AmpC ?-lactamases but also cause by chromosomal ampC promotor or attenuator gene mutations in our hostiptal.