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Objective To Screen the carbapenem-resistant acinetobacter baumannii so as to understand the drug resistance,and to analyze the outer membrane protein expression between the carbapenem-resistant Acinetobacter baumannii and the sensitive strains.Methods The twofold agar dilution method was used to screen the carbapenem-resistant acinetobacter baumannii.PCR technique was used to detect the outer membrane protein-encoding gene carO.SDS-PAGE was used to analyze the expression of outer membrane protein.Results 32 strains were carbapenem-resistant acinetobacter baumannii separated from 1 10 strains,which were 100% sensitive to polymyxin B and were resistant to other drugs with different degrees O.They all carried carO gene.There were different protein bands between the carbapenem-resistant strains and the sensitive strains,in which two protein bands of 50× 10 3 and 22×10 3 were recognized as outer membrane proteins through the analysis of SDS-PAGE.Conclusion Drug resistance of acinetobacter baumannii is serious,and the outer membrane protein expression between the carbapenem-resistant strains and the sensitive strains presents discrepancy.
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OBJECTIVE:To clone,prokaryotic express and characterize the TEM-type ?-lactamase produced by Enterobacter cloacae clinical isolate EC002. METHODS: The drug susceptibility of Enterobacter cloacae clinical isolate EC002 was detected by agar double dilution,double disk screening and confirmatory test were employed to detect the ESBLs. The isoelectric point (pI) of enzyme was detected by isoelectric focusing electrophoresis (IEF),the genes were coded by PCR amplification enzyme,and the prokaryotic expression and phenotype of the TEM-type ?-lactamase were detected. RESULTS: Enterobacter cloacae EC002 were resistant to most of the ?-lactamases. Positive results were noted for the phenotype identification and plasmid conjugation test. IEF showed that Enterobacter cloacae EC002 produced two ?-lactamases with pI value at 8.7 and 5.4 respectively,which were confirmed to be CTX-M-22 and a new TEM-subtype ?-lactamase by DNA sequencing,and the phenotype of the expressed enzyme of the cloned strains was non-ESBLs. The TEM-type ?-lactamase was named as TEM-141 by GenBank. CONCLUSION: The TEM-141 produced by Enterobacter cloacae EC002 was a new type of plasmid-mediated broad-spectrum ?-lactamase.
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OBJECTIVE To study the molecular mechanism of the drug-resistance of Gram-negative bacilli to the third generation of cephalosporins.METHODS MICs of 13 ?-lactams to the eleven Gram-negative bacilli clinical isolates were detected with the standard agar dilution technique.K-B disc confirmatory method was conducted to determine the ESBLs phenotype of the eleven isolates.The ESBLs encoding genes were analyzed by using PCR.RESULTS The eleven isolates were all resistant to the third generation of cephalosporins(MIC≥64 ?g/ml).Disk confirmatory test showed that 10 isolates produced ESBLs.The hydrolytic activity of the ESBLs from the 10 isolates to cefoperazone and cefamandole was very high.However,the hydrolytic activity of the ESBLs from the 10 isolates to ceftazidime was very low.CONCLUSIONS The enzyme activities and the genes of extended-spectrum ?-lactamases from 10 Gram-negative bacilli clinical isolates are preliminarily analyzed.These results provide the basis for further study on the molecular mechanism of the drug-resistence of Gram-negative bacilli.
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OBJECTIVE To study the resistant characteristics and the resistant mechanisms of Gram-negative bacilli(G-) to ?-lactam antibiotics in the local nosocomial infections.METHODS The effects of efflux pump inhibitor on MICs were determined.Phenotypes and isoelectric points of ?-lactamases(Bla) were determined.Bla Genes were amplified and sequenced.RESULTS Of all tested isolates,the resistant rates to the most antibiotics were high.Besides 10.5% isolates with the efflux pumps,all tested isolates produced Bla,among which extended spectrum ?-lactamases(ESBLs),cephalosporin ?-lactamase(AmpC Bla) and metallo-?-lactamase were responsible for 42.1%,17.5% and 7.0% isolates,respectively.The complete nucleotide sequences of the ampC genes in 8 Enterobacter cloacae isolates had very high homology with the ampC gene in E.cloacae ECLC074.CONCLUSIONS The production of Bla is the main resistant mechanism of G-to ?-lactam antibiotics.ESBLs are the most frequent Bla.All of the ampC genes of AmpC Bla-producing E.cloacae originate from the ampC gene in E.cloacae ECLC074.Imipenen is the best choice for the treatment of the infections caused by multidrug resistant G-.Piperacillin/tazobactam and cefoperazone/sulbactam can be used to treat the infections caused by drug-resistant non-fermentative bacilli.Amikacin is effective to treat the infections caused by AmpC Bla-producing E.cloacae.
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OBJECTIVE:To study the characteristics of ceftazidime-resistant?-lactamase produced by Escherichia coli.METHODS:The types of2strains of drug fast?-lactamase produced by Escherichia coli were determined initially by K-B slip diffusion method and ampholine electrophoresis method;The plasmid was extracted by alkaline lysis and the PCR ampli?fication and sequencing were conducted;?-Lactamase was counter-extracted by saturated ammonium sulfate,filtrated by Sephadex G-75gel and purified by DE-52anion exchange chromatography;The molecular weight of which was determined by SDS-PAGE and the enzyme kinetics parameters of?-lactamas were determined by ultraviolet spectrophotometry.RE?SULTS:The2strains produced a super-broad spectrum?-lactamase(CTX-M-1V)with isoionic point at8.7and the molecular weight at29kDa,which can hydrolyze cefotaxim and aztreonam but imipenem and which was sensitive to sulbactam(IC 50 =94nmol/L)and tazobactam(IC 50 =5nmol/L).CONCLUSION:CTX-M-1V is a CTX-M type super-broad spectrum?-lactamase sensitive to suppressants.
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OBJECTIVE:To observe the relationship between gyrA gene mutations of the clinical isolates of Pseudomonas aeruginosa and quinolone resistance and to evaluate the feasibility of analyzing gyrA gene mutations using PCR-RFLP-SSCP.METHODS:With gyrA gene order of the clinical isolates of Pseudomonas aeruginosa taken as target sequence,gyrA gene mutations in strain ATCC 27853 and 16 clinical isolates of Pseudomonas aeruginosa were analyzed contrastively using PCR,PCR-RFLP,PCR-SSCP,and DNA sequencing.RESULTS:Of the total 8 ciprofloxacin resistant Pseudomonas aeruginosa,6 strains showed single point ACC→ ATC mutation in the gyrA gene at codon 83,leading to amino acid substitution of Thr83→Ile.SacⅡ digestion fragment of the PCR amplified products in gyrA gene was in line with the sequencing results.SSCP showed that the banding patterns of all strains were different from that of strain ATCC 27853 except 2 strains.CONCLUSION:The molecular mechanism of the quinolone resistant Pseudomonas aeruginosa isolated from clinics was manifested as mutations in the gyrA gene at codon 83.The results showed that PCR-RFLP-SSCP is a rapid and accurate method for the detection of basyl variation in gyrA in quinolone resistant Pseudomonas aeruginosa.
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AIM: To study the inhibitory effects of 10-23 deoxyribozyme (10-23 DRz) on Escherichia coli ?-lactamase gene expression. METHODS: According to the gene sequence of Escherichia coli ?-lactamase gene blashv-5, 10-23 DRz and antisense oligonucleotides (As-ODN) were designed and synthesized. 10-23 DRz, As-ODN or control oligonucleotides were respectively introduced into Escherichia coli by the method of electroporation. Following electroporation, bacterial viability in liquid medium contained ceftazidime was detected, bacterial ?-lactamase expression was analysed by using IEF-PAGE and the ?-lactamase band was measured with gel documentation-analyzing system. RESULTS: A_ 600 in 10-23 DRz transfected Escherichia coli was lower compared with that in As-ODN transfected Escherichia coli (P