[Objective] Ceftiofur was as the substrate to induce the standard strain of Escherichia coli（E.coli）to be the drug-resistance one.The resistant mechanism of E.coli to ceftiofur was studied.[Method] The sub-inhibitor...[Objective] Ceftiofur was as the substrate to induce the standard strain of Escherichia coli（E.coli）to be the drug-resistance one.The resistant mechanism of E.coli to ceftiofur was studied.[Method] The sub-inhibitory concentration method was used to induce the standard strains C83907 and C83845.After they were induced for 10 generations,the double disc synergy test（DDST）,NCCLS（National Committee for Clinical Laboratory Standards）confirmatory test and PCR amplification were used to detect the extend spectrum β-lactamases（ESBLs）.The two fold dilution method was used to measure the minimal inhibitory concentration（MIC）of cetiofur to the strain which produced ESBLs.For the drug-resistance strain which produced ESBLs,the two fold dilution method was used to measure the minimal inhibitory concentrations of different proportions of cetiofur and tazobactam sodium.[Result] After they were induced 15 generations,MIC value of ceftiofur to the induced bacteria was during 8-10 μg/ml,and ESBLs was detected.MICs of cetiofur combining tazobactam sodium（the mass ratio was 1∶1-8∶1）to Escherichia coli produced ESBLs reduced 20-22 times than that of cetiofur.[Conclusion] The main mechanism of pathogenic Escherichia coli resistance to ceftiofur was that which produced ESBLs.展开更多
In the present study, 27 multi-drug resistant strains of Pseudomonas aeruginosa were isolated from clinical specimens in our hospital from Jan 2005 to Nov 2005, in which the resistant genes encoding β-lactamase inclu...In the present study, 27 multi-drug resistant strains of Pseudomonas aeruginosa were isolated from clinical specimens in our hospital from Jan 2005 to Nov 2005, in which the resistant genes encoding β-lactamase including TEM, SHV, OXA, PER, VEB, GES, CARB, IMP, VIM, SPM, GIM, DHA and OprD2 were tested by PCR amplification and sequenced by DNA sequencer. It was found that the detection rates of blaVEB, blaGES and blaCARB genes in these 27 isolates of P. aeruginosa were 11.1%, 11.1% and 48.1%, respectively, but almost the oprD2 gene was lacked （92.6%）. In addition, the resistant genes encoding TEM, SHV, OXA, PER, IMP, VIM, SPM, GIM and DHA β-lactamase were all not found. It was also demonstrated that the sequence of blaVEB gene appeared to be identical to that of the blaVEB-1 （AY536743）, while the blaGES and blaCARB genes shared 99% identity with blaGES-1 （AY219651） and blaCARB-3 （S46063） genes. From these observations, it is evident that P. aeruginosa carrying the blarEs, blaGES and blaCARB resistant genes isolated in our hospital confers the resistance to β- lactams, and the loss of the oprD2 gene may be the important cause to develop resistance to imipenem in P. aeruginosa.展开更多
An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were c...An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer （pH 7.8） containing different concentrations of NaC1 is used as elution agent. The corresponding breakthrough characteristics and elution behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The maximum purity of the obtained lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of high-purit lysozyme from chicken egg white.展开更多
The antimicrobial susceptibility testing was performed with Kirby-Bauer disc diffusion and agar diffusion methods, and the crude β-lactamase was extracted by sonication with its isoelectric point （pI） determined wi...The antimicrobial susceptibility testing was performed with Kirby-Bauer disc diffusion and agar diffusion methods, and the crude β-lactamase was extracted by sonication with its isoelectric point （pI） determined with isoelectric focusing, and purified by two steps of chromatography. The genome DNA fragments of bacterial strains were amplified with PCR and subjected to sequencing. The kinetic parameters for β-lactamase were detected by spectrophoto metric method. It was found that the bacterial strains isolated from clinical specimens were resistant to penicillin, ceftazidine, cefotaxime and azitreonam, but sensitive to imipenem and cefoxitin, in which two resistant strains to ceftazidine were found to produce a single extended spectrum β-lactamase（ESBL） with pI value of 8.7. Results of cloning and sequencing of the β-lactamase encoding gene showed that this gene was similar to blactx-m-l with 6 point mutations including 3 silent mutations. The amino acid sequence derived from the nucleic acid data indicated that this enzyme was distinct from β-lactamse CTX-M-1 by 3 amino acids, i.e. Val-80→Ala, Asp-117→Asn and Ser-143→Ala（CTX-M-Ⅳ）. Molecular weight of this enzyjne was 29 kDa. Kinetic analysis of the partially purified β-lactamase confirmed that this enzyine was ＇able to hydrolyze cefotaxime and aztreonanl, but not to imipenem. In addition, the the β-lactamase was well inhibited by sulbactam（IC50 94 nM） and tazobactam（IC50 5 nM）. It is concluded that CTX-M-Ⅳ is a CTX-M-type extended spectrum β-lactamase.展开更多
The subject of this study was to survey prevalence Beta lactamase enzyme in Bacillus species isolated from foods in Isfahan city in Iran. This is a laboratory study performed during 2009-2010 years in Isfahan. In this...The subject of this study was to survey prevalence Beta lactamase enzyme in Bacillus species isolated from foods in Isfahan city in Iran. This is a laboratory study performed during 2009-2010 years in Isfahan. In this study, 150 samples of food, including juice ketchup, mayonnaise,展开更多
With the continuous emergence and rapid spread of multidrug-resistant and extensively-drug-resistant Mycobacterium tuberculosis strains, it is imperative to develop novel therapies against this bacterium. The intrins...With the continuous emergence and rapid spread of multidrug-resistant and extensively-drug-resistant Mycobacterium tuberculosis strains, it is imperative to develop novel therapies against this bacterium. The intrinsic β-lactam resistance of M. tuberculosis is primarily due to the production of an Ambler class-A β-lactamase BlaC, which limits the application of β-lactam antibiotics in the treatment of tuberculosis. Therefore, the inhibitors of BlaC could be novel anti-tuberculosis drug synergistic agents to recover the sensibility of M. Tuberculosis to the β-lactam antibiotics. In the present study, BlaC of M. tuberculosis was expressed and purified to establish a screening model of the BlaC inhibitors. The screening conditions were determined, and the screening model was evaluated to fit for the high throughput screening. A total of 22 BlaC inhibitors were screened out from 26 400 compound samples with a positive rate of 0.083%. Taken together, our findings lay the foundation for the discovery of novel anti-tuberculosis drug synergistic agents in clinic.展开更多
Objective： To investigate the mechanism of carbapenem resistance and the occurrence ofplasmid-mediated quinolone resistance determinants qnr and aac（6＇）-Ib-cr in a clinical isolate of Enterobacter cloacae. Methods...Objective： To investigate the mechanism of carbapenem resistance and the occurrence ofplasmid-mediated quinolone resistance determinants qnr and aac（6＇）-Ib-cr in a clinical isolate of Enterobacter cloacae. Methods： An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction （PCR）, and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins （OMPs） were examined by urea-sodium dodecyl sulfate- polyacrylamide gel electrophoresis （Urea-SDS-PAGE）. Results： Minimum inhibitory concentrations （MICs） of imipenem, mer- openem, and ertapenem for ZY 106 were 2, 4, and 16 pg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-β-lactamase and CTX-M-3 extended-spectrum β-1actamase, and E. coli transconjugant produced IMP-1. Plasrnid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrSl-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZYI06 lacked an OMP of approximately 38 kDa. Conclusion： It is the first IMP-l-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blalMP and qnrS genes as well. The blalMP-1, blaCTX-M-3, and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and mero- penern susceptibility in E. cloacae.展开更多
基金Supported by Education Department of Henan Province Item(2006230004)Henan Science and Technology Agency Item(072102130009)~~
文摘[Objective] Ceftiofur was as the substrate to induce the standard strain of Escherichia coli（E.coli）to be the drug-resistance one.The resistant mechanism of E.coli to ceftiofur was studied.[Method] The sub-inhibitory concentration method was used to induce the standard strains C83907 and C83845.After they were induced for 10 generations,the double disc synergy test（DDST）,NCCLS（National Committee for Clinical Laboratory Standards）confirmatory test and PCR amplification were used to detect the extend spectrum β-lactamases（ESBLs）.The two fold dilution method was used to measure the minimal inhibitory concentration（MIC）of cetiofur to the strain which produced ESBLs.For the drug-resistance strain which produced ESBLs,the two fold dilution method was used to measure the minimal inhibitory concentrations of different proportions of cetiofur and tazobactam sodium.[Result] After they were induced 15 generations,MIC value of ceftiofur to the induced bacteria was during 8-10 μg/ml,and ESBLs was detected.MICs of cetiofur combining tazobactam sodium（the mass ratio was 1∶1-8∶1）to Escherichia coli produced ESBLs reduced 20-22 times than that of cetiofur.[Conclusion] The main mechanism of pathogenic Escherichia coli resistance to ceftiofur was that which produced ESBLs.
文摘In the present study, 27 multi-drug resistant strains of Pseudomonas aeruginosa were isolated from clinical specimens in our hospital from Jan 2005 to Nov 2005, in which the resistant genes encoding β-lactamase including TEM, SHV, OXA, PER, VEB, GES, CARB, IMP, VIM, SPM, GIM, DHA and OprD2 were tested by PCR amplification and sequenced by DNA sequencer. It was found that the detection rates of blaVEB, blaGES and blaCARB genes in these 27 isolates of P. aeruginosa were 11.1%, 11.1% and 48.1%, respectively, but almost the oprD2 gene was lacked （92.6%）. In addition, the resistant genes encoding TEM, SHV, OXA, PER, IMP, VIM, SPM, GIM and DHA β-lactamase were all not found. It was also demonstrated that the sequence of blaVEB gene appeared to be identical to that of the blaVEB-1 （AY536743）, while the blaGES and blaCARB genes shared 99% identity with blaGES-1 （AY219651） and blaCARB-3 （S46063） genes. From these observations, it is evident that P. aeruginosa carrying the blarEs, blaGES and blaCARB resistant genes isolated in our hospital confers the resistance to β- lactams, and the loss of the oprD2 gene may be the important cause to develop resistance to imipenem in P. aeruginosa.
基金Supported by the National lqatural Science Foundation of China （21036005, 20876145）, the Science and Technology Cooperation Project between China-Europe Country＇s Governments from the Ministry of Science and Technology of China （1017） and the Natural Science Foundation of Zhejiang Provincial （Y4080326）.
文摘An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer （pH 7.8） containing different concentrations of NaC1 is used as elution agent. The corresponding breakthrough characteristics and elution behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The maximum purity of the obtained lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of high-purit lysozyme from chicken egg white.
文摘The antimicrobial susceptibility testing was performed with Kirby-Bauer disc diffusion and agar diffusion methods, and the crude β-lactamase was extracted by sonication with its isoelectric point （pI） determined with isoelectric focusing, and purified by two steps of chromatography. The genome DNA fragments of bacterial strains were amplified with PCR and subjected to sequencing. The kinetic parameters for β-lactamase were detected by spectrophoto metric method. It was found that the bacterial strains isolated from clinical specimens were resistant to penicillin, ceftazidine, cefotaxime and azitreonam, but sensitive to imipenem and cefoxitin, in which two resistant strains to ceftazidine were found to produce a single extended spectrum β-lactamase（ESBL） with pI value of 8.7. Results of cloning and sequencing of the β-lactamase encoding gene showed that this gene was similar to blactx-m-l with 6 point mutations including 3 silent mutations. The amino acid sequence derived from the nucleic acid data indicated that this enzyme was distinct from β-lactamse CTX-M-1 by 3 amino acids, i.e. Val-80→Ala, Asp-117→Asn and Ser-143→Ala（CTX-M-Ⅳ）. Molecular weight of this enzyjne was 29 kDa. Kinetic analysis of the partially purified β-lactamase confirmed that this enzyine was ＇able to hydrolyze cefotaxime and aztreonanl, but not to imipenem. In addition, the the β-lactamase was well inhibited by sulbactam（IC50 94 nM） and tazobactam（IC50 5 nM）. It is concluded that CTX-M-Ⅳ is a CTX-M-type extended spectrum β-lactamase.
文摘The subject of this study was to survey prevalence Beta lactamase enzyme in Bacillus species isolated from foods in Isfahan city in Iran. This is a laboratory study performed during 2009-2010 years in Isfahan. In this study, 150 samples of food, including juice ketchup, mayonnaise,
基金Fundamental Research Funds for Central Public Welfare Research Institutes(Grant No.2015PT350001)National Major Scientific and Technological Special Project for “Significant New Drugs Development”(Grant No.2015ZX09102007-009)
文摘With the continuous emergence and rapid spread of multidrug-resistant and extensively-drug-resistant Mycobacterium tuberculosis strains, it is imperative to develop novel therapies against this bacterium. The intrinsic β-lactam resistance of M. tuberculosis is primarily due to the production of an Ambler class-A β-lactamase BlaC, which limits the application of β-lactam antibiotics in the treatment of tuberculosis. Therefore, the inhibitors of BlaC could be novel anti-tuberculosis drug synergistic agents to recover the sensibility of M. Tuberculosis to the β-lactam antibiotics. In the present study, BlaC of M. tuberculosis was expressed and purified to establish a screening model of the BlaC inhibitors. The screening conditions were determined, and the screening model was evaluated to fit for the high throughput screening. A total of 22 BlaC inhibitors were screened out from 26 400 compound samples with a positive rate of 0.083%. Taken together, our findings lay the foundation for the discovery of novel anti-tuberculosis drug synergistic agents in clinic.
文摘Objective： To investigate the mechanism of carbapenem resistance and the occurrence ofplasmid-mediated quinolone resistance determinants qnr and aac（6＇）-Ib-cr in a clinical isolate of Enterobacter cloacae. Methods： An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction （PCR）, and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins （OMPs） were examined by urea-sodium dodecyl sulfate- polyacrylamide gel electrophoresis （Urea-SDS-PAGE）. Results： Minimum inhibitory concentrations （MICs） of imipenem, mer- openem, and ertapenem for ZY 106 were 2, 4, and 16 pg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-β-lactamase and CTX-M-3 extended-spectrum β-1actamase, and E. coli transconjugant produced IMP-1. Plasrnid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrSl-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZYI06 lacked an OMP of approximately 38 kDa. Conclusion： It is the first IMP-l-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blalMP and qnrS genes as well. The blalMP-1, blaCTX-M-3, and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and mero- penern susceptibility in E. cloacae.
