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
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.
