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Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli.
Acta Pharmacol Sin. 2008 Feb; 29(2):217-25.AP

Abstract

AIM

The aim of the present study was to study the phenotypic and molecular characterization of 5 novel CTX-M-beta-1actamases carried by 5 Klebsiella pneumoniae isolates and 3 Escherichia coli isolates collected from 4 hospitals in Hefei, China.

METHODS

The purified PCR products were ligated with pGEM-Teasy vectors, expressed, and sequenced. The complete genes of the CTX-M-beta-lactamases were ligated with the pHSG398 vector to express prokaryotic recombinant proteins. Plasmids were extracted by rapid alkaline lysis protocol, and the PCR method was performed to determine whether the prokaryotic expression was successful or not. Antimicrobial susceptibility was tested and the phenotypes of transformants were determined according to criteria recommended by the Clinical and Laboratory Standards Institute. The kinetic parameters of enzymes were confirmed. The isoelectric points (pI) were determined by isoelectric focusing assay. Pulsed-field gel electrophoresis and plasmid profiling were performed.

RESULTS

The PCR products had 1101 nucleotides and were determined as CTX-M-46, CTX-M-47, CTX-M-48, CTX-M-49, and CTX-M-50. All strains were resistant to cefotaxime, but most of them were susceptible or intermediate to ceftazidime. The phenotypes of novel enzymes were determined as extended-spectrum-beta-lactamases (ESBL). Penicillin G, cephalothin, cefuroxime, and cefotaxime were determined to good substrates, whereas ceftazidime hydrolysis was not detected. The pI of the 5 novel CTX-M-beta-lactamases were 8.0. CTX-M-derivatives could be the multiplex genesis in our area.

CONCLUSION

This is the first report of these 5 novel plasmid-mediated CTX-M ESBL produced from China in the world. Molecular typing reveals notably different origin in genes encoding different CTX-M variants of 8 strains.

Authors+Show Affiliations

Department of Infectious Diseases, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

18215351

Citation

Cheng, Jun, et al. "Phenotypic and Molecular Characterization of 5 Novel CTX-M Enzymes Carried By Klebsiella Pneumoniae and Escherichia Coli." Acta Pharmacologica Sinica, vol. 29, no. 2, 2008, pp. 217-25.
Cheng J, Ye Y, Wang YY, et al. Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli. Acta Pharmacol Sin. 2008;29(2):217-25.
Cheng, J., Ye, Y., Wang, Y. Y., Li, H., Li, X., & Li, J. B. (2008). Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli. Acta Pharmacologica Sinica, 29(2), 217-25. https://doi.org/10.1111/j.1745-7254.2008.00736.x
Cheng J, et al. Phenotypic and Molecular Characterization of 5 Novel CTX-M Enzymes Carried By Klebsiella Pneumoniae and Escherichia Coli. Acta Pharmacol Sin. 2008;29(2):217-25. PubMed PMID: 18215351.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Phenotypic and molecular characterization of 5 novel CTX-M enzymes carried by Klebsiella pneumoniae and Escherichia coli. AU - Cheng,Jun, AU - Ye,Ying, AU - Wang,Ying-ying, AU - Li,Hui, AU - Li,Xu, AU - Li,Jia-bin, PY - 2008/1/25/pubmed PY - 2009/5/5/medline PY - 2008/1/25/entrez SP - 217 EP - 25 JF - Acta pharmacologica Sinica JO - Acta Pharmacol Sin VL - 29 IS - 2 N2 - AIM: The aim of the present study was to study the phenotypic and molecular characterization of 5 novel CTX-M-beta-1actamases carried by 5 Klebsiella pneumoniae isolates and 3 Escherichia coli isolates collected from 4 hospitals in Hefei, China. METHODS: The purified PCR products were ligated with pGEM-Teasy vectors, expressed, and sequenced. The complete genes of the CTX-M-beta-lactamases were ligated with the pHSG398 vector to express prokaryotic recombinant proteins. Plasmids were extracted by rapid alkaline lysis protocol, and the PCR method was performed to determine whether the prokaryotic expression was successful or not. Antimicrobial susceptibility was tested and the phenotypes of transformants were determined according to criteria recommended by the Clinical and Laboratory Standards Institute. The kinetic parameters of enzymes were confirmed. The isoelectric points (pI) were determined by isoelectric focusing assay. Pulsed-field gel electrophoresis and plasmid profiling were performed. RESULTS: The PCR products had 1101 nucleotides and were determined as CTX-M-46, CTX-M-47, CTX-M-48, CTX-M-49, and CTX-M-50. All strains were resistant to cefotaxime, but most of them were susceptible or intermediate to ceftazidime. The phenotypes of novel enzymes were determined as extended-spectrum-beta-lactamases (ESBL). Penicillin G, cephalothin, cefuroxime, and cefotaxime were determined to good substrates, whereas ceftazidime hydrolysis was not detected. The pI of the 5 novel CTX-M-beta-lactamases were 8.0. CTX-M-derivatives could be the multiplex genesis in our area. CONCLUSION: This is the first report of these 5 novel plasmid-mediated CTX-M ESBL produced from China in the world. Molecular typing reveals notably different origin in genes encoding different CTX-M variants of 8 strains. SN - 1671-4083 UR - https://www.unboundmedicine.com/medline/citation/18215351/Phenotypic_and_molecular_characterization_of_5_novel_CTX_M_enzymes_carried_by_Klebsiella_pneumoniae_and_Escherichia_coli_ L2 - https://doi.org/10.1111/j.1745-7254.2008.00736.x DB - PRIME DP - Unbound Medicine ER -