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Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons.
BMC Infect Dis. 2009 Jan 22; 9:7.BI

Abstract

BACKGROUND

Plasmid encoded blaCTX-M enzymes represent an important sub-group of class A beta-lactamases causing the ESBL phenotype which is increasingly found in Enterobacteriaceae including Klebsiella spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.

METHODS

Multiple displacement amplified DNA derived from 20 K. pneumoniae and 34 K. oxytoca clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of blaCTX-M and blaOXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.

RESULTS

Nine out of 20 K. pneumoniae clinical isolates had a blaCTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 K. oxytoca clinical isolates. Molecular identification and differentiation between blaCTX-M and blaOXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. In silico analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located blaOXY and K1-genes in Klebsiella spp. and K1-like genes in other Enterobacteriaceae.

CONCLUSION

The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of blaCTX-M, and blaOXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.

Authors+Show Affiliations

Clinical Microbiology-LMC, University Hospital S-581 85 Linköping, Sweden. hanmo@ibk.liu.seNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

19161622

Citation

Monstein, Hans-Jürg, et al. "Molecular Identification of CTX-M and blaOXY/K1 Beta-lactamase Genes in Enterobacteriaceae By Sequencing of Universal M13-sequence Tagged PCR-amplicons." BMC Infectious Diseases, vol. 9, 2009, p. 7.
Monstein HJ, Tärnberg M, Nilsson LE. Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons. BMC Infect Dis. 2009;9:7.
Monstein, H. J., Tärnberg, M., & Nilsson, L. E. (2009). Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons. BMC Infectious Diseases, 9, 7. https://doi.org/10.1186/1471-2334-9-7
Monstein HJ, Tärnberg M, Nilsson LE. Molecular Identification of CTX-M and blaOXY/K1 Beta-lactamase Genes in Enterobacteriaceae By Sequencing of Universal M13-sequence Tagged PCR-amplicons. BMC Infect Dis. 2009 Jan 22;9:7. PubMed PMID: 19161622.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons. AU - Monstein,Hans-Jürg, AU - Tärnberg,Maria, AU - Nilsson,Lennart E, Y1 - 2009/01/22/ PY - 2008/08/05/received PY - 2009/01/22/accepted PY - 2009/1/24/entrez PY - 2009/1/24/pubmed PY - 2009/3/19/medline SP - 7 EP - 7 JF - BMC infectious diseases JO - BMC Infect Dis VL - 9 N2 - BACKGROUND: Plasmid encoded blaCTX-M enzymes represent an important sub-group of class A beta-lactamases causing the ESBL phenotype which is increasingly found in Enterobacteriaceae including Klebsiella spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment. METHODS: Multiple displacement amplified DNA derived from 20 K. pneumoniae and 34 K. oxytoca clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of blaCTX-M and blaOXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer. RESULTS: Nine out of 20 K. pneumoniae clinical isolates had a blaCTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 K. oxytoca clinical isolates. Molecular identification and differentiation between blaCTX-M and blaOXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. In silico analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located blaOXY and K1-genes in Klebsiella spp. and K1-like genes in other Enterobacteriaceae. CONCLUSION: The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of blaCTX-M, and blaOXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced. SN - 1471-2334 UR - https://www.unboundmedicine.com/medline/citation/19161622/Molecular_identification_of_CTX_M_and_blaOXY/K1_beta_lactamase_genes_in_Enterobacteriaceae_by_sequencing_of_universal_M13_sequence_tagged_PCR_amplicons_ L2 - https://bmcinfectdis.biomedcentral.com/articles/10.1186/1471-2334-9-7 DB - PRIME DP - Unbound Medicine ER -