Published in:
Open Access 01-12-2009 | Research article
Molecular identification of CTX-M and bla OXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons
Published in: BMC Infectious Diseases | Issue 1/2009
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Background
Plasmid encoded
bla
CTX-M enzymes represent an important sub-group of class A β-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
bla
CTX-M and
bla
OXY/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
bla
CTX-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
bla
CTX-M and
bla
OXY/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
bla
OXY 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
bla
CTX-M, and
bla
OXY/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.