Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

Background: Plasmid encoded (CTX)-C-bla-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 (CTX)-C-bla-M and (OXY)-O-bla/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 (CTX)-C-bla-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 (CTX)-C-bla-M and (OXY)-O-bla/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 (OXY)-O-bla 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 (CTX)-C-bla-M, and (OXY)-O-bla/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.