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

Hans-Jürg Monstein,Maria Tärnberg,Lennart E Nilsson

doi:10.1186/1471-2334-9-7

Abstract

BackgroundPlasmid encoded blaCTX-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.MethodsMultiple 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.ResultsNine 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.ConclusionThe 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.

Highlights

Plasmid encoded blaCTX-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
Plasmid encoded blaCTX-M enzymes represent an important sub-group of class-A β-lactamases which hydrolyse broad-spectrum β-lactam antibiotics causing an extended spectrum β-lactamase (ESBL) phenotype, which is increasingly found in enterobacterial species including Klebsiella [1,2]
We report on the use of a simple, accurate and universal CTX-M PCR amplification and sequencing assay well suited for high-throughput analysis

Summary

Introduction

Plasmid encoded blaCTX-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. Plasmid encoded blaCTX-M enzymes represent an important sub-group of class-A β-lactamases which hydrolyse broad-spectrum β-lactam antibiotics causing an extended spectrum β-lactamase (ESBL) phenotype, which is increasingly found in enterobacterial species including Klebsiella [1,2]. Due to constitutive expression of a chromosomal class A β-lactamases [5], Klebsiella oxytoca was shown to have a high level resistance to ceftriaxone and cefotaxime [6]. This class A β-lactamase was named K1 [7] and later on referred to as KOXY [8] or blaOXY [6]. Sequence diversity of the chromosomally located K. oxytoca K1-gene and the existence of discrete groups of blaOXY-1 and blaOXY-2 genes has been described in detail [5,9]

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Discussion

Conclusion