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


CONCLUSIONS
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.
Monstein
Hans-Jürg
HJ
Clinical Microbiology-LMC, University Hospital S-581 85 Linköping, Sweden. hanmo@ibk.liu.se
Tärnberg
Maria
M
Nilsson
Lennart E
LE
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007710:Klebsiella Infections / Q000382:microbiology; D041121:Klebsiella oxytoca / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D001618:beta-Lactamases