Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer resistance.
Standard clinical procedures for pathogen resistance identification are laborious and usually require 2 days of cultivation before the resistance can be determined unequivocally. In contrast, clinicians and patients face increasing threats from antibiotic-resistant pathogenic bacteria in terms of their frequencies and levels of resistance. A major class of microbial resistance stems from the occurrence of beta-lactamases, which, if mutated, can cause the severe extended-spectrum beta-lactamase (ESBL) or inhibitor-resistant TEM (IRT) phenotype, which cause resistance to extended-spectrum cephalosporins, monobactams, and beta-lactamase inhibitors. We describe an oligonucleotide microarray for identification of the single nucleotide polymorphisms (SNPs) of 96% of the TEM beta-lactamase variants described to date which are related to the ESBL and/or IRT phenotype. The target DNA, originating from Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae cells isolated from clinical samples, was amplified and fluorescently labeled by PCR with consensus primers in the presence of cyanine 5-labeled nucleotides. The total assay, including PCR, hybridization, and image analysis, could be performed in 3.5 h. The microarray results were validated by standard clinical procedures. The microarray outperformed the standard procedures in terms of assay time and the depth of information provided. In conclusion, this array offers an attractive option for the identification and epidemiologic monitoring of TEM beta-lactamases in the routine clinical diagnostic laboratory.
Grimm
Verena
V
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, Stuttgart, Germany.
Ezaki
Satoshi
S
Susa
Milorad
M
Knabbe
Cornelius
C
Schmid
Rolf D
RD
Bachmann
Till T
TT
D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D016972:Enterobacter cloacae / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases