Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.
OBJECTIVE
Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M.


METHODS
Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive).


RESULTS
The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates.


CONCLUSIONS
This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.
Cohen Stuart
J
J
Department of Medical Microbiology, University Medical Centre Utrecht, The Netherlands. j.cohenstuart@umcutrecht.nl
Dierikx
C
C
Al Naiemi
N
N
Karczmarek
A
A
Van Hoek
A H A M
AH
Vos
P
P
Fluit
A C
AC
Scharringa
J
J
Duim
B
B
Mevius
D
D
Leverstein-Van Hall
M A
MA
D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012680:Sensitivity and Specificity; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D001618:beta-Lactamases