Rapid detection of TEM, SHV and CTX-M extended-spectrum β-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis

• Published in: Journal of antimicrobial chemotherapy 2010-07, Vol.65 (7), p.1377-1381
• Main Authors: Cohen Stuart, J., Dierikx, C., Al Naiemi, N., Karczmarek, A., Van Hoek, A. H. A. M., Vos, P., Fluit, A. C., Scharringa, J., Duim, B., Mevius, D., Leverstein-Van Hall, M. A.
• Format: Article
• Language: English
• Subjects: Antibiotics. Antiinfectious agents. Antiparasitic agents; Bacterial Proteins - genetics; Bacteriological Techniques - methods; beta-Lactam Resistance; beta-Lactamases - genetics; Biological and medical sciences; DNA; DNA, Bacterial - genetics; Enterobacteriaceae - drug effects; Enterobacteriaceae - enzymology; Enterobacteriaceae - genetics; Enterobacteriaceae Infections - microbiology; ESBL detection; ESBLs; Humans; Ligase Chain Reaction - methods; Medical sciences; Microarray Analysis - methods; Oligonucleotide Probes - genetics; Pharmacology. Drug treatments; Sensitivity and Specificity
• ISSN: 0305-7453; 1460-2091
• DOI: 10.1093/jac/dkq146

Objectives Fast and adequate detection of extended-spectrum β-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 β-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.