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

J. Cohen Stuart, C.M. Dierikx, N. Al Naiemi, A. Karczmarek, A. Hoek, P. Vos, A.C. Fluit, J. Scharringa, B. Duim, D.J. Mevius, M.A. Leverstein-van Hall

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Abstract

Objectives Fast and adequate detection of extended-spectrum ß-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 ß-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.
Original languageEnglish
Pages (from-to)1377-1381
JournalJournal of Antimicrobial Chemotherapy
Volume65
Issue number7
DOIs
Publication statusPublished - 2010

Keywords

  • resistance

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    Cohen Stuart, J., Dierikx, C. M., Al Naiemi, N., Karczmarek, A., Hoek, A., Vos, P., Fluit, A. C., Scharringa, J., Duim, B., Mevius, D. J., & Leverstein-van Hall, M. A. (2010). Rapid detection of TEM, SHV and CTX-M extended-spectrum ß-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. Journal of Antimicrobial Chemotherapy, 65(7), 1377-1381. https://doi.org/10.1093/jac/dkq146