Development of a Luminex xTAG® assay for cost-effective multiplex detection of β-lactamases in Gram-negative bacteria

The objective of this study was to design and validate a genotyping method for multiplex identification of ESBLs and carbapenemases in Gram-negative bacilli. This assay had to be (i) superior to traditional (multiplex) PCR/sequencing-based tests in turn-around time, gene coverage and the ability to detect multiple variants of the same allele, and (ii) significantly more cost-effective than commercial microarrays and WGS. The targeted beta-lactamases include ESBLs (CTX-M families and subtypes, ESBL and non-ESBL SHV- and TEM-likes, OXA-1/2/7-likes, PER, VEB, GES), plasmid-mediated cephalosporinases (CMY, MOX, FOX, ACC, DHA, MIR/ACT) and carbapenemases (OXA-48, NDM, KPC, VIM, IMP). A modular multiplex oligonucleotide ligation-PCR procedure was used, with read-out on a Luminex MAGPIX(A (R)) platform. We designed 46 xTAG(A (R))-compatible probes targeting beta-lactamase alleles and allele variants, and one probe targeting a conserved 16S rRNA region serving as a DNA extraction control. The assay was optimized using a collection of 48 reference strains and further validated using 105 foodborne ESBL-producing Escherichia coli isolates. The specificity and selectivity of the test are 100% and 99.4%, respectively. Multiple variants of the same allele were successfully discriminated, as exemplified by five E. coli strains encoding both bla(TEM-1) and bla(TEM-52) genes. The turn-around time from single colony to result is 5 h and total consumable costs remained < a,not sign5 per sample. We designed and validated the first Luminex-compatible genotyping assay that reliably and rapidly identifies a broad range of ESBL, pAmpC and carbapenemase producers in culture.