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 β-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(®) platform. We designed 46 xTAG(®)-compatible probes targeting β-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 blaTEM-1 and blaTEM-52 genes. The turn-around time from single colony to result is 5 h and total consumable costs remained <€5 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.
Division of Bacterial Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Pieter-Jan
PJ
Ceyssens
Division of Foodborne Pathogens, Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Cristina
C
Garcia-Graells
Division of Bacterial Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Frédéric
F
Fux
Division of Foodborne Pathogens, Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Nadine
N
Botteldoorn
Division of Bacterial Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Wesley
W
Mattheus
Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Véronique
V
Wuyts
Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Sigrid
S
De Keersmaecker
Division of Foodborne Pathogens, Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium.
Katelijne
K
Dierick
Division of Bacterial Diseases, Scientific Institute of Public Health (WIV-ISP), 1050 Brussels, Belgium sophie.bertrand@wiv-isp.be.
Sophie
S
Bertrand
D001431:Bacteriological Techniques / Q000191:economics / Q000379:methods*; D003362:Cost-Benefit Analysis; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods*; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics
D012336:RNA, Ribosomal, 16S; D001618:beta-Lactamases