Purpose:
Molecular characterization of antimicrobial resistance determinants is recommended to monitor the mechanisms underlying antimicrobial resistance in indicator bacteria such as Escherichia coli. An affordable all-in-one alternative to whole genome sequencing is welcome to conduct the monitoring of critical antimicrobial resistance determinants in large collections of indicator or pathogenic bacteria in a one-health context

Methods:
Here we present a rapid genetic screening system based on a home-made multiplex Ligase Chain Reaction (LCR) assay using padlock probes coupled with a MagPlex-TAG beads hybridization and analysis on a Luminex® 200 TM platform. This 55-plex array, called the AMR-ARRAY, has been developed to detect the main antibiotic resistance determinants against β-lactams, (fluoro)quinolones, colistin, aminoglycosides and macrolides in E. coli, Shigella and Salmonella spp and can be used for other Enterobacteriaceae.

Results:
The mix of 55 probes was validated with available reference strains carrying the corresponding resistance gene or mutation. The array performed successfully on both reference and field strains. Four hundred eighty-one strains resistant to β-lactams, (fluoro)quinolones and/or colistin isolated in Belgium from food-producing animals (179 E. coli), food chain (117 E. coli), human samples (66 Salmonella spp. and 100 Shigella spp.) and 19 EURL-AR reference strains (9 E. coli and 10 Salmonella spp.) were assayed with a reduced version of this array (41-plex targeting β-lactams, (fluoro)quinolones and colistin resistance markers). Results were compared with phenotypic resistance profiles determined for the animal strains and with sequence-inferred resistance profiles determined for the other strains (using Sanger sequencing or PCR as reference method). The array performed well in detecting critical genetic determinants underlying antimicrobial resistance since 97.8% (175/179) of the food-producing animal strains yielded bead array profiles fully compatible with phenotypic resistance. For the genotype comparison, the array has a good performance too. The bead array profiles matched Sanger or PCR genotypes for 95.7% (112/117) of the food chain isolates, 97.6% (162/166) of the human isolates and 94.7% (18/19) of the inter-laboratory tests strains, giving a global genotypic concordance of 96.7% (292/302).

Conclusion:
We have developed in-house a 55-plex LCR/Luminex® MagPlex-TAG bead array able to detect the main antibiotic resistance determinants against β-lactams, (fluoro)quinolones, colistin, aminoglycosides and macrolides in Gram-negative bacteria. The performance of a reduced version of this array (41-plex targeting β-lactams, (fluoro)quinolones and colistin resistance markers) was evaluated with a collection of 481 field strains and a global concordance of 97.1% (467/481) was observed between the bead-array results and results obtained either by susceptibility testing or by genotyping using Sanger sequencing or PCR. The evaluation of the performance of the 55-plex AMR-ARRAY with field strains resistant to aminoglycosides and macrolides is ongoing.