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We describe an in\u2010house bead array targeting AMR genes of Gram\u2010positive bacteria and allowing their rapid detection all at once at a reduced cost. A total of 41 AMR probes were designed to target genes frequently associated with resistance to tetracycline, macrolides, lincosamides, streptogramins, pleuromutilins, phenicols, glycopeptides, aminoglycosides, diaminopyrimidines, oxazolidinones and particularly shared among <jats:italic>Enterococcus<\/jats:italic> and <jats:italic>Staphylococcus<\/jats:italic> spp. A collection of 124 enterococci and 62 staphylococci isolated from healthy livestock animals through the official Belgian AMR monitoring (2018\u20132020) was studied with this array from which a subsample was further investigated by whole\u2010genome sequencing. The array detected AMR genes associated with phenotypic resistance for 93.0% and 89.2% of the individual resistant phenotypes in enterococci and staphylococci, respectively. Although linezolid is not used in veterinary medicine, linezolid\u2010resistant isolates were detected. These were characterized by the presence of <jats:italic>optrA<\/jats:italic> and <jats:italic>poxtA<\/jats:italic>, providing cross\u2010resistance to other antibiotics. Rarer, vancomycin resistance was conferred by the <jats:italic>vanA<\/jats:italic> or by the <jats:italic>vanL<\/jats:italic> cluster. Numerous resistance genes circulating among <jats:italic>Enterococcus<\/jats:italic> and <jats:italic>Staphylococcus<\/jats:italic> spp. were detected by this array allowing rapid screening of a large strain collection at an affordable cost. Our data stress the importance of interpreting AMR with caution and the complementarity of both phenotyping and genotyping methods. This array is now available to assess other One\u2010Health AMR reservoirs.<\/jats:p>","DOI":"10.1002\/mbo3.1341","type":"journal-article","created":{"date-parts":[[2022,12,26]],"date-time":"2022-12-26T07:05:36Z","timestamp":1672038336000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["An in\u2010house 45\u2010plex array for the detection of antimicrobial resistance genes in Gram\u2010positive bacteria"],"prefix":"10.1002","volume":"12","author":[{"given":"Carole","family":"Kowalewicz","sequence":"first","affiliation":[{"name":"Veterinary Bacteriology, Sciensano Ixelles Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Timmermans","sequence":"additional","affiliation":[{"name":"Veterinary Bacteriology, Sciensano Ixelles Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Fretin","sequence":"additional","affiliation":[{"name":"Veterinary Bacteriology, Sciensano Ixelles Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pierre","family":"Wattiau","sequence":"additional","affiliation":[{"name":"Veterinary Bacteriology, Sciensano Ixelles Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5610-2127","authenticated-orcid":false,"given":"C\u00e9cile","family":"Boland","sequence":"additional","affiliation":[{"name":"Veterinary Bacteriology, Sciensano Ixelles Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,12,25]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkab137"},{"key":"e_1_2_11_3_1","first-page":"D517","article-title":"(2020). 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