Detection of ARGs from Gram-Negative Bacteria in Positive Blood Cultures Using a Microarray-Based System: Towards a Molecular Antibiotic Susceptibility Assay

<b>Background/Objectives</b>: Antimicrobial resistance (AMR) represents a major global health challenge, driving the need for rapid and accurate diagnostic tools. Novel molecular assays, including multiplex PCR and DNA microarray-based systems, have emerged to detect antimicrobial resistance genes (ARGs) alongside bacterial identification. <b>Methods</b>: In this study, we evaluated the performance of the HybriSpot12 PCR AUTO (HS12a) system and the MDR Direct Flow Chip (MDR-FC) Kit-an automatic microarray assay based on reverse hybridization-for the detection of ARGs directly from positive blood culture (PBC) samples. A total of 111 Gram-negative bacterial isolates (92 Enterobacterales, 14 <i>Acinetobacter baumannii</i>, and 6 <i>Pseudomonas</i> spp.), previously characterized by whole-genome sequencing (WGS), were each used to generate a PBC, which was then analyzed with the HS12a/MDR-FC assay. <b>Results</b>: We demonstrated perfect agreement for the detection of macrolide resistance genes across all bacterial species and high agreement for genes conferring resistance to sulfonamides and β-lactams. In contrast, aminoglycoside resistance genes showed only moderate agreement, with minor discrepancies observed in <i>Klebsiella pneumoniae</i> and <i>Escherichia coli</i>, largely attributable to specific SNP variations. <b>Conclusions:</b> The HS12a/MDR-FC assay includes 51 ARGs, though not all were represented in our isolate set, and some false negatives were observed. Despite these limitations, its broad coverage and rapid turnaround remain advantageous compared to other rapid assays with fewer targets. Future refinements should aim at broader gene coverage, inclusion of key mutations, and detection of emerging variants, making this approach a promising tool for rapid AMR surveillance and antimicrobial stewardship.