Abstract
<jats:p>The dissemination of antibiotic-resistant bacterial pathogens within healthcare systems represents a critical and growing threat to immunocompromised and high-risk patients. Current genomic surveillance strategies are constrained by prolonged turnaround time and the limitations of short-read sequencing technologies for resolving resistance determinants in their genomic context. Here, we performed a systematic and species-resolved benchmark of real-time Oxford Nanopore Technologies (ONT) sequencing from an antibiotic stewardship perspective. A collection of ten clinical isolates from each ESKAPE pathogen species was subjected to whole genome sequencing using both Illumina short-read and ONT long-read platforms; ONT libraries were sequenced using R9.4.1 flow cells. Nanopore-only assemblies generated at cumulative hourly sequencing intervals were compared against Illumina-only and hybrid assemblies, while Nanopore reads were simultaneously evaluated for the detection of antibiotic resistance genes and mutations using a read-based analytical approach. Our results show that real-time Nanopore de novo assemblies reliably recapitulate the antibiotic resistance repertoire identified in hybrid assemblies, achieving concordance exceeding 95% for most species, in some cases within 4-6 hours of sequencing initiation. Performance was influenced by species-specific genome characteristics, sequencing yield, and assembly quality, with Pseudomonas aeruginosa requiring longer sequencing time to achieve comparable sensitivity. Collectively, these findings support real-time Nanopore assembly-based workflows as a practical and informative approach for species-resolved antibiotic resistance surveillance in clinical and public health settings.</jats:p>