Version 2: Hybrid Illumina-Nanopore assembly improves identification of multilocus sequence types and antimicrobial resistance genes of Staphylococcus aureus isolated from Vermont dairy farms: comparison to Illumina-only and R9.4.1 nanopore-only assemblies

Antimicrobial resistance (AMR) in Staphylococcus aureus is a pressing public health challenge with significant implications for the dairy industry, encompassing bovine mastitis concerns and potential zoonotic threats. To delve deeper into the resistance mechanisms of S. aureus, this study employed a hybrid whole genome assembly approach that synergized the precision of Illumina with the continuity of Oxford Nanopore. A total of 62 isolates, collected from multiple sources from Vermont dairy farms, were sequenced using the GridION Oxford Nanopore R9.4.1 platform and the Illumina platform, and subsequently processed through our specialized bioinformatics pipeline. Our analyses showcased the hybrid-assembled genome's superior completeness compared to Oxford Nanopore (R9.4.1)-only or Illumina-only assembled genomes. Furthermore, the hybrid assembly accurately determined multilocus sequence typing (MLST) strain types across all isolates. The comprehensive probe for antibiotic resistance genes (ARGs) using databases like CARD, Resfinder, and MEGARES 2.0 characterized AMR in S. aureus isolates from Vermont dairy farms, and revealed the presence of notable resistance genes, including beta-lactam genes blaZ, blaI, and blaR. In conclusion, the hybrid assembly approach emerges as a tool for uncovering the genomic nuances of S. aureus isolates collected from multiple sources on dairy farms. Our findings offer a pathway for detecting AMR gene prevalence and shaping AMR management strategies crucial for safeguarding human and animal health.