Optimizing Sample Preparation for Direct Metagenomic Nanopore Sequencing to Enable Rapid Pathogen and Antimicrobial Resistance Profiling in Bovine Mastitis

Long-read metagenomic sequencing enables the rapid, culture-independent, and accurate identification of causative pathogens and antimicrobial resistance (AMR) profiles, supporting targeted antibiotic use and minimizing the spread of resistance. However, its application to mastitis milk is challenging due to the complex milk matrix, low bacterial load, and high somatic cell content. This study first tested three sample treatment approaches, combining centrifugation, gradient centrifugation, and fat fraction treatment with Tween 20 and Citric acid. Four DNA extraction kits (Blood and Tissue, Molysis Complete5, HostZero, and SPINeasy Host depletion) were compared, focusing on their ability to remove host DNA and enrich bacterial DNA for long-read sequencing with Oxford Nanopore technologies. Our results demonstrate that simple centrifugation is an effective method for concentrating bacterial cells, eliminating the need for additional chemical treatments. The HostZero kit consistently delivered higher DNA yields, better DNA integrity, and host DNA depletion. Using nanopore sequencing, both Gram-positive and Gram-negative mastitis pathogens, as well as their antimicrobial resistance (AMR) genes, were accurately detected, matching the results from culture-based isolate sequencing. qPCR can help to assess the proportion of host and bacterial DNA, however it may not exactly reflect the pathogen and host read distribution in sequencing. Overall, this study supports the potential of direct metagenomic sequencing as a rapid, culture-free approach for identifying mastitis pathogens and their resistance profiles.