A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk

Introduction: Rapid and accurate diagnosis of causative pathogens in mastitis would minimize the imprudent use of antibiotics and, therefore, reduce the spread of antimicrobial resistance. Whole genome sequencing offers a unique opportunity to study the microbial community and antimicrobial resistance (AMR) in mastitis. However, the complexity of milk samples and the presence of a high amount of host DNA in milk from infected udders often make this very challenging. Methods: Here, we tested 24 bovine milk samples (18 mastitis and six non-mastitis) using four different commercial kits (Qiagens' DNeasy((R)) PowerFood((R)) Microbial, Norgens' Milk Bacterial DNA Isolation, and Molzyms' MolYsis (TM) Plus and Complete5) in combination with filtration, low-speed centrifugation, nuclease, and 10% bile extract of male bovine (Ox bile). Isolated DNA was quantified, checked for the presence/absence of host and pathogen using PCR and sequenced using MinION nanopore sequencing. Bioinformatics analysis was performed for taxonomic classification and antimicrobial resistance gene detection. Results: The results showed that kits designed explicitly for bacterial DNA isolation from food and dairy matrices could not deplete/minimize host DNA. Following using MolYsis (TM) Complete 5 + 10% Ox bile + micrococcal nuclease combination, on average, 17% and 66.5% of reads were classified as bovine and Staphylococcus aureus reads, respectively. This combination also effectively enriched other mastitis pathogens, including Escherichia coli and Streptococcus dysgalactiae. Furthermore, using this approach, we identified important AMR genes such as Tet (A), Tet (38), fosB-Saur, and blaZ. We showed that even 40 min of the MinION run was enough for bacterial identification and detecting the first AMR gene. Conclusion: We implemented an effective method (sensitivity of 100% and specificity of 92.3%) for host DNA removal and bacterial DNA enrichment (both gram-negative and positive) directly from bovine mastitis milk. To the best of our knowledge, this is the first culture- and amplification-independent study using nanopore-based metagenomic sequencing for real-time detection of the pathogen (within 5 hours) and the AMR profile (within 5-9 hours), in mastitis milk samples. These results provide a promising and potential future on-farm adaptable approach for better clinical management of mastitis.