Field-deployable molecular workflow for detection, resistance screening, and genotyping of Staphylococcus aureus in bovine mastitis

Staphylococcus aureus (S. aureus) is a common cause of cow-associated bovine mastitis and continues to pose a challenge in dairy herds. Early and accurate diagnosis is essential for effective control and treatment of the infection. This study developed a field-adapted molecular workflow combining a simple DNA extraction method with a colourimetric loop-mediated isothermal amplification (LAMP) assay targeting the nuc gene for S. aureus detection, and a second LAMP assay targeting the mecA gene to identify methicillin resistance. LAMP results were benchmarked against PCR using 14 milk samples confirmed as S. aureus-positive by culture and MALDI-TOF MS. A secondary aim of the study was to investigate the genetic diversity of S. aureus isolates from cases of bovine mastitis in Australia by analysis of the spa gene. The spa genotyping was carried out using PCR followed by high-resolution melt (HRM) curve analysis, supported by a mathematical model based on genotype confidence percentage (GCP) values and validated by DNA sequencing. The nuc-LAMP assay demonstrated 89.5 % sensitivity and 100 % specificity relative to PCR, while the mecA-LAMP assay showed complete agreement with PCR in identifying methicillin-susceptible isolates. Among 14 positive milk samples, nine distinct spa types were identified by sequencing, reflecting high genetic diversity. The GCP-based model provided a reproducible and objective method for assigning spa types, even when melt curve differences were subtle. This integrated, low-cost approach offers a practical solution for point-of-care mastitis diagnostics and antimicrobial resistance monitoring using LAMP, particularly suited to resource-limited and remote farm settings, followed by laboratory-based spa genotyping through HRM for strain characterisation.