Genotypic and phenotypic characterization of

Acute mastitis is a common infection during lactation, primarily caused by Staphylococcus aureus, a bacterium known for its ability to form biofilms within mammary ducts and develop antibiotic resistance. This study aimed to genomically characterize S. aureus strains isolated from women with acute mastitis and healthy asymptomatic women to better understand how S. aureus strains transition from harmless components of the human milk microbiota to pathogenic agents responsible for mastitis. Whole-genome sequencing was performed on nine S. aureus strains-six from women with mastitis and three from healthy women-followed by in silico analyses of core and accessory genes, resistome, virulome, mobilome, and secondary metabolite synthesis to identify genes related to virulence, antibiotic resistance, biofilm formation, and mobile genetic elements such as plasmids, bacteriophages, and pathogenicity islands. Antimicrobial resistance profiles were evaluated using Sensititre EUST2 plates, antimicrobial activity by an agar diffusion method, biofilm formation in 96-well plates, and siderophore production with the Chrome Azurol S assay. Results showed that complete bacteriophage genomes were only present in S. aureus strains isolated from mastitis cases. Some virulence genes, including fnbB and cna, were absent in strains from healthy women. Both types of S. aureus strains exhibited biofilm formation capacity, with mastitis-associated strains SA4 and SA5 being the highest biofilm producers. Similarly, although all strains secreted siderophores, SA4 and SA55 exhibited the strongest siderophore production, indicating a link between this trait and virulence in mastitis-associated strains. The analysis of key genomic features, including virulence factors, resistance genes, and biofilm-forming capabilities, revealed some mechanisms by which S. aureus contributes to the pathogenesis of mastitis.IMPORTANCEAcute mastitis is a widespread infection in lactating women, and its main cause, Staphylococcus aureus, has developed resistance to antibiotics, making treatment challenging. The ability of this bacterium to form biofilms complicates its eradication from the mammary glands. Understanding the genomic and phenotypic characteristics of S. aureus strains associated with mastitis, compared to those isolated from asymtomatic women, is critical for developing better treatment strategies. This study provides new insights into the genetic features, such as virulence factors, antibiotic resistance profiles, and presence of bacteriophages, that make S. aureus strains pathogenic in mastitis. It also highlights the potential of biofilm formation and siderophore production as key factors in mastitis progression. These findings could guide the development of novel therapeutic approaches, such as targeted therapies or probiotics, which can more effectively treat mastitis and reduce reliance on antibiotics, ultimately improving maternal and infant health.