Interaction of primary mammary bovine epithelial cells with biofilmforming staphylococci associated with subclinical bovine mastitis

Background: Staphylococci are recognized worldwide as one of the most important etiological agents of bovine mastitis due to their virulence factors such as their ability to penetrate inside mammary epithelial cells and their ability to form biofilm. Aims: The objectives of this study were to establish a model of primary mammary epithelial cells originating from the secretory tissue of the bovine udder in order to evaluate the invasion ability of 42 staphylococci isolated from subclinical bovine mastitis cases. Methods: Two techniques were used to establish a model of primary mammary epithelial cells, the explant technique and the enzymatic method. Biofilm formation was detected using a quantitative spectrophotometric assay. When compared with the enzymatic digestion method, the epithelial cells obtained by the explant technique grew faster and reached quickly to confluence. Results: The results showed that 60% of Staphylococcus aureus isolates (n=12) were able to invade the epithelial cells and 72.7% of coagulase negative staphylococci (CNS) isolates were invasive (n=16). Staphylococcus xylosus isolates showed higher invasion values compared to S. aureus isolates and nonbiofilm forming staphylococci were able to invade primary epithelial cells, but no significant difference was found between the internalization capabilities of biofilm positive and negative isolates. Conclusion: The results show that the explant technique is a valuable method for developing primary epithelial cells without damaging the cells, and provides new insights regarding the ability of staphylococci to penetrate inside primary mammary epithelial cells.