Investigation of biofilm-associated genes and biofilm formation in Non- <i>aureus Staphylococcus</i> (NAS) isolated from cow’s milk

The isolation of non-<i>aureus Staphylococcus</i> (NAS) from the milk of both healthy cows and cows with mastitis has been frequently reported. However, there are few in-depth studies regarding their virulence profile and the ability of these microorganisms to form biofilms. Therefore, this research aimed to evaluate the biofilm formation capacity of NAS isolates from Brazilian milk. In this work, 309 NAS isolates were subjected to the Congo Red Agar (CRA) phenotypic test. Next, genotypic characterization was carried out by screening the <i>bap</i>, <i>ica</i>A, <i>ica</i>D, and MSCRAMMs genes: <i>bbp</i>, <i>cna</i>, <i>ebps</i>, <i>eno</i>, <i>fib</i>, <i>fnb</i>A, <i>fnb</i>B, <i>clf</i>A and <i>clf</i>B. Finally, ten isolates that presented the highest frequency of the genes analysed were selected to evaluate their ability to form biofilm on stainless-steel discs. t The number of cells (log10 CFU/cm²) in the biofilms was assessed at three time periods (24 h, 48 h, and 72 h) at a temperature of 25 °C. 35 NAS (11.32%) produced biofilms in the CRA test. Genotypic analysis showed the <i>eno</i> (38.5%) and <i>bap</i> (27.5%) genes were the most prevalent. In the analysis of biofilm formation on stainless steel, the factor 'growth time' had no significant effect on cell numbers. All selected isolates formed biofilm on stainless steel, and cell numbers were estimated to be in the 5.94 to 9.10 log10 CFU/m² range. These results provide evidence that NAS isolated from milk may represent a risk to human and animal health since they carry several virulence genes and demonstrate the ability to form biofilms.