CHARACTERIZATION OF BIOFILM FORMATION BY Staphylococcus aureus ISOLATED FROM BOVINE MASTITIS CASES

Staphylococcus aureus (S. aureus) strains are related with a large range of infectious diseases in humans and animals. Treatments with conventional antibimicrobials are failing due to microorganism resistance. In dairy cattle, S. aureus is the main pathogen enrolled in mastitis ethiology, and causes great economic losses. Nowadays its eradication is considered impossible. A survival mechanism by the bacteria is the development of biofilm, a collective structure made by bacterium growth formed with exopolysaccharides. As a result, the biofilm acts as a cement material between bacteria, and functions like a wall against the antimicrobials. S. aureus can produce biofilm, which is related to bacterial resistance . The objective of the present study was to analyze the biofilms production of 10 S. aureus strains, isolated from bovine mastitis cases. Biofilms were developed on 96-well microtiter for 24 hours, and quantified by adapting the crystal violet (CV) staining method by Stepanovic´ et al. 2000. For fixing, it were added 200 µL of 99 % methanol to each well containing adhered cells or biofilms treated with essential oils, as previously described. After 15 minutes, the methanol was removed, and the plates were allowed to dry at room temperature. Then it were added 200 µL of crystal violet stain (CV; 1 % v/v) to all wells. After 5 minutes, CV excess was removed and plates were washed in water. Finally, 230 µL of acetic acid (33 % v/v) were added to the wells to dissolve CV stain, and the absorbance was measured at 630 nm. All assays were performed in triplicate on three separate occasions. Using the adhesion classification criteria for biofilm parameters, proposed by Stepanovic´ et al. 2000, four groups were stablished, according to its optical relative density. Only two strains were identified among the isolates with moderate adherence; the remaining were classified as weak adherences. However, the low rate of adhesion could be related with the incubation time, then new analysis could be necessary to provide better characterization of the biofilm production. This preliminary study will ground future analysis about the essential oils action against S. aureus biofilm producers’ strains, especially those enrolled in bovine mastitis.