Quantification and Comparison of Different Biofilm Components from Methicillin-Susceptible Staphylococcus aureus...

As we previously demonstrated that tranexamic acid (TXA), an antifibrinolytic, showed an antibacterial effect alone and in combination with vancomycin and gentamicin, we now wanted to analyze its own efficacy using new, different fluorescent staining reagents that target different components of the biofilm matrix and compare which one quantifies biofilm reduction better. A 10⁸ cfu/mL suspension of the Staphylococcus aureus (ATCC29213) strain was placed into the wells of a 24-multiwell plate covered with glass slides coated with 10% poly-L-lysine under agitation for 24 h at 37 °C. After 3 washes with PBS, wells were treated with either TXA 10 mg/mL or sterile water and incubated for 24 h at 37 °C. After three washes with PBS, the density area of the following biofilm components was calculated using confocal laser scanning microscopy: extracellular proteins (Sypro Ruby), α-extracellular polysaccharides (ConA-Alexa fluor 633), α or β-extracellular polysaccharides (GS-II-Alexa fluor 488), bacterial DNA (PI), and eDNA (TOTO^®-1). We observed a statistically significant reduction in the occupied area by all components of the S. aureus biofilm (p < 0.001) after TXA 10 mg/mL treatment, compared to the positive control. All biofilm components' reduction percentages reached ≥90.0%. We demonstrated that TXA reduced both bacteria and extracellular matrix components of S. aureus biofilm by using five different stain reagents, with all being equally valid for quantification.