Evaluation of Drug Activity Against Staphylococcal Biofilms – Settings of Conditions Leading to Reproducible and Robust Biofilm Formation in vitro

Abstract

Staphylococcus aureus(SA)andepidermidis(SE) are the most common pathogens of the genus Staphylococcus, causing biofilm-associated infections. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections. A variety of protocols for in vitro drug activity testing against biofilms has been introduced. However, there are often fundamental differences. In our pilot study, we developed optimal conditions for staphylococcal biofilm formation on plastic pegs in order to set a methodology for an evaluation of the antibiofilm activity of candidate molecules. The convenience of the plastic pegs lies in their removability from the lid for easy access to multiple equivalent biofilms, and in possibility of in situ detection and quantification by confocal laser microscopy. For the purpose of enhancement in staphylococcal biofilm formation, the impact of peg surface modification with 3 different coating materials was studied as well. An increase of biofilm biomass was evaluated by crystal violet staining method. The basic precondition for obtaining relevant and reproducible data regarding antibiofilm activity is the formation of robust biofilms with typical attributes such as the presence of a biofilm matrix. In our study, in vitro conditions revealed that we fully met the preconditions for the SA and methicillin-resistant SA strains. In conclusion, we demonstrated statistically significant enhancement of biofilm formation in all studied staphylococcal strains, including either strong biofilm producer phenotype (SA, methicillin-resistant SA) and weak biofilm producer phenotype (SE). Supported by the SVV Project No. 260549 and by the Czech Science Foundation project No. 20-19638Y.