A microtiter plate assay for Staphylococcus aureus biofilm quantification at various pH levels and hydrogen peroxide supplementation.

Biofilm formation is the leading cause of the pathogenesis of S. aureus associated with biomaterial infections. In S. aureus polysaccharide intercellular adhesin (PIA) was encoded by icaA and icaD genes. Production of PIA is currently responsible for staphylococcal biofilm development. In this study, S. aureus strains isolated from auricular infection (n = 46) and S. aureus ATCC 25923 were phenotyped and genotyped. Slime production was assessed using Congo red agar plate assay. In order to determine the biofilm formation capacity at various pH levels of the studied S. aureus strains, microtiter plate assay was performed. The strains were grown in medium adjusted at various pH levels (3, 5, 7, 9 and 12) and medium supplemented with hydrogen peroxide 3% (v/v). Qualitative biofilm production of S. aureus revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were icaA and icaD positive. Quantitative biofilm showed that biofilm production depended on the pH value of the medium. At highly acidic (pH 3) and alkaline (pH 12) levels, biofilm formation was lower, while at pH 7 the adhesion was moderate. In addition, the cells adhered weakly after 3% hydrogen peroxide supplementation. Our results suggest that pH was a stress factor that led some S. aureus strains to produce the biofilm.