Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

Abstract

Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25-2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.

Figure 1

Syto9 quantification of 3 biofilm forming isolates of S. aureus treated with different concentrations of ethanol or chloramine T. Strong biofilm former (B+++), moderate biofilm former (B++), and weak biofilm former (B+) were treated with increasing concentrations of ethanol (a) or chloramine T (b) and biofilm quantity was determined by Syto9 labeling. Graphs show biofilm levels in samples cultivated with disinfectants versus controls (samples cultivated without disinfectants). Columns represent mean values of fluorescence and vertical bars represent 95% confidence intervals regarding the means.

Figure 2

Representative SEM images of 48 h biofilm formed by B+ (1053) isolate in medium (control), 1.25% ethanol, or 2500 μg/mL chloramine T. Arrows: extracellular matrix.