Changes in the Expression of Biofilm-Associated Surface Proteins inStaphylococcus aureusFood-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.

Formation of Staphylococcus aureus Biofilm in the Presence of Sublethal Concentrations of Disinfectants Studied via a Transcriptomic Analysis Using Transcriptome Sequencing (RNA-seq)

“…As reported previously, sublethal concentrations of some disinfectants can lead to enhanced biofilm formation, probably due to the adaptation of bacteria to these low-level stress conditions (19). Cincarova et al determined the concentration ranges of EtOH and ChT that significantly enhanced biofilm formation of strong or weak S. aureus biofilm formers using Syto9 labeling (19). The effects of both tested disinfectants were higher in weak biofilm formers, and the sublethal concentrations with the strongest stimulating activity were approximately 2.5% EtOH and 2,500 g/ml ChT (19).…”

“…As reported previously, sublethal concentrations of some disinfectants can lead to enhanced biofilm formation, probably due to the adaptation of bacteria to these low-level stress conditions (19). Cincarova et al determined the concentration ranges of EtOH and ChT that significantly enhanced biofilm formation of strong or weak S. aureus biofilm formers using Syto9 labeling (19).…”

“…Cincarova et al determined the concentration ranges of EtOH and ChT that significantly enhanced biofilm formation of strong or weak S. aureus biofilm formers using Syto9 labeling (19). The effects of both tested disinfectants were higher in weak biofilm formers, and the sublethal concentrations with the strongest stimulating activity were approximately 2.5% EtOH and 2,500 g/ml ChT (19). With reference to the work of Cincarova et al, we also chose to determine the effects of 2.5% EtOH and 2,500 g/ml ChT on a weak S. aureus biofilm former.…”

“…First, the strength of biofilm formation was determined according to a procedure described previously (44). For further analysis, the weak biofilm former Bϩ (internal collection number 1053; isolated from knives used in slaughtering) was chosen, because according to previous measurements the highest induction of biofilm formation caused by ethanol or chloramine T was observed in weak isolates (19). The S. aureus biofilm was grown in Trypticase soy broth (TSB) (Oxoid Ltd., Hampshire, England) supplemented with 1% NaCl (Penta, Chrudim, Czech Republic) and 1% D-glucose (Penta).…”

“…The growth of S. aureus biofilms can be enhanced by some processing methods encountered in the food industry, such as suboptimal temperatures, improper disinfection, or a combination of salt and glucose (18). Sublethal concentrations of disinfectants were described to promote the expression of virulence factors involved in biofilm growth (13,19). To investigate this issue in more depth, we formed biofilms from S. aureus strains treated with sublethal concentrations of commonly used food industry disinfectants (EtOH, ChT, and a combination of the two [EtOH_ChT]).…”