Evidence for icaADBC-independent biofilm development mechanism in methicillin-resistant Staphylococcus aureus clinical isolates

Abstract

Synthesis of a polysaccharide adhesin by icaADBC-encoded enzymes is currently the best-understood mechanism of staphylococcal biofilm development. In four methicillin-resistant Staphylococcus aureus isolates, environmental activation of icaADBC did not always correlate with increased biofilm production. Moreover, glucose-mediated biofilm development in these isolates was icaADBC independent. Apparently, an environmentally regulated, ica-independent mechanism(s) of biofilm development exists in S. aureus clinical isolates.

FIG. 1.

Comparison of biofilm and ica operon expression in S. aureus isolates. (A) Biofilm formation in tissue culture-treated 96-well plates by S. aureus isolates BH1CC, BH1S, BH1P, BH1Y in BHI medium or in BHI medium supplemented with 4% NaCl or 1% glucose. Biofilm values are the optical densities at 492 nm (OD₄₉₂) after staining with crystal violet and are the means of at least three independent assays. Standard deviations were less that 25% (data not shown). (B) Comparative measurement of icaA and gyrB (control) transcription in the S. aureus isolate BH1CC. RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD₆₀₀ of 4.0 in BHI medium or in BHI medium supplemented with 4% NaCl, 1% glucose, or 4% NaCl and 1% glucose. (C) Comparative measurement of icaA transcription in the S. aureus isolates BH1S, BH1P, and BH1Y. RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD₆₀₀ of 4.0 in BHI medium or in BHI medium supplemented with 4% NaCl or 1% glucose. Measurement of gyrB (control) transcription in BH1S, BH1P, BH1Y was performed to confirm equal loading of RNA (data not shown). Gluc, glucose.

FIG. 2.

Comparison of biofilm environmental regulation in S. aureus strains BH1CC, BH1CC Δica::tet, RN4220, and RN4220 Δica::tet. (A) Biofilm formation by S. aureus BH1CC and BH1CC Δica::tet in BHI medium or in BHI medium supplemented with 4% NaCl or 1% glucose. (B) Biofilm formation by S. aureus RN4220 and RN4220 Δica::tet in BHI medium or in BHI medium supplemented with 4% NaCl or 1% glucose. Biofilm values are OD₄₉₂ readings after staining with crystal violet and are the means of at least six independent assays. Standard deviations were less that 25% (data not shown). Gluc, glucose.