Purpose: The objective of this study was to elucidate the relation between NaCl and icaA gene, and the role of the icaA gene in biofilm formation by Staphylococcus aureus.
Methods: The icaA-deletion mutant of S. aureus ATCC13565 was constructed with temperature-sensitive vector, pIMAY. The wild type and ΔicaA mutant type of S. aureus ATCC13565 were cultured in tryptic soy broth (TSB) plus 0%, 2%, 4%, and 6% NaCl at 35°C for 24 h. A hundred microliter portions of the cultures were plated on tryptic soy agar plus 0%, 2%, 4%, and 6%, followed by incubation at 35°C for 24 h. The colonies on the plates were collected and suspended in phosphate buffer saline to OD600 = 0.1, and 20 μl of the suspensions were inoculated in 230 μl TSB plus 0%, 2%, 4%, and 6% NaCl concentration in a 96-well microtiter plate, followed by incubation at 35°C for 9 h. After supernatants of these cultures were discarded, crystal violet was added in each well to quantify biofilm. To visualize the biofilm formation of both S. aureus types at 0%, 2%, 4%, and 6% NaCl, confocal laser scanning microscope (CLSM) was used.
Results: ΔicaA mutant type of S. aureus ATCC13565 was prepared with pIMAY vector. The microtiter plate assay and CLSM showed that the ΔicaA mutant produced less biofilm at higher NaCl concentrations compared to that of S. aureus ATCC13565 wild type, but biofilm formation by the wild type increased as NaCl concentration increased (p< 0.05).
Significance: These results indicate that NaCl causes icaA upregulation, increasing biofilm formation of S. aureus.