Introduction: Staphylococcus aureus is a major cause of chronic infections and causes particular problems in relation to implanted prostheses. Biofilm formation on abiotic surfaces affords bacteria innate protection from opsonophagocytosis and antibiotic agents and complicates the eradication of infection from bone and implanted prostheses. Increased concentrations of sodium, the major extracellular cation, have previously been implicated in increased biofilm formation in Staphylococcus aureus. In this study we demonstrate that increased concentrations of potassium, the major intracellular cation, also causes a significant increase in biofilm formation. Furthermore we also show that halide stress also leads to a primary increase in penicillin resistance in Staphylococcus aureus. Finally we demonstrate that pbp4, a key gene in cell wall synthesis, is down-regulated under sodium and potassium stress.

Methods: Staphylococcus aureus ATCC 9144 was cultured in broth supplemented with variable amounts of potassium chloride and sodium chloride. Biofilm formation was investigated in 96-well microtiter plates using a standard technique. Antibiotic resistance was investigated using graduated E-test strips. Gene transcription was assessed using RT-PCR.

Results: There was a positive correlation between biofilm formation and increased concentrations of sodium and potassium. Biofilm formation was noted to be even greater under potassium stress than under sodium stress. Sodium stress also lead to a five-fold increase in penicillin resistance in naïve Staphylococcus aureus cells. A key gene involved in cell wall production (pbp4) was down-regulated under sodium (p = 0.03) and potassium (p = 0.03) stress.

Discussion: Cellular injury or insult can lead to cell necrosis and lysis. The intracellular concentration of potassium is 30 times higher than that of the surrounding extracellular fluid. Hence, cell necrosis leads to markedly increased local concentrations of potassium. These experiments show that an increase in potassium concentration leads to an increase in biofilm formation. This suggests that biofilm formation and hence infection of implanted prostheses may be more likely in areas of major tissue trauma such as large resections and revisions. Furthermore, cellular stress leads to increased resistance to penicillin, a cell wall active antibiotic, in naïve cells which may nullify prophylaxis and complicate bacterial eradication in vivo. Finally we postulate a link between the experimental rise in penicillin resistance and the down-regulation of pbp4 demonstrated by RT-PCR under the same halide stresses.