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.

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.

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.

Results: There was a positive correlation between bio-film 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.

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 pros-theses may be more likely in areas of major tissue trauma such as large resections and revisions. Furthermore, cellular stress leads to increased antibiotic resistance in naïve cells which may nullify prophylaxis and complicate bacterial eradication in vivo.

A phenomenon of methicillin resistance in methicillin sensitive Staphylococcus aureus has been noted in organisms living in biofilm induced by the state of cell wall deficiency. The rate and the amount of biofilm formed by the cell wall deficient organisms far exceeds that of cell wall patent organisms. Once removed from the biofilm the S. aureus had the same sensitivities of the original organism. Cell wall deficient organisms outside the biofilm did not demonstrate the methicillin resistance. A known laboratory strain (ATCC 9144) was induced into a cell wall deficient state and allowed to form biofilm. The rate of formation and amount formed was compared with that formed by cell wall patent organisms. Before inducing cell wall deficiency sensitivity to methicillin was demonstrated using standard microbiological technique. Using an oxacillin containing plate as a culture medium: the biofilm, cell wall deficient organisms and the cell wall competent organisms were inoculated onto separate media. Organisms from the biofilm were isolated and grown free of the biofilm on blood agar. Any growth on the oxacillin containing plate would demonstrate methicillin resistance. There was no growth on the plates containing the cell wall competent or cell wall deficient organisms. There was however growth on the plate inoculated with bio-film, however when organisms were isolated from the biofilm, there was no growth on the media. Antibiotic sensitivities of the original inoculant and the organisms isolated from the biofilm were the same. The biofilm, induced as a result of cell wall deficiency, offers a form of structural protection to the Staphylococcus aureus without altering the resistance pattern of organism. Standard microbiological techniques would therefore report the organism as methicillin sensitive, however clinically the organism may behave as a methicillin resistant organism. The state of cell wall deficiency encourages the formation of biofilm in S. aureus. In-vitro the state of cell wall deficiency is induced using high osmolality media or sub-lethal doses of cell wall active antibiotics. Both these states are found in clinical practice.

The use of sub-lethal doses of cell wall active antibiotics to induce cell wall deficiency in S aureus has been described. Cell Wall Deficient S aureus show an increased in-vitro ability to form biofilm. Cephalosporins(cell wall active antibiotics.) are commonly used at time of arthroplasty surgery as antimicrobial prophylaxis. Adherence is fundamental step in biofilm formation.

The adherence of cell wall deficient S aureus versus ‘wild type’ S aureus to glass was investigated. Slides comparing the two types of organisms were analysed using fluoroscopy and J-image software. The ability to adhere to plastic was investigated using a micro-titre based absorption test. In a third investigation a centrifugal force was used to quantify the adherence ability of the cell wall deficient organisms to the glass slides.

The cell wall deficient organisms demonstrated an increased ability to adhere to glass compared to the ‘wild type’. After exposure, there was on average twenty times more cell wall deficient organisms per unit area compared to the ‘wild-type’. The micro-titre plates were similar. After incubation, the absorption of each well was measured. Compared to the ‘wild type’ there was a significantly increased absorption in wells containing the cell wall deficient organisms. Showing an increased ability to adhere to plastic. The third technique quantified the ability to adhere using a centrifugal force. The slides were exposed to ‘wild type’ and cell wall deficient organisms, however before staining they were placed in a centrifuge. On analysis there were five cell wall deficient S aureus per field of view, compared to 0.5 ‘wild-type’.

An increased ability of cell wall deficient S aureus to adhere to surfaces has been shown. Adherence is fundamental to biofilm formation. The significance to orthopaedics is that the inadequate use of Cephalosporins at time of operation may be facilitating chronic infections.