Introduction: Staphylococcal bacteria, especially the coagulase negative Staphylococci, are responsible for the majority of orthopaedic device related infection. These infections are sub acute, and may not present for months or years following surgery. The virulence of these bacteria is related to their ability to form biofilm, a protective slime which allows them to survive the effects of the host immune system and antimicrobial therapy. Treatment of biofilm based infection almost always necessitates removal of the implant.

Recent work has identified environmental stimuli which induce biofilm formation in Staphylococci. These include stressors such as high temperature, high osmolarity, anaerobiosis, nutrient depletion, salt, ethanol and subinhibitory concentrations of certain antimicrobial drugs. Given the ability of these bacteria to survive the “respiratory burst” from the cells of the mononuclear-macrophage system, we hypothesised that oxidative stress may be one such promoter of biofilm formation by Staphylococci.

Methods and Materials: Staphylococcus epidermidis CSF41498 and Staphylococcus aureus RN422O were selected for study as these are known biofilm forming organisms. Hydrogen peroxide (H₂O₂) was used as an oxidizing agent.

Bacteria were incubated for 24 hours at 37°C in Brain-Heart Infusion (BHI, Oxoid) containing progressively weaker concentrations of H₂O₂ to determine a Minimal Inhibitory Concentration (M.I.C.) for the representative strains. Bacterial viability was assessed by measuring the optical density of the incubated culture using a cell density meter (Ultraspec 10, Amersham Biosciences).

The bacteria were then grown as a biofilm on a 96 well microtitre plate (Nunc) in the presence of subinhibitory concentrations of H₂O₂, using pure BHI as a control. Semiquantative determination of biofilm formation was performed by washing the plates, staining the adherent cells with crystal violet, and measuring the light absorbance of the adherent stained cells at 492 nm using a Multiskan plate reader (Flow Laboratories).

Results: The M.I.C. of H₂O₂ was 18 mM for both Staphylococcus epidermidis CSF41498 and Staphylococcus aureus RN422O. Concentrations of H₂O₂ of 16 mM and below had no normal bacterial growth and replication.

There was no difference in biofilm formation by Staphylococcus epidermidis csf41498 in the presence of 15 mM H₂O₂ when compared to that of the control. However, H₂O₂ had a significant inhibitory effect on biofilm formation by Staphylococcus aureus RN422O, even at a concentration well below the M.I.C.

Conclusion: We conclude that oxidative stress may have an antibiofilm action on certain Staphylococcal species, which is independent from its bactericidal effect, and which is manifest at a concentration below the M.I.C. for that species.