Accurate quantitation of antibiotic penetration through staphylococcal biofilms

Limited antimicrobial penetration is an important mechanism underlying antimicrobial resistance of biofilms and has often been incorrectly quantitated. We adopted a rationalized antibiotic agar-diffusion model to accurately interpret experimental results of a widely-accepted biofilm penetration assay, and to determine drug-related physiochemical properties impacting antimicrobial biofilm penetration.

Staphylococcal reference strains and eight conventional antibiotics were selected for this study. A well-established biofilm penetration assay based on disk diffusion and colony biofilms was used. Sizes of the zone of inhibition (ZOI) were converted to concentrations of antibiotics, using linear regressions of squared radii of the ZOI on the natural logarithm of antibiotic concentrations. Biofilm penetration ratios were calculated by comparing concentrations of antibiotics reaching the agar surface with or without biofilm barriers. Multiple regression analysis was performed to assess the impact of antibiotic physicochemical properties, such as surface charge, on their biofilm penetration.

Ciprofloxacin and oxacillin showed great capacities in penetrating staphylococcal biofilms. Rifampicin penetrated biofilms at low rates of ∼20 %. Aminoglycosides showed strain- and agent-specific penetration ratios, with tobramycin showing the least penetration (17.8 % for Staphylococcus aureus and 35.6 % for Staphylococcus epidermidis) and kanamycin presenting good penetration (∼82.3 %) against S. aureus biofilms. Surface charges of antibiotics at neutral and acidic conditions were important for their biofilm penetration.

Accurate quantitation of antibiotic biofilm penetration can be achieved using the transformed linear regression between the ZOI and antibiotic concentrations. Mathematical evidence was provided to support the importance of surface charge of antimicrobials on their biofilm penetration.