Abstract
The aim of the present study was to assess the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL100 (EUD) microparticles against mature biofilms of polysaccharide intercellular adhesin-positive S. epidermidis.
The effect of plain, daptomycin- and vancomycin-loaded PMMA and PMMA-EUD microparticles on S. epidermidis biofilms was assessed by isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). Biofilms were grown for 48h onto poly-urethane pieces of fixed dimensions. Each sample was washed with PBS in order to remove planktonic bacteria and incubated for 24h with different concentrations of acrylic microparticles (20–1.25 mg/mL). The minimal biofilm inhibitory concentration (MBIC) of the antibiotic-loaded particles was defined as the lowest concentration of particles that was able to prevent heat flow associated to the recovery of the biofilms. After incubation with the microparticles, sessile cocci were hybridized with the pan-bacterial EUB338-FITC and the staphylococci-specific STAPHY-FICT probes and stained with DAPI. Biofilm structure and metabolic state were characterized by fluorescence microscopy.
According to the IMC results, plain PMMA-particles showed no effect on S. epidermidis biofilms, whereas PMMA-EUD-microparticles negatively influenced the recovery of the biofilm probably due to the highly positive charge of these particles. The MBIC of daptomycin-loaded PMMA-microparticles was 20 mg/mL, whereas vancomycin-loaded PMMA microparticles were not able to inhibit biofilm recovery. Adding EUD to the formulation reduced the MBIC of daptomycin-loaded microparticles to 1.25 mg/mL, corresponding to a 16-fold reduction. Regarding the vancomycin-loaded microparticles, EUD caused a further decrease of their antibiofilm activity. The FISH micrographs corroborated the IMC results and provided additional insights on the antibiofilm effect of these carriers. According to FISH, daptomycin-loaded PMMA-EUD microparticles were responsible for the most pronounced reduction in biofilm mass. In addition, FISH showed that both PMMA and PMMA-EUD microparticles were able to attach to the biofilms.
Adding EUD to the formulations proved to be a powerful strategy to improve daptomycin-loaded microparticles antibiofilm activity. In addition, the combination of IMC and FISH was essential in order to fully assess the effect of polymeric microparticles on sessile S. epidermidis. Although the present study enabled gaining further insights on this subject, the nature of these interactions remains unclear. However, this may be a crucial aspect for the enhancement of antibiofilm activity of antibiotic-loaded polymeric microcarriers against mature biofilms.
This work was supported by the Portuguese government (Fundação para a Ciência e a Tecnologia) and FEDER (grant SFRH/BD/69260/2010 and research project EXCL/CTM-NAN/0166/2012) and strategic project PEst-OE/SAU/UI4013/2011.
