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The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.

We used Raman microspectroscopy to investigate in situ the spatial distribution of the biomass in Streptococcus mutans biofilms. We used the CH stretching band to probe the organic matter and the area of the OH stretching band as an internal intensity standard, the biofilms being highly hydrated. The size of the biofilm regions that were mapped was 300 x 300 microm. We also recorded, in the confocal mode, the z profiles describing the biomass distribution as a function of depth in the biofilms. In our growth conditions, the biofilm is described as an approximately 75 microm thick mat completely covering the surface and includes columnar clusters with a diameter of approximately 100 microm surrounded by voids filled with water. Raman mapping was also used to examine the diffusion of HOD and polyethylene glycol with a molar mass of 10,000 (PEG-10k) in the biofilms. This study establishes that HOD can diffuse practically everywhere in the biofilms but that the penetration of PEG-10k is limited. There is a correlation between the restricted penetration of the macromolecule and the biomass content in the different regions of the biofilms. The method presented here provides a convenient approach to determine the diffusion of molecules, including antibacterials, in bacterial biofilms.