Characterization of the diffusion of polyethylene glycol in Streptococcus mutans biofilms by Raman microspectroscopy

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.