(665a) Colloidal Microstructure of Bacterial Biofilms By Direct Visualization With Confocal Microscopy

Microscale cellular clustering and intercellular separation of surface adherent biofilms of the opportunistic pathogen Staphylococcus epidermidis are found to be well-described by colloidal measures such as the cluster distribution and the radial distribution function.  These measures are found to depend significantly on the environmental stress or antibiotic stress present as the biofilm grows under conditions of steady flow.  Bacterial biofilms are bacterial cells encapsulated in an extracellular polymeric substance consisting of polysaccharides, DNA, and proteins.  Since bacterial size is on the order of 1 μm, we view bacterial biofilms as a colloidal composite material with the bacterial cells as the particles and the extracellular matrix as a viscoelastic hydrogel.  Here we probe the cellular microstructure of S. epidermidis biofilms at varying environmental stress conditions by combining high-resolution confocal microscopy with colloidal image analysis techniques that identify individual cells.  The microstructural properties characterized are the local number density, the cluster distribution, the radial distribution function, and the fractal dimension of biofilm image volumes.  Environmental stressors of high osmotic pressure (776 mM NaCl) and sub-lethal antibiotic dose (1.9 μg/mL vancomycin) decreased the average bacterial local number density 10-fold.  Density phenotypes associated with low, medium, and high local number densities were identified in unstressed biofilms, while environmentally stressed biofilms only contained medium- and high-density phenotypes.  All biofilms considered exhibited short-range clustering on length scales associated with cell division (~ 1 μm).  However, on length scales of ~ 6 μm, biofilms with high-, and medium-density phenotypes contained longer range connectivity and structures characteristic of densely packed disordered colloidal suspensions, while low-density phenotype biofilms contained clusters of bacteria with fractal structure (d_f=1.7±0.1).  This clustering and connectivity has implications for the mechanical properties of biofilms, similar to how the statistical configuration of particles determines the elasticity of colloidal gels.