Urinary tract infections (UTIs) are one of the most common human infections and primarily caused by uropathogenic Escherichia coli (UPEC). Increasing UPEC resistance to antimicrobials is a major global health threat, compounded by the ability of UPEC to form biofilms during bladder infections. How UPEC biofilm growth confers resistance to antimicrobials, however, remains un-elucidated. Biofilms are complex communities of microorganisms encased in matrices of extracellular polymeric substances (EPS), made of biomolecules such as proteins, extracellular DNA, polysaccharides, and lipids. Here, we focused on elucidating the role of major components of UPEC biofilm EPS, namely, curli amyloid fibers and polysaccharide cellulose, in controlling biofilm properties to understand why biofilm cells remain viable during antimicrobial treatment. We used single-plane illumination microscopy combined with fluorescence correlation spectroscopy (SPIM-FCS) to quantify diffusion of differently sized molecules in UPEC biofilms. This method generated three-dimensional diffusion coefficient maps with high spatial and temporal resolution, allowing precise measurements across biofilm regions. Our results indicated that curli-positive biofilms exhibited strongly reduced and spatially uneven diffusion, consistent with a molecular sieving effect that blocked larger molecules from moving through the dense network. In contrast, cellulose had little measurable impact on diffusion. To understand tolerance of biofilms against antimicrobial peptides, we used SPIM-FCS to measure peptide movement. Compared to neutral peptides, positively charged peptides aggregated strongly and had significantly reduced diffusion within UPEC biofilms. This behavior indicates charged-based binding to the biofilm matrix, reducing peptide penetration and thus contributing to survival of biofilm-encased cells. The combined effect of charged-based interactions and molecular sieving leads to a significantly different distribution and mobility of differently charged peptides, providing a mechanism to explain why cationic antimicrobial peptides have reduced activity in UPEC biofilms that contain curli as a major EPS component.
Kulkarni et al. (Sun,) studied this question.