Role of Cell Appendages in Initial Attachment and Stability of E. coli on Silica Monitored by Nondestructive TIRF Microscopy

Total internal reflection fluorescence (TIRF) microscopy was used to investigate initial attachment and stability of wild-type, curli-deficient (ΔcsgA), flagella-deficient (ΔflhDC), and type-1 fimbriae-deficient (Δfim) mutant E. coli strains. Suspended bacteria were injected into a flow cell where they deposited on a silica coverslip, and images were acquired over a 2 min period. TIRF microscope image analysis revealed that curli- and flagella-deficient mutants attached closer to the surface and required a longer time to find their equilibrium position (i.e., bond maturation) as compared to the wild-type and fimbriae-deficient mutants. Analysis of the change in bacterial surface area over the 2 min period also indicated that curli- and flagella-deficient mutants have less initial stability than the wild-type and fimbriae-deficient mutants, evidenced by their fluctuating position at equilibrium. TIRF observations at the microscopic level were complemented macroscopically using quartz crystal microbalance with dissipation (QCM-D) and sand-packed column experiments, which support the distinctive behavior observed at the microscopic scale. For each mutant strain, as fluorescence intensity increased in TIRF, the negative frequency shift in QCM-D (related to the attached mass of bacteria) also increased. Packed-column experiments indicated that curli- and flagella-deficient mutants exhibited a characteristically different attachment behavior and more retention as compared to the wild-type and fimbriae-deficient strains. This study utilized a new approach to understand bacterial attachment/detachment and provides new insights into the role of various appendages on initial attachment and stability.