Weak rolling adhesion enhances bacterial surface colonization

Abstract

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as Escherichia coli when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by E. coli strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.

FIG. 1.

Once-through, shear- and temperature-controlled parallel-plate flow chamber system.

FIG. 2.

Adhesion of E. coli to the 3M surface. (Upper panel) Increased shear stress reduced the number of bacteria that transfer from solution to the surface after 5 min for FimH-wt E. coli and FimH-hi E. coli. (Lower panel) When already adherent bacteria were switched from low shear to the indicated level of shear stress, increased shear stress decreased the fraction of adherent bacteria that are rolling (moving at least 1 μm in 20 seconds.) The remainder of bacteria bound in a stationary manner (moved less than 1 μm in the same time). Error bars represent exact 68% confidence intervals, comparable to 1 standard deviation.

FIG. 3.

Surface colonization after 3 h. (A) FimH-wt E. coli cells grown at moderate shear stress (0.2 Pa). (B) FimH-wt E. coli cells grown at 2 Pa, which causes a switch to stationary adhesion. (C) FimH-hi E. coli cells grown at 0.2 Pa. (D) FimH-med E. coli cells grown at moderate shear stress (0.2 Pa). In all cases, approximately 10 bacteria were attached at the start of the growth conditions. (E) Quantification of the increase (n-fold) in surface area covered by the bacteria in the experiments shown in panels A through D. The complete videos for panels A through C are available online (see Videos S1 through S3 in the supplemental material.).

FIG. 4.

High-resolution images of dynamic changes in colonization. FimH-wt (A and B) and FimH-hi (C and D) E. coli bacteria at 0.2 Pa. Surfaces contain one initially adherent bacteria (A and C) or none (B and D). FimH-wt bacteria spread diffusely, while FimH-hi bacteria remain close to their initial location.

FIG. 5.

Quantification of FimH-hi microcolony growth. (A) The symbols show the average areas of the tight microcolonies formed by FimH-hi (n = 4 for each symbol). The solid line shows an exponential expansion in area, with the time constant taken directly from the measured doubling time of 21 min. The arrow indicates the point at which colonies fall below their predicted growth rate; this occurs by approximately 4 doubling times, when the colonies have about 16 cells. (B) The symbols show the average radii of the microcolonies (n = 3, with both x and y dimensions used). The solid line shows a linear increase in radius at the rate of 0.12 μm/min. Error bars indicate standard errors of the mean.

FIG. 6.

Surface colonization after 8 h. (A) FimH-wt E. coli variant grown at 0.5 Pa. (B) FimH-hi E. coli grown at 0.5 Pa. (C) Quantification of the increase (n-fold) in surface area colonized for FimH-hi and FimH-wt E. coli. In each case, one bacterium was seeded per field of view. A shear stress of 0.5 Pa, slightly higher than that used in the previous moderate shear experiments, was used to minimize the reattachment of any detached bacteria.

FIG. 7.

Effect of inhibitor on surface colonization. (A) 5 mM αMM inhibitor (gray symbols) was added to the medium during growth of FimH-wt or FimH-hi E. coli cells for 3 hours at 0.2 Pa. The fraction of surface area covered was calculated and normalized to the value at the start of the experiment. The results are compared to those from the same experiments without inhibitor (open or solid symbols). The remaining panels show the appearance of the surface with FimH-wt (B) and FimH-hi (C) E. coli cells grown for 3 h with the inhibitor. Compared to the equivalent condition without inhibitor as shown in the lower panel of Fig. 2, the compact microcolonies of FimH-hi bacteria are well spread.