Bacterial Surface Appendages Modulate the Antimicrobial Activity Induced by Nanoflake Surfaces on Titanium

Bioinspired nanotopography is a promising approach to generate antimicrobial surfaces to combat implant-associated infection. Despite efforts to develop bactericidal 1D structures, the antibacterial capacity of 2D structures and their mechanism of action remains uncertain. Here, hydrothermal synthesis is utilized to generate two 2D nanoflake surfaces on titanium (Ti) substrates and investigate the physiological effects of nanoflakes on bacteria. The nanoflakes impair the attachment and growth of Escherichia coli and trigger the accumulation of intracellular reactive oxygen species (ROS), potentially contributing to the killing of adherent bacteria. E. coli surface appendages type-1 fimbriae and flagella are not implicated in the nanoflake-mediated modulation of bacterial attachment but do influence the bactericidal effects of nanoflakes. An E. coli ΔfimA mutant lacking type-1 fimbriae is more susceptible to the bactericidal effects of nanoflakes than the parent strain, while E. coli cells lacking flagella (ΔfliC) are more resistant. The results suggest that type-1 fimbriae confer a cushioning effect that protects bacteria upon initial contact with the nanoflake surface, while flagella-mediated motility can lead to elevated membrane abrasion. This finding offers a better understanding of the antibacterial properties of nanoflake structures that can be applied to the design of antimicrobial surfaces for future medical applications.


Figure S1 .Figure S2 .
Figure S1.Representative AFM images of NF1 and NF2.Data were obtained by AFM and analyzed by Gwyddion and Fiji.

Figure S3 .
Figure S3.(a) Total number of E. coli cells following 6 h or 24 h incubation on nanoflake or control surfaces based on fluorescence intensity.(b) Number of viable E. coli cells following 6 h or 24 h incubation on nanoflake or control surfaces.CFU values are given as mean ± standard deviation.*** P < 0.001 relative to control, as determined by one-way ANOVA with Tukey HSD post hoc test, n = 3.This figure corresponds to Figure 2b in the main text.

Figure S4 .
Figure S4.Representative SEM images of E. coli on Ti nanoflake surfaces following 6 h incubation.

Figure S5 .
Figure S5.Characterization of E. coli biofilms formed on control, NF1 and NF2 surfaces after 24 h incubation.Quantification of total biofilm biomass based on Thioflavin S stain.Data are presented as mean ± SD. * P <0.05 or ** P<0.01 relative to control, as determined by one-way ANOVA with Tukey HSD post hoc test, n = 3.

Figure S6 .
Figure S6.Visualization of parent, ΔfimA and fimA+ E. coli strains (top row) or parent, ΔfliC and fliC+ E. coli strains (bottom row) following negative staining and TEM.Red arrows indicate type-1 fimbriae while blue arrows indicate flagella.

Figure S7 .
Figure S7.Total number of E. coli fimbrial mutant cells following 6 h (a) or 24 h (c) incubation on nanoflake or control surfaces based on fluorescence intensity.(b,d) Numbers of viable bacteria after 6 h (b) or 24 h (d) incubation on nanoflake or control surfaces.CFU values are given as mean ± standard deviation.* P < 0.05, ** P < 0.01, *** P < 0.001 relative to control, as determined by one-way ANOVA with Tukey HSD post hoc test, n = 3.This figure corresponds to Figure 5c and 5d in the main text.

Figure S8 .
Figure S8.Characterization of E. coli parent, ΔfimA and ΔfimA+ biofilms formed on control or NF1 surfaces after 24 h incubation.Quantification of total biofilm biomass based on Thioflavin S stain.Data are presented as mean ± SD. * P <0.05 or ** P<0.01 relative to control, as determined by one-way ANOVA with Tukey HSD post hoc test, n = 3.

Figure S9 .
Figure S9.Total E. coli flagella mutant cells following 6 h (a) or 24 h (c) incubation on nanoflake or control surfaces based on fluorescence intensity.(b,d) Numbers of viable E. coli flagella mutants following 6 h (b) or 24 h (d) incubation on nanoflake or control surfaces.CFU values are given as mean ± standard deviation.* P < 0.05, ** P < 0.01, *** P < 0.001 relative to control, as determined by one-way ANOVA with Tukey HSD post hoc test, n = 3.This figure corresponds to Figure 7c and Figure 7d in the main text.

Figure S10 .
Figure S10.Characterization of E. coli parent, ΔfliC and ΔfliC+ biofilms formed on control or NF1 surfaces after 24 h incubation.Quantification of total biofilm biomass based on Thioflavin S stain.Data are presented as mean ± SD. * P <0.05 or ** P<0.01 relative to control, as determined by one-way ANOVA with Tukey HSD post hoc test, n = 3.

Table S2 .
Plasmids and primers used for bacterial mutagenesis