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. 2022 Oct 3;119(41):e2209699119. doi: 10.1073/pnas.2209699119

Fig. 2.

Fig. 2.

Enhanced mechanical resistance and antimicrobial tolerance of surface-attached interkingdom assemblage. (A) Schematic diagram of the microfluidic device used to test the mechanical resistance of surface-attached assemblages. (B) Time-lapse confocal imaging of the cellular response to the surface detachment force. Left image (0 Pa) illustrates surface-attached biostructures prior to shear stress exposure. Center and Right images show the same biostructures after being exposed to intermediate (10 Pa) or high shear stress (20 Pa). Green, S. mutans; cyan, C. albicans. (Scale bar, 20 μm.) (C) Relative remaining biovolume of the surface-attached biostructures after applying different shear stress. For interkingdom assemblages, the total biovolume of S. mutans and C. albicans was calculated. (D) Relative mechanical resistance of surface-attached assemblages after exposed to high shear stress (20 Pa), which is defined as the ratio of remaining biovolume on the surface to the original biovolume. (E) Schematic diagram of the experimental setup for the real-time antimicrobial killing assay for surface-attached assemblages, using 100 µg/mL chlorhexidine. (F) Time-lapse confocal image of antimicrobial killing. Dead cells (bacteria and fungi) are visualized using Toto-3 iodide (in red). Left image illustrates biostructures prior to antimicrobial exposure. Green, S. mutans; cyan, C. albicans. Images on the Right show the real-time killing profile (red channel only) within the same surface-attached biostructure. White solid lines indicate the bacterial clusters within aggregated S. mutans or interkingdom assemblage. Green, S. mutans; cyan, C. albicans; red, dead cells (S. mutans and C. albicans). (Scale bar, 20 μm.) (G) Reduction of S. mutans and C. albicans CFU in different biostructures after 5-min antimicrobial treatment. (H) Survival rate for each species in the biostructures after 5-min treatment. Abbreviation: Agg C.a., aggregated C. albicans; Agg S.m., aggregated S. mutans; Assembl, interkingdom assemblages. *P < 0.05 by one-way analysis of variance with Dunnett's multiple-comparison test (C and D) or by Student’s t test (G and H).