Evaluation of extracellular polymeric substances matrix volume, surface roughness and bacterial adhesion property of oral biofilm

Abstract

Background/purpose: Oral biofilms are highly structured bacterial colonies embedded in a highly hydrated extracellular polymeric substances (EPS) matrix. This study aimed to investigate the characteristics of oral biofilm at different stages of maturation.

Materials and methods: Oral multispecies biofilms were grown anaerobically from plaque bacteria on collagen coated hydroxyapatite discs in brain heart infusion broth for one and three weeks. The volume of live bacteria and EPS matrix of the biofilms were determined by using corresponding fluorescent probes and confocal laser scanning microscopy. Atomic force microscopy (AFM) was used to quantitatively probe and correlate cell surface adhesion force of biofilms. The surface roughness was quantified in terms of the root mean square average of the height deviations. Adhesion was measured from force-distance data for the retraction of the cell from the surface.

Results: The volume of live bacteria and EPS of 3-week-old biofilms was higher than 1-week-old biofilms. The surface roughness value in 1-week-old biofilms was significantly higher than that in 3-week-old biofilms. AFM force-distance curve results showed that the adhesion force at the cell-cell interface was significantly more at-tractive than those at bacterial cells surface of both stages biofilms. Adhesion forces between the AFM tip and the surface of bacterial cell were fairly constant, whereas the cell-cell interface experienced greater adhesion forces in the biofilm's development.

Conclusion: As oral biofilms become mature, EPS volume and cell-cell adhesion forces increase while the surface roughness decreases.

Keywords: Atomic force microscopy; Bacterial adhesion; Biofilm; Extra-cellular polymeric substances; Surface roughness.

Figure 1

Biovolume of EPS and live bacteria of 1-week-old and 3-week-old biofilms groups. Different superscript uppercase letters indicate a significant difference between the two groups. Abbreviation: EPS, extracellular polymeric substances.

Figure 2

Representative CLSM images of biofilm, live bacteria and EPS in 1-week-old and 3-week-old biofilms groups (Green: live bacteria; Red: EPS). Abbreviations: CLSM, confocal laser scanning microscope; EPS, extracellular polymeric substances.

Figure 3

Typical 2D (A and C) and 3D (B and D) AFM images of one-week-old biofilms; (A) low-magnification topographic image (8 × 8 μm) showing typical surface profile of 2D AFM image and (B) 3D AFM image; (C) high-magnification topographic image (2 × 2 μm) showing microbial cells inside the biofilm matrix in image A and B (arrow) of 2D AFM image and (D) 3D AFM image: X-1 (cell surface), X-2 (cell-cell interface); (E) the corresponding force-distance curves at location X-1 (cell surface); and (F) X-2 (cell-cell interface) which adhesion force marked with ‘e’. Abbreviations: 2D, two dimensional; 3D, three dimensional; AFM, atomic force microscopy.

Figure 4

Typical 2D (A and C) and 3D (B and D) AFM images of three-week-old biofilms; (A) Low-magnification topographic image (8 × 8 μm) showing typical surface profile of 2D AFM image and (B) 3D AFM image; (C) high-magnification topographic image (2 × 2 μm) showing microbial cells inside the biofilm matrix in image A and B (arrow) of 2D AFM image and (D) 3D AFM image: X-1 (cell surface), X-2 (cell-cell interface); (E) the corresponding force-distance curves at location X-1 (cell surface); and (F) X-2 (cell-cell interface) which adhesion force marked with ‘e’. Abbreviations: 2D, two dimensional; 3D, three dimensional; AFM, atomic force microscopy.

Figure 5

Schematic diagram drawing showing 1-week-old and 3-week-old biofilms regarding the EPS volume (blue arrow), surface roughness (green arrow), cell surface (brown arrow), and intercellular interactions (red arrow). Abbreviation: EPS, extracellular polymeric substances. Created with BioRender.com.