Figure 1.
Dynamics of the morphogenesis, 3D architecture development and microbial population shifts of mixed-species biofilms.
(A) Representative 3D rendering images of mixed-species biofilms at distinct time points. Images (a–c) represent biofilms formed after the introduction of 1% (w/v) glucose. Images (d–f) represent biofilms formed in the presence of 0.1% (w/v) sucrose. Images (g–i) represent biofilms formed after the introduction of 1% (w/v) sucrose. The EPS channel is depicted in red, while the cells are depicted in green. At the upper left of each panel, the two channels are displayed separately, while the merged image is displayed at the bottom right. Lateral (side) views of each biofilm are displayed at the bottom left, while a magnified (close-up) view of each small box depicted in the merged image is positioned in the upper right corner of each panel. (B) The data are mean values ± s.d. (n = 30). The asterisks (*) indicate that the each of the values (EPS and bacterial biomass) in the 1% sucrose group are significantly different from others (P<0.05, Tukey-Krammer HSD). (C) Bacterial species [S. mutans (102 CFU/ml), A. naeslundii (106 CFU/ml), and S. oralis (107 CFU/ml)] were inoculated in the culture medium supplemented with 0.1% (w/v) sucrose until 29 h for establishment of the initial biofilm community [S. oralis (5.6±3.5×106 CFU), A. naeslundii (9.4±6.5×104 CFU) and S. mutans (8.8±2.5×103 CFU)]. The biofilms were then challenged with an environmental change by introducing 1% (w/v) sucrose (B-2) or 1% (w/v) glucose (B-3). Viable populations of S. mutans, S. oralis, and A. naeslundii recovered from the biofilms were counted (number of CFU recovered per biofilm) over time, and the proportion of each strain at each time point was calculated based on CFU data. Microbial population at 115 h in: 1% sucrose (1.1±0.2×1011 CFU of S. mutans; 2.1±2.9×109 CFU of S. oralis; 1.7±0.4×107 CFU of A. naeslundii), 0.1% sucrose (6.7±0.8×109 CFU of S. mutans; 1.4±1.9×1010 CFU of S. oralis; 9.3±1.3×109 CFU of A. naeslundii), and 1% glucose (2.6±1.9×109 CFU of S. mutans; 3.2±0.4×1010 CFU of S. oralis). The data are mean values ± s.d. (n = 12). The asterisks (*) indicate that the values from each strain are significantly different from each other (P<0.05).
Figure 2.
Expression of Streptococcus mutans EPS-associated genes and proteins during mixed-species biofilm development.
(A) This panel shows RT-qPCR analysis of gtfB, gtfC, gtfD, fruA, ftf and dexA gene expression by S. mutans in mixed-species biofilms at specific time points after introduction of 1% (w/v) sucrose. The data shown are mean values ± s.d. (n = 12). The asterisks (*) indicate that the expression level of gtfB, gtfC (at 67 h) is significantly different from other time points (P<0.05, Tukey-Krammer HSD). (B) This panel displays RT-qPCR analysis of gtfB, gtfC, and dexA gene expression by S. mutans in single- or mixed-species biofilm at 67 h, after introduction of 1% (w/v) sucrose. The data shown are mean values ± s.d. (n = 12). The expression level of the specific S. mutans genes is significantly different between single- and mixed-species biofilms (P<0.05). (C) This panel contains MudPIT analysis of expression of EPS-related proteins by S. mutans in mixed-species biofilms at specific time points after introduction of 1% (w/v) sucrose (n = 2). Spectral counts were used for quantification of proteins in each of the time points tested; standard deviations for protein data are not shown in the table. (D) This panel depicts MudPIT analysis of the abundance of GtfB, GtfC, and DexA by S. mutans in single- or mixed-species biofilms at 67 h after introduction of 1% (w/v) sucrose (n = 2). Spectral counts were used for quantification of proteins in each of the time points tested; standard deviations for protein data are not shown in the table.
Figure 3.
Mixed-species biofilms formed with S. mutans ΔgtfBC::kan or with parental strain UA159 treated with mutanase.
The biofilms were formed in the presence of the gtfBC null mutant (A) or parental strain UA159 (B) with 1% (w/v) sucrose. Selected areas in (A) and (B) show detailed views of separated and merged confocal images of bacterial cells (green) and EPS (red). The biomass values of EPS and bacterial cells in the biofilms were calculated using COMSTAT. The data shown are mean values ± s.d. (n = 15).
Figure 4.
Structural arrangement between EPS and bacterial cells during the assembly of surface-attached EPS-microcolony complex.
(A) This figure gives representative images of 3D renderings of mixed-species biofilms after introduction of 1% (w/v) sucrose. Panel (a) shows the dynamic evolution of surface-attached microcolonies over time. Panels (b–m) show cross sectional images of selected area for close-up views of the structural organization of EPS (red) and bacterial cells (green) during the development of an EPS-microcolony complex. The arrows indicate EPS bridging (i, j) and providing support for aggregation of multiple microcolonies (l, m). (B) The amount of co-localization between bacteria and EPS was calculated using DUOSTAT. The graph shows the percentage of bacteria and EPS colocalized within the biofilm over time (mean values ± s.d; n = 15). The asterisks (*) indicate that the values from each time point are significantly different from each other (P<0.05, Tukey-Krammer HSD).
Figure 5.
Three-dimensional pH mapping of intact mixed-species biofilm.
(A) This figure displays representative images of the pH profile throughout the biofilm 3D architecture and the spatial distribution of pH within the selected EPS-microcolony complex in mixed-species biofilms formed after introduction of 1% sucrose. Dark areas are indicative of regions of low pH, while white or light areas are indicative of regions of pH that are close to neutral, as indicated by the scale bar. The EPS channel is depicted in red, while the cells are depicted in green. Arrows indicate various acidic pH regions within the microcolony and at the sHA interface. Red arrows indicate acidic pH regions (i) across the structure and (ii) at the microcolony/sHA interface. Yellow arrows indicate pH close to neutral at the microcolony/fluid phase interface. White arrows indicate the corresponding pH landmarks in the merged panel. (B) This panel shows temporal changes of in situ pH across the EPS-microcolony complex after the biofilm was exposed to sodium phosphate-based buffer (pH 7.0) for a total of 120 min.
Figure 6.
In situ pH mapping at the EPS-microcolony complex and sHA interface.
(A) This figure shows representative cross sectional images (bird's eye view) at the sHA surface. White marks indicate the areas with surface-attached microcolonies (in the bacteria channel; depicted in green) while yellow marks show the corresponding area in the pH channel; the orange mark indicate an area without surface-attached microcolony. Dark areas are indicative of regions of low pH, while white or light areas are indicative of regions of pH that are close to neutral. (B) This table shows the pH values at the sHA interface for areas with and without surface-attached microcolonies after 30 min incubation in sodium phosphate-based buffer (pH 7.0). The data shown are mean values ± s.d. (n = 90). The asterisks (*) indicate that the differences between the two conditions are statistically significant (P<0.05, Tukey-Krammer HSD).
Figure 7.
Relationship of in situ pH at sHA surface with size of surface-attached EPS-microcolony complexes.
This figure depicts mixed-species biofilms (67 h) formed after the introduction of 1% (w/v) sucrose, which were processed for measurement of in situ pH at the interface between sHA surface and microcolonies. All the pH values were sorted by (A) the diameter and (B) height (distance from surface to fluid-phase) of the surface-attached microcolonies. The data shown are values from three separate experiments (n = 90). Correlation analysis showed that there is a linear relationship between pH and microcolony diameter (Pearson's test, P<0.0001, r-square 0.259) and height (P<0.0001, r-square 0.367).
Figure 8.
Time-lapse imaging of bacterial viability after exposure of the biofilm to 0.12% (v/v) chlorhexidine (CHX).
Panel (A) shows the relative populations of live (SYTO 9-labeled; depicted in green) and dead (propidium iodide-labeled; depicted in red) cells over time in the mixed-species biofilm formed with the parental strain (A-1; cells within EPS-microcolony complex and A-2; cells outside EPS-microcolony complex) and the ΔgtfBC::kan mutant (A-3) after exposure to CHX. Panel (B) shows the survival rate of live cells. The data shown are mean values ± s.d. (n = 6). The asterisks (*) indicate that the differences between the conditions tested (cells within EPS-microcolony complex vs. cells outside EPS-microcolony complex or cells not forming such structures, i.e. biofilm formed with ΔgtfBC::kan) are statistically significant (P<0.05).