Dietary sugars modulate bacterial-fungal interactions in saliva and inter-kingdom biofilm formation on apatitic surface

Abstract

Bacteria and fungi can interact to form inter-kingdom biofilms in the oral cavity. Streptococcus mutans and Candida albicans are frequently detected in saliva and in dental biofilms associated with early childhood caries (tooth-decay), a prevalent oral disease induced by dietary sugars. However, how different sugars influence this bacterial-fungal interaction remains unclear. Here, we investigate whether specific sugars affect the inter-kingdom interaction in saliva and subsequent biofilm formation on tooth-mimetic surfaces. The microbes were incubated in saliva containing common dietary sugars (glucose and fructose, sucrose, starch, and combinations) and analyzed via fluorescence imaging and quantitative computational analyses. The bacterial and fungal cells in saliva were then transferred to hydroxyapatite discs (tooth mimic) to allow microbial binding and biofilm development. We found diverse bacterial-fungal aggregates which varied in size, structure, and spatial organization depending on the type of sugars. Sucrose and starch+sucrose induced the formation of large mixed-species aggregates characterized by bacterial clusters co-bound with fungal cells, whereas mostly single-cells were found in the absence of sugar or in the presence of glucose and fructose. Notably, both colonization and further growth on the apatitic surface were dependent on sugar-mediated aggregation, leading to biofilms with distinctive spatial organizations and 3D architectures. Starch+sucrose and sucrose-mediated aggregates developed into large and highly acidogenic biofilms with complex network of bacterial and fungal cells (yeast and hyphae) surrounded by an intricate matrix of extracellular glucans. In contrast, biofilms originated from glucose and fructose-mediated consortia (or without sugar) were sparsely distributed on the surface without structural integration, growing predominantly as individual species with reduced acidogenicity. These findings reveal the impact of dietary sugars on inter-kingdom interactions in saliva and how they mediate biofilm formation with distinctive structural organization and varying acidogenicity implicated with human tooth-decay.

Keywords: C. albicans; EPS; S. mutans; inter-kingdom aggregate; saliva; sucrose.

Figure 1

Sugar-mediated inter-kingdom aggregates in human saliva. (A) Using fluorescent staining and confocal microscopy, aggregates formed by S. mutans and C. albicans were found in saliva supplemented with different dietary sugars, which varied in size, structure, and spatial organization depending on the type of sugars. (B) EPS α-glucan matrix were found in sucrose-mediated and starch+sucrose-mediated aggregates, but were not detected in aggregates formed in the presence of other dietary sugars, or no sugar. Green, S. mutans; purple, C. albicans; blue, EPS α-glucans. G+F: glucose+fructose. Scale bar length=25 µm.

Figure 2

Binding of bacterial and fungal cells onto tooth-mimetic hydroxyapatite surface in saliva supplemented with different dietary sugars. (A-F), representative images (z projection) showing surface-bound bacterial and fungal cells on the surface. The sugar condition is indicated to the left of the images. In each panel, the left image shows a large field of view (640 μm × 640 μm; scale bar = 100 μm) and the right image shows a magnified view (as shown using dotted box in the left image; scale bar = 25 µm). Green, S. mutans; purple, C. albicans. Arrowheads, sucrose or starch+sucrose-mediated inter-kingdom aggregates attached on the surface. G+F: glucose+fructose.

Figure 3

Biofilms formed by surface-bound inter-kingdom aggregates. (A–C), confocal images of the 19h biofilms originated from aggregates mediated by different sugars, as indicated above the images. In each panel, the upper image shows the z projection of a large representative field of view (640 μm × 640 μm; scale bar = 100 μm). The middle images show individual fluorescence channels. The lower images are three-dimensional rendering of the confocal image illustrating the spatial structure of C. albicans and S. mutans, and EPS matrix. Green, S. mutans; purple, C. albicans; Red, EPS α-glucans.

Figure 4

Results of biochemical and microbiological assays. (A) Total biomass (measure as dry-weight in mg) of 19h biofilms originated from different sugar-mediated aggregates. (B) Counts of S. mutans (CFU) within the biofilms (C) Counts of C. albicans (CFU) within the biofilms. Bars denote the mean and vertical lines denote standard deviation. G+F: glucose+fructose. Groups with distinct capital letter are statistically different (p<0.05 by one-way ANOVA with Tukey test)" by: Groups whose means are followed by different upper case letters differ statistically by ANOVA followed by Tukey test (p<0.05).

Figure 5

Acidogenic profile of biofilms originated from different sugar-mediated aggregates. Results of glycolytic pH drop assays (glucose-fermenting). 19h biofilms were removed from the culture medium and transferred into a buffer solution containing glucose as the only carbon source. pH of the buffer was measured every 10 min. pH of the fresh culture medium was measured every 2h. Dotted box indicates the data used for proton production analysis (as shown in Table 1 ). G+F: glucose+fructose.