Fig. 1.

Interkingdom microbial assemblages in saliva attach to surfaces as structured cell groups. (A) Using fluorescent staining and confocal microscopy, native fungal–bacterial assemblages are found in saliva of patients with early childhood caries, but not in healthy individuals. (B) S. mutans and C. albicans were found in high levels in the diseased plaque. Data are presented as median with interquartile range. *P < 0.05 by Mann–Whitney U test. (C) Interkingdom assemblages and surface colonization is recapitulated using an in vitro model based on human saliva and hydroxyapatite surfaces, to mimic the tooth enamel. (D) Spatially structured S. mutans and C. albicans assemblage on the tooth-mimetic surface. (E) Surface-colonized assemblage using different fluorescent markers, as indicated underneath the images. EPS: extracellular α-glucan matrix produced by S. mutans. Asterisk: inner core harboring a mix of C. albicans and S. mutans. Arrowheads: peripheral areas containing mostly Candida hypha covered with bacterial-derived EPS. (F) Spatial distribution of S. mutans and C. albicans within the assemblage. Lines correspond to mean and shaded region to SD of n = 4 independent replicates. (G) Schematic diagram describing the spatial arrangement of the two species inside the assemblage, based on the computational image analysis results from F and H. (H) Spatial organization of the fungal and bacterial species relative to the surface. Lines correspond to mean and shaded area to SD of n = 4 independent replicates. (I) Confocal images of initial surface colonizers, for WT–WT assemblages and different WT–mutant assemblages (using C. albicans ΔΔefg1 or S. mutans ΔgtfBC mutants). (Scale bars, 10 μm.)