Veillonella dispar biofilm colonization depends on Streptococcus mutans GtfB activity

Objective: The aim of this study was to investigate the effect of GTF-deficiences in Streptococcus mutans glucan production on Veillonella dispar's ability to colonize dual in vitro biofilms. Methods: GTF knockout mutants (gtfB^-C^-, gtfB^-D^-, gtfC^-D^-, gtfB^-C^-D^-) were constructed by standard procedures. Dual biofilms consisting of V. dispar and either a GTF-mutant or the wild-type strain of S. mutans were grown on hydroxyapatite disks (anaerobic, with and without sucrose, 64.5 h, 37°C; "Zurich feeding model"). Microorganisms were stained with Yo-Pro-1. Additionally, V. dispar was stained by fluorescent in situ hybridization using a species-specific 16S rRNA probe. Confocal laser scanning microscopy assessed biofilm structure at 10 randomly selected positions using two biofilms from different experiments. Multiple optical scans per site were used to estimate volumes of both total bacterial cells and V. dispar in the biofilm. Results: In biofilms containing the wild-type strain the bacterial volume fraction was 51% (total biofilm volume=bacteria+EPS+voids=100%) and the mean biofilm thickness was 33 µm. In biofilms containing the gtfC^-D^--mutant, the volume occupied by bacteria decreased to 32% and the biofilm thickness to 22.9 µm. However, the volume ratio of S. mutans and V. dispar in these biofilms was almost identical, namely 2.8 and 3.0, respectively. In contrast, in biofilms containing the gtfB^-D^--, gtfB^-C^--, or gtfB^-C^-D^--mutant, the bacterial volume fraction dropped below 6%, the biofilm thickness below 5 µm, and the volume ratios were 9.1, 54.5 and 158.6, respectively, indicating a very low colonization level of Veillonella. When the growth medium contained glucose instead of sucrose biofilm formation was reduced to thin non-confluent biofilms not exceeding a thickness of 11 µm. Conclusions: It appears that the colonization level of V. dispar depends on GTF B. This enzyme diffuses from S. mutans cells, attaches on Veillonella and produces a glucan fostered colonization.