Fluorescence lectin binding analysis of carbohydrate components in dental biofilms grown in situ in the presence or absence of sucrose

Abstract

Carbohydrate components, such as glycoconjugates and polysaccharides, are constituents of the dental biofilm matrix that play an important role in biofilm stability and virulence. Exopolysaccharides in Streptococcus mutans biofilms have been characterized extensively, but comparably little is known about the matrix carbohydrates in complex, in situ-grown dental biofilms. The present study employed fluorescence lectin binding analysis (FLBA) to investigate the abundance and spatial distribution of glycoconjugates/polysaccharides in biofilms (n = 306) from 10 participants, grown in situ with (SUC) and without (H2O) exposure to sucrose. Biofilms were stained with 10 fluorescently labeled lectins with different carbohydrate specificities (AAL, ABA, ASA, HPA, LEA, MNA-G, MPA, PSA, VGA and WGA) and analyzed by confocal microscopy and digital image analysis. Microbial composition was determined by 16S rRNA gene sequencing. With the exception of ABA, all lectins targeted considerable matrix biovolumes, ranging from 19.3% to 194.0% of the microbial biovolume in the biofilms, which illustrates a remarkable variety of carbohydrate compounds in in situ-grown dental biofilms. MNA-G, AAL, and ASA, specific for galactose, fucose, and mannose, respectively, stained the largest biovolumes. AAL and ASA biovolumes were increased in SUC biofilms, but the difference was not significant due to considerable biological variation. SUC biofilms were enriched in streptococci and showed reduced abundances of Neisseria and Haemophilus spp., but no significant correlations between lectin-stained biovolumes and bacterial abundance were observed. In conclusion, FLBA demonstrates the presence of a voluminous biofilm matrix comprising a variety of different carbohydrate components in complex, in situ-grown dental biofilms.

Keywords: 16S rRNA sequencing; biofilm; confocal laser scanning microscopy; extracellular polymeric substances; fluorescence lectin binding analysis; glycoconjugates; in situ.