IADR Abstract Archives
Examination of the Dental Biofilm Matrix by Fluorescence Lectin-Binding Analysis
Objectives: Fluorescently labeled lectins are powerful tools to characterize glycoconjugates in the biofilm matrix. This study aimed to investigate the ability of 76 fluorescently labeled lectins to visualize and quantify extracellular glycoconjugates in dental biofilm.
Methods: Pooled supragingival plaque samples were initially screened with fluorescein isothiocyanate (FITC)-labeled lectins using Confocal Laser Scanning Microscopy. Fluorescence lectin-binding analysis (FLBA) was then applied to test 11 selected lectins derived from the screening on 48-h in situ grown biofilms in absence of sucrose. Five FITC-labeled lectins (Aleuria aurantia lectin, Calystega sepiem, Lycopersicon esculentum, Morniga G, and Helix pomatia) and a nucleic acid stain (Syto 60) were used for biovolume quantification of EPS-glycoconjugates and bacteria (n= 45). Bacterial composition of biofilms was analyzed by next generation sequencing (NGS). Additionally, different staining combinations of up to three suitable lectins and a nucleic acid stain (Syto 60 or Dapi) were used on in situ grown biofilms.
Results: Eleven FLBA-tested-lectins were able to stain glycoconjugates within different oral biofilm communities with good binding pattern. However, further tests with in situ biofilms indicated four “universally applicable” lectins: Aleuria aurantia lectin, Calystega sepiem, Lycopersicon esculentum, and Morniga G as well as two lectins with more selective binding patterns (Helix pomatia and Vicia graminea). The NGS results of pooled biofilms revealed greater taxonomic richness, while most in situ biofilms showed species diversities, representing natural dental biofilms. The application of several lectins was possible when suitable combinations were selected. Quantified biovolumes were significantly different between all tested lectins when applied on in situ biofilms of the same subject (Kruskal-Wallis).
Conclusions: Fluor-conjugated lectins enable the characterization and quantification of oral biofilm EPS-glycoconjugates. Furthermore, application of double or triple staining using different lectins in combination with general nucleic acid stains may be recommended for examining the EPS-glycoconjugate architecture of fully hydrated oral biofilms.
Methods: Pooled supragingival plaque samples were initially screened with fluorescein isothiocyanate (FITC)-labeled lectins using Confocal Laser Scanning Microscopy. Fluorescence lectin-binding analysis (FLBA) was then applied to test 11 selected lectins derived from the screening on 48-h in situ grown biofilms in absence of sucrose. Five FITC-labeled lectins (Aleuria aurantia lectin, Calystega sepiem, Lycopersicon esculentum, Morniga G, and Helix pomatia) and a nucleic acid stain (Syto 60) were used for biovolume quantification of EPS-glycoconjugates and bacteria (n= 45). Bacterial composition of biofilms was analyzed by next generation sequencing (NGS). Additionally, different staining combinations of up to three suitable lectins and a nucleic acid stain (Syto 60 or Dapi) were used on in situ grown biofilms.
Results: Eleven FLBA-tested-lectins were able to stain glycoconjugates within different oral biofilm communities with good binding pattern. However, further tests with in situ biofilms indicated four “universally applicable” lectins: Aleuria aurantia lectin, Calystega sepiem, Lycopersicon esculentum, and Morniga G as well as two lectins with more selective binding patterns (Helix pomatia and Vicia graminea). The NGS results of pooled biofilms revealed greater taxonomic richness, while most in situ biofilms showed species diversities, representing natural dental biofilms. The application of several lectins was possible when suitable combinations were selected. Quantified biovolumes were significantly different between all tested lectins when applied on in situ biofilms of the same subject (Kruskal-Wallis).
Conclusions: Fluor-conjugated lectins enable the characterization and quantification of oral biofilm EPS-glycoconjugates. Furthermore, application of double or triple staining using different lectins in combination with general nucleic acid stains may be recommended for examining the EPS-glycoconjugate architecture of fully hydrated oral biofilms.