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Detection of bacterial populations in denture plaque using DGGE

Coulthwaite, Lisa and Smith, Philip W. and Higham, Susan M. and Verran, Joanna (2006) Detection of bacterial populations in denture plaque using DGGE. [Conference or Workshop Item]

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Abstract

Objectives: The aim of this study was to use the culture independent technique, Denaturing Gradient Gel Electrophoresis (DGGE) to characterise the bacterial populations in denture plaque and to investigate the differences in composition from sites seen to fluoresce in vivo under Quantitative Light-induced Fluorescence (QLF) conditions. Methods: In a clinical study 40 removable PMMA dentures were screened for fluorescent plaque deposits under QLF conditions. Plaque samples taken were suspended in reduced transport fluid and frozen at -20 ºC. Total genomic DNA was extracted from the plaque samples and from pure cultures of Porphyromonas gingivalis, Prevotella intermedia, P. melaninogenica, Fusobacterium nucleatum, Actinomyces naeslundii, A. viscosus, A. israelii, Streptococcus sanguis, S. oralis, S. salivarius and Peptostreptococcus micros. Extracted DNA from denture plaque samples and pure cultures was amplified by PCR using universal primers for the 16S rRNA gene. DNA fragments were separated by DGGE to produce bacterial community profiles ‘fingerprints'. Banding pattern profiles were analysed for band composition and number, representing species present and species diversity within each plaque sample. Partial funding from Reckitt Benckiser. Results: Differences were seen in DGGE community profiles from red and green fluorescing sites. This indicated the presence of different bacterial species between sites. No single band was present in all samples, indicating no single species was present in all samples. Species diversity was much lower than expected and was not related to the fluorescence of the plaque samples. Red fluorescing microorganisms are not always cultured from red fluorescing sites, thus the microorganisms responsible for fluorescence in vivo may be dependent on the presence of other species which may be viable but non-cultivable. Conclusion: This study showed differences in the bacterial populations present in red and green fluorescent plaque on dentures, supporting results obtained from culture studies, where some obligate anaerobic species were associated with red fluorescence.

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