Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of XXXX bacteria in oral biofilms.

@entity6586 @entity10688 @entity291 @entity880
BACKGROUND: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of @entity880 bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. RESULTS: As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized @entity291 enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only @entity10688 and bacteria of the @entity10688 casei group. The most cariogenic biofilm contained two orders of magnitude higher @entity10688 cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas @entity6586 was detected in only one sample in very low numbers. CONCLUSIONS: Application of these new group- and species-specific FISH probes to oral biofilm-forming @entity880 bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.
