The phylum Synergistetes in gingivitis and necrotizing ulcerative gingivitis.

The clinical manifestation of necrotizing ulcerative gingivitis (NUG) is distinct from that of common gingivitis in that it is characterized by local necrosis of the gingival tissues, rapid onset, pain and extensive bleeding. The phylum Synergistetes is a novel bacterial phylum consisting of Gram-negative anaerobes, with evidence of presence in biofilms associated with periodontal and endodontic infections. To date, the involvement of members of this phylum in NUG has not been investigated. This study aimed to evaluate the presence and levels of known human oral Synergistetes bacterial clusters in dental plaque from patients with NUG and compare them with those found in gingivitis. Marginal dental plaque samples from 21 NUG and 21 gingivitis patients were analysed quantitatively by fluorescent in situ hybridization and microscopy for members of two oral Synergistetes clusters (A and B) and for Jonquetella anthropi. Synergistetes cluster A bacteria were detected in all samples but at higher levels (9.4-fold) and proportions (2.5-fold) in NUG patients than in gingivitis patients. However, with regard to Synergistetes cluster B bacteria, there were no differences between NUG and gingivitis patients. J. anthropi was detected in only half of the samples and at lower levels than the other taxa. In conclusion, these data demonstrate that Synergistetes cluster A bacteria, but not cluster B bacteria or J. anthropi, are more strongly associated with NUG than with gingivitis.

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

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 lactic acid 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 bovine 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 Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans 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 lactic acid 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.

Uncultivated Tannerella BU045 and BU063 are slim segmented filamentous rods of high prevalence but low abundance in inflammatory disease-associated dental plaques.

Uncultivated clones BU045 and BU063 and Tannerella forsythia, a 'consensus periodontal pathogen', are the closest known relatives within the genus Tannerella. They have been described to inhabit different ecological niches of the human oral cavity. In this study, fluorescent in situ hybridization (FISH) and immunofluorescence were combined to investigate the prevalence and abundance of BU045 and BU063 in comparison to T. forsythia in plaques from gingivitis, necrotizing ulcerative gingivitis (NUG) and chronic periodontitis. Phylotype-specific FISH probes identified BU045 and BU063 as elongated thin rods with a segmented structure. Two structurally similar and previously unknown, rare phylotypes (127+ and 997+) were also identified due to partial 16S rRNA sequence identity with T. forsythia. In gingivitis, NUG and periodontitis patients, BU045, BU063, 127+, 997+ and T. forsythia were detected with prevalences of 50/83/71/14 and 81%, 100/100/86/17 and 53%, and 100/100/12/0 and 100%, respectively. Supragingivally, colonization density of all five organisms was generally low, rarely exceeding 0.1% of the total biota. In periodontal pocket samples, however, cell numbers of T. forsythia, but not of the uncultivable phylotypes, were greatly elevated. Our data demonstrate that Tannerella phylotypes BU045, BU063, 127+ and 997+ consist of long slim rods with segments, which, with respect to FISH stainability, often behaved as independent units. The phylotypes are frequent but low-level colonizers of various periodontal disease-associated plaques. Their apparent inability to proliferate to high density seems to exclude any relevance for the pathogenesis of periodontal diseases.

A combined immunofluorescence and fluorescent in situ hybridization assay for single cell analyses of dental plaque microorganisms.

A IF-FISH protocol was developed to characterize discrete cell populations within complex dental plaque samples. Optimizing and shortening the 3-step IF procedure and including RNase inhibitor in all IF reagents enabled the combination of both techniques and thus the simultaneous detection and enumeration of bacterial populations with distinct 16S rRNA sequences and/or surface markers of interest.