Molecular methods for diagnosis of odontogenic infections.

PURPOSE: Historically, the identification of microorganisms has been limited to species that could be cultured in the microbiology laboratory. The purpose of the present study was to apply molecular techniques to identify microorganisms in orofacial odontogenic infections (OIs). MATERIALS AND METHODS: Specimens were obtained from subjects with clinical evidence of OI. To identify the microorganisms involved, 16S rRNA sequencing methods were used on clinical specimens. The name and number of the clones of each species identified and the combinations of species present were recorded for each subject. Descriptive statistics were computed for the study variables. RESULTS: Specimens of pus or wound fluid were obtained from 9 subjects. A mean of 7.4 ± 3.7 (standard deviation) species per case were identified. The predominant species detected in the present study that have previously been associated with OIs were Fusobacterium spp, Parvimonas micra, Porphyromonas endodontalis, and Prevotella oris. The predominant species detected in our study that have not been previously associated with OIs were Dialister pneumosintes and Eubacterium brachy. Unculturable phylotypes accounted for 24% of the species identified in our study. All species detected were obligate or facultative anaerobes. Streptococci were not detected. CONCLUSIONS: Molecular methods have enabled us to detect previously cultivated and not-yet-cultivated species in OIs; these methods could change our understanding of the pathogenic flora of orofacial OIs.

Defining the normal bacterial flora of the oral cavity.

More than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity. Our purposes were (i) to utilize culture-independent molecular techniques to extend our knowledge on the breadth of bacterial diversity in the healthy human oral cavity, including not-yet-cultivated bacteria species, and (ii) to determine the site and subject specificity of bacterial colonization. Nine sites from five clinically healthy subjects were analyzed. Sites included tongue dorsum, lateral sides of tongue, buccal epithelium, hard palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary anterior vestibule, and tonsils. 16S rRNA genes from sample DNA were amplified, cloned, and transformed into Escherichia coli. Sequences of 16S rRNA genes were used to determine species identity or closest relatives. In 2,589 clones, 141 predominant species were detected, of which over 60% have not been cultivated. Thirteen new phylotypes were identified. Species common to all sites belonged to the genera Gemella, Granulicatella, Streptococcus, and Veillonella. While some species were subject specific and detected in most sites, other species were site specific. Most sites possessed 20 to 30 different predominant species, and the number of predominant species from all nine sites per individual ranged from 34 to 72. Species typically associated with periodontitis and caries were not detected. There is a distinctive predominant bacterial flora of the healthy oral cavity that is highly diverse and site and subject specific. It is important to fully define the human microflora of the healthy oral cavity before we can understand the role of bacteria in oral disease.

Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics.

Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.