Molecular analysis of the oral microbiota of dental diseases

Abstract: Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p<0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p<0.05, ECC), Veillonella parvula (p<0.01), Veillonella atypica (p<0.05), and Veillonella sp. HOT-780 (p<0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p <0.05), and Porphyromonas gingivalis (p<0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p<0.05), and visible dental plaque (p<0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p<0.01) by oligonucleotide DNA probes, and P. gingivalis (p<0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p<0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p<0.05). P. gingivalis (p<0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.