The Caries Microbiome: Implications for Reversing Dysbiosis.

The oral microbiome plays a critical role in maintaining oral health. Frequent dietary carbohydrate intake can lead to dysbiosis of the microbial community from overproduction of acid with selection for increases in acidogenic, acid-tolerant bacteria. Knowledge of the caries-associated microbiome is key in planning approaches to reverse the dysbiosis to achieve health. For risk assessment and treatment studies, it would be valuable to establish whether microbial monitoring requires assay of multiple species or whether selected key species would suffice. Early investigations of the oral microbiota relied on culture-based methods to determine the major bacteria in health and disease. Microbial monitoring using gene probes facilitated study of larger populations. DNA probe methods confirmed and expanded the importance of transmission of bacteria from mother to infant and association of preselected species, including mutans streptococci and lactobacilli with caries in larger populations. 16S ribosomal RNA (rRNA) probes confirmed the wide diversity of species in oral and caries microbiomes. Open-ended techniques provide tools for discovery of new species, particularly when strain/clone identification includes gene sequence data. Anaerobic culture highlighted the caries association of Actinomyces and related species. Scardovia wiggsiae, in the Actinomyces/Bifidobacterium family, and several Actinomyces species have the cariogenic traits of acid production and acid tolerance. Next-generation sequencing combined with polymerase chain reaction methods revealed a strong association with mutans streptococci in a high caries population with poor oral hygiene and limited access to care. A population with a lower caries experience generally had lower or no Streptococcus mutans detection but harbored other acidogenic taxa in the microbiome. Study of the microbiome suggests a role for the assay of selected putative cariogenic species in more aggressive diseases. For many populations with caries progression, however, assay of multiple species will likely be warranted to determine the caries profile of the population and/or individuals under study.

Hypercementosis Associated with ENPP1 Mutations and GACI.

Mineralization of bones and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. This study for the first time analyzed the effect of decreased PPi on dental development in individuals with generalized arterial calcification of infancy (GACI) due to loss-of-function mutations in the ENPP1 gene. Four of the 5 subjects reported a history of infraocclusion, overretained primary teeth, ankylosis, and/or slow orthodontic tooth movement, suggesting altered mineral metabolism contributing to disrupted tooth movement and exfoliation. All subjects had radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. High-resolution micro-computed tomography (micro-CT) analyses of extracted primary teeth from 3 GACI subjects revealed 4-fold increased cervical cementum thickness ( P = 0.00007) and a 23% increase in cementum density ( P = 0.009) compared to age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 mutant mouse molars revealed 4-fold increased acellular cementum thickness ( P = 0.002) and 5-fold increased cementum volume ( P = 0.002), with no changes in enamel or dentin. Immunohistochemistry identified elevated ENPP1 expression in cementoblasts of human and mouse control teeth. Collectively, these findings reveal a novel dental phenotype in GACI and identify ENPP1 genetic mutations associated with hypercementosis. The sensitivity of cementum to reduced PPi levels in both human and mouse teeth establishes this as a well-conserved and fundamental biological process directing cementogenesis across species (ClinicalTrials.gov NCT00369421).

Microbiota of severe early childhood caries before and after therapy.

Severe early childhood caries (ECC) is difficult to treat successfully. This study aimed to characterize the microbiota of severe ECC and evaluate whether baseline or follow-up microbiotas are associated with new lesions post-treatment. Plaque samples from 2- to 6-year-old children were analyzed by a 16S rRNA-based microarray and by PCR for selected taxa. Severe-ECC children were monitored for 12 months post-therapy. By microarray, species associated with severe-ECC (n = 53) compared with caries-free (n = 32) children included Slackia exigua (p = 0.002), Streptococcus parasanguinis (p = 0.013), and Prevotella species (p < 0.02). By PCR, severe-ECC-associated taxa included Bifidobacteriaceae (p < 0.001), Scardovia wiggsiae (p = 0.003), Streptococcus mutans with bifidobacteria (p < 0.001), and S. mutans with S. wiggsiae (p = 0.001). In follow-up, children without new lesions (n = 36) showed lower detection of taxa including S. mutans, changes not observed in children with follow-up lesions (n = 17). Partial least-squares modeling separated the children into caries-free and two severe-ECC groups with either a stronger bacterial or a stronger dietary component. We conclude that several species, including S. wiggsiae and S. exigua, are associated with the ecology of advanced caries, that successful treatment is accompanied by a change in the microbiota, and that severe ECC is diverse, with influences from selected bacteria or from diet.

Cultivable anaerobic microbiota of severe early childhood caries.

Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen.

Clonal analysis of the microbiota of severe early childhood caries.

BACKGROUND/AIMS: Severe early childhood caries is a microbial infection that severely compromises the dentition of young children. The aim of this study was to characterize the microbiota of severe early childhood caries. METHODS: Dental plaque samples from 2- to 6-year-old children were analyzed using 16S rRNA gene cloning and sequencing, and by specific PCR amplification for Streptococcus mutans and Bifidobacteriaceae species. RESULTS: Children with severe caries (n = 39) had more dental plaque and gingival inflammation than caries-free children (n = 41). Analysis of phylotypes from operational taxonomic unit analysis of 16S rRNA clonal metalibraries from severe caries and caries-free children indicated that while libraries differed significantly (p < 0.0001), there was increased diversity than detected in this clonal analysis. Using the Human Oral Microbiome Database, 139 different taxa were identified. Within the limits of this study, caries-associated taxa included Granulicatella elegans (p < 0.01) and Veillonella sp. HOT-780 (p < 0.01). The species associated with caries-free children included Capnocytophaga gingivalis (p < 0.01), Abiotrophia defectiva (p < 0.01), Lachnospiraceae sp. HOT-100 (p < 0.05), Streptococcus sanguinis (p < 0.05) and Streptococcus cristatus (p < 0.05). By specific PCR, S. mutans (p < 0.005) and Bifidobacteriaceae spp. (p < 0.0001) were significantly associated with severe caries. CONCLUSION: Clonal analysis of 80 children identified a diverse microbiota that differed between severe caries and caries-free children, but the association of S. mutans with caries was from specific PCR analysis, not from clonal analysis, of samples.