Residual Bacteriome after Chemomechanical Preparation of Root Canals in Primary and Secondary Infections.

INTRODUCTION: Secondary infections may be linked to the presence of residual microorganisms within dental root canals. The purpose of this study was to investigate the bacterial composition of primary and secondary root canal infections before and after chemomechanical treatment. METHODS: Samples were collected before chemomechanical preparation (S1) and before obturation (S2) from 19 subjects (10 primary and 9 secondary infections). DNA was extracted, and the V3/V4 region of the 16S ribosomal RNA gene was amplified using the 347 F/803R primers and paired-end sequenced using the MiSeq (Illumina, San Diego, CA) instrument. RESULTS: Sequencing analysis yielded partial 16S ribosomal RNA gene sequences that were taxonomically classified into 10 phyla and 143 genera. The most prevalent phyla in the S1 and S2 samples were Firmicutes and Bacteroides; however, when comparing between sample groups, Proteobacteria seem to have been enriched in secondary infections. The dominant genera in the primary S1 samples were Bacillus, Streptococcus, and Prevotella, whereas Bacillus, Streptococcus, and Selenomonas dominated the secondary infection S1 samples. Bacillus and Marinilactibacillus were the most dominant genera in the primary and secondary S2 samples. The mean number of operational taxonomic units per sample was 32,656 (±12,124 SD) and 37,113 (±16,994 SD) in the S1 and S2 samples, respectively. Alpha and beta diversities presented the same pattern within samples from both groups. CONCLUSIONS: Great interindividual variations in the bacterial composition of the root canal biofilms were observed. There was no difference in the bacterial composition before and after treatment, although some genera survived and seem to be part of a residual microbiome. Our findings revealed a high diversity of the bacterial communities present in root canal infections after chemomechanical treatment, although the majority of the taxa detected were in low abundance.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries.

A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries

Abstract A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

Enrichment of periodontal pathogens from the biofilms of healthy adults.

Periodontitis is associated with shifts in the balance of the subgingival microbiome. Many species that predominate in disease have not been isolated from healthy sites, raising questions as to the origin of these putative pathogens. The study aim was to determine whether periodontal pathogens could be enriched from pooled saliva, plaque and tongue samples from dentally-healthy adult volunteers using growth media that simulate nutritional aspects of the inflamed subgingival environment. The microbiome was characterised before and after enrichment using established metagenomic approaches, and the data analysed bioinformatically to identify major functional changes. After three weeks, there was a shift from an inoculum in which Streptococcus, Haemophilus, Neisseria, Veillonella and Prevotella species predominated to biofilms comprising an increased abundance of taxa implicated in periodontitis, including Porphyromonas gingivalis, Fretibacterium fastidiosum, Filifactor alocis, Tannerella forsythia, and several Peptostreptococcus and Treponema spp., with concomitant decreases in health-associated species. Sixty-four species were present after enrichment that were undetectable in the inoculum, including Jonquetella anthropi, Desulfovibrio desulfuricans and Dialister invisus. These studies support the Ecological Plaque Hypothesis, providing evidence that putative periodontopathogens are present in health at low levels, but changes to the subgingival nutritional environment increase their competitiveness and drive deleterious changes to biofilm composition.

Insulin-Like Growth Factor Axis Expression in Dental Pulp Cells Derived From Carious Teeth.

The insulin-like growth factor (IGF) axis plays an important role in dental tissue regeneration and most components of this axis are expressed in human dental pulp cells (DPCs). In our previous study, we analyzed IGF axis gene expression in DPCs and demonstrated a novel role of IGF binding protein (IGFBP)-2 and -3 in coordinating mineralized matrix formation in differentiating DPCs. A more recent study from our laboratory partially characterized dental pulp stem cells from teeth with superficial caries (cDPCs) and showed that their potential to differentiate odontoblasts and/or into osteoblasts is enhanced by exposure to the mild inflammatory conditions characteristic of superficial caries. In the present study, we examine whether changes apparent in IGF axis expression during osteogenic differentiation of healthy DPCs are also apparent in DPCs derived from carious affected teeth.

Gene expression of bacterial collagenolytic proteases in root caries.

Objective: It is unknown whether bacteria play a role in the collagen matrix degradation that occurs during caries progression. Our aim was to characterize the expression level of genes involved in bacterial collagenolytic proteases in root biofilms with and without caries. Method: we collected samples from active cavitated root caries lesions (RC, n = 30) and from sound root surfaces (SRS, n = 10). Total microbial RNA was isolated and cDNA sequenced on the Illumina Hi-Seq2500. Reads were mapped to 162 oral bacterial reference genomes. Genes encoding putative bacterial collagenolytic proteases were identified. Normalization and differential expression analysis was performed on all metatranscriptomes (FDR<10-3). Result: Genes encoding collagenases were identified in 113 bacterial species the majority were peptidase U32. In RC, Streptococcus mutans and Veillonella parvula expressed the most collagenases. Organisms that overexpressed collagenolytic protease genes in RC (Log2FoldChange>8) but none in SRS were Pseudoramibacter alactolyticus [HMPREF0721_RS02020; HMPREF0721_RS04640], Scardovia inopinata [SCIP_RS02440] and Olsenella uli DSM7084 [OLSU_RS02990]. Conclusion: Our findings suggest that the U32 proteases may be related to carious dentine. The contribution of a small number of species to dentine degradation should be further investigated. These proteases may have potential in future biotechnological and medical applications, serving as targets for the development of therapeutic agents.