Dental Caries Etiopathogenesis: Microbial Composition, Functional Activity and Host Recognition

For decades, the sugar-fermenting, acidogenic species Streptococcus mutans has been considered the main causative agent of dental caries and most diagnostic and therapeutic strategies have been targeted toward this microorganism. However, the DNA- and RNA-based studies from carious lesions reported in this thesis, have uncovered an extraordinarily diverse ecosystem where S. mutans accounts only for a tiny fraction of the bacterial community. This supports the concept that consortia formed by multiple microorganisms act collectively, probably synergistically, to initiate and expand the carious lesion. The data also show that these microbial consortia are different between individuals, between the affected tissue, and even between different lesions from the same individual. Thus, antimicrobial therapies are not expected to be effective in the treatment of caries and other polymicrobial diseases that do not follow Koch's postulates, and that I propose cannot be considered infectious diseases in classical terms. In addition, the data also indicate a prominent role for the immune system in caries risk, suggesting that therapies directed towards stimulating immunological competence should be explored. Based on the results from this Thesis, I propose that dental caries is a dysbiotic polymicrobial disease caused by pathobionts

Revealing microbial recognition by specific antibodies

Background: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system. Results: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin. Conclusions: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases

Potential implications of SARS-CoV-2 oral infection in the host microbiota

The oral cavity, as the entry point to the body, may play a critical role in the pathogenesis of SARS-CoV-2 infection that has caused a global outbreak of the coronavirus disease 2019 (COVID-19). Available data indicate that the oral cavity may be an active site of infection and an important reservoir of SARS-CoV-2. Considering that the oral surfaces are colonized by a diverse microbial community, it is likely that viruses have interactions with the host microbiota. Patients infected by SARS-CoV-2 may have alterations in the oral and gut micro-biota, while oral species have been found in the lung of COVID-19 patients. Furthermore, interactions between the oral, lung, and gut microbiomes appear to occur dynamically whereby a dysbiotic oral microbial community could influence respiratory and gastrointest-inal diseases. However, it is unclear whether SARS-CoV-2 infection can alter the local home-ostasis of the resident microbiota, actively cause dysbiosis, or influence cross-body sites interactions. Here, we provide a conceptual framework on the potential impact of SARS- CoV-2 oral infection on the local and distant microbiomes across the respiratory and gastro-intestinal tracts (‘oral-tract axes’), which remains largely unexplored. Studies in this area could further elucidate the pathogenic mechanism of SARS-CoV-2 and the course of infection as well as the clinical symptoms of COVID-19 across different sites in the human host

Impact of the repurposed drug thonzonium bromide on host oral-gut microbiomes

Drug repurposing is a feasible strategy for the development of novel therapeutic applications. However, its potential use for oral treatments and impact on host microbiota remain underexplored. Here, we assessed the influences of topical oral applications of a repurposed FDA-approved drug, thonzonium bromide, on gastrointestinal microbiomes and host tissues in a rat model of dental caries designed to reduce cross-contamination associated with coprophagy. Using this model, we recapitulated the body site microbiota that mirrored the human microbiome profile. Oral microbiota was perturbed by the treatments with specific disruption of Rothia and Veillonella without affecting the global composition of the fecal microbiome. However, disturbances in the oral-gut microbial interactions were identified using nestedness and machine learning, showing increased sharing of oral taxon Sutterella in the gut microbiota. Host-tissue analyses revealed caries reduction on teeth by thonzonium bromide without cytotoxic effects, indicating bioactivity and biocompatibility when used orally. Altogether, we demonstrate how an oral treatment using a repurposed drug causes localized microbial disturbances and therapeutic effects while promoting turnover of specific oral species in the lower gut in vivo

Stimulated and unstimulated saliva samples have significantly different bacterial profiles.

Epidemiological studies use saliva on a regular basis as a non-invasive and easy-to-take sample, which is assumed to be a microbial representative of the oral cavity ecosystem. However, comparative studies between different kinds of saliva samples normally used in microbial studies are scarce. The aim of the current study was to compare oral microbiota composition between two different saliva samples collected simultaneously: non-stimulated saliva with paper points and stimulated saliva collected after chewing paraffin gum. DNA was extracted from saliva samples of ten individuals, then analyzed by 16S rRNA pyrosequencing to describe bacterial diversity. The results demonstrate significant differences between the microbiota of these two kinds of saliva. Stimulated saliva was found to contain an estimated number of species over three times higher than unstimulated saliva. In addition, bacterial composition at the class and genus level was radically different between both types of samples. When compared to other oral niches, both types of saliva showed some similarity to tongue and buccal mucosa, but they do not correlate at all with the bacterial composition described in supra- or sub-gingival dental plaque, questioning their use in etiological and epidemiological studies of oral diseases of microbial origin

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 (FDR8) 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

Dental biofilm: ecological interactions in health and disease.

BACKGROUND: The oral microbiome is diverse and exists as multispecies microbial communities on oral surfaces in structurally and functionally organized biofilms. AIM: To describe the network of microbial interactions (both synergistic and antagonistic) occurring within these biofilms and assess their role in oral health and dental disease. METHODS: PubMed database was searched for studies on microbial ecological interactions in dental biofilms. The search results did not lend themselves to systematic review and have been summarized in a narrative review instead. RESULTS: Five hundred and forty-seven original research articles and 212 reviews were identified. The majority (86%) of research articles addressed bacterial-bacterial interactions, while inter-kingdom microbial interactions were the least studied. The interactions included physical and nutritional synergistic associations, antagonism, cell-to-cell communication and gene transfer. CONCLUSIONS: Oral microbial communities display emergent properties that cannot be inferred from studies of single species. Individual organisms grow in environments they would not tolerate in pure culture. The networks of multiple synergistic and antagonistic interactions generate microbial inter-dependencies and give biofilms a resilience to minor environmental perturbations, and this contributes to oral health. If key environmental pressures exceed thresholds associated with health, then the competitiveness among oral microorganisms is altered and dysbiosis can occur, increasing the risk of dental disease