Presence of host and bacterial-derived collagenolytic proteases in carious dentin: a systematic review of ex vivo studies.

Introduction and aim: The presence of host collagenases in the degradation of the protein matrix at later stages of carious dentin lesions development, as well as the potential involvement of bacterial collagenases, have been suggested but lack conclusive evidence. This study aims to conduct a systematic review to comprehensively assess the profile of host and bacterial-derived collagenolytic proteases in both root and coronal dentin carious lesions. Methods: The search was performed in eight databases and the grey literature. Studies evaluating ex vivo dentin, extracted teeth, or biofilms from natural caries lesions were included. The methodological quality of studies was assessed using the Joanna Briggs Institute tool. Synthesis of the results and the certainty of evidence were performed following the Synthesis without Meta-analysis (SWiM) checklist and GRADE approach for narrative synthesis, respectively. Results: From 935 recovered articles, 18 were included. Although the evidence was very uncertain, it was possible to suggest that 1) MMP-2, MMP-9, MMP-13, and CT-B may be increased in carious dentin when compared to sound dentin; 2) there is no difference in MMP-2 presence, while MMP-13 may be increased in root when compared to coronal carious dentin; 3) there is no difference of MMP-2 and MMP-9 expression/activity before and after cavity sealing; 4) MMP-8 may be increased in the dentin before cavity sealing compared to dentin after cavity sealing; 5) there is no difference of MMP-20 in irradiated vs. non-irradiated carious dentin. MMP-20 probably reduces in carious outer dentin when compared to carious inner dentin (moderate certainty). Genes encoding bacterial collagenolytic proteases and protein-degrading bacteria were detected in coronal and root carious lesions. Conclusion: Trends in the direction of the effect were observed for some collagenolytic proteases in carious dentin, which may represent a potential target for the development of new treatments. (Protocol register-PROSPERO: CRD42020213141).

Meta-analyses on the Periodontal Archaeome.

Recently, we have published a scoping review on the oral archaeome, showing that these microorganisms inhabit various oral niches, including periodontal sites. In order to reinforce the importance of the Archaea domain and alert the scientific community about the importance of inter-domain relationships in oral dysbiosis, we have performed meta-analyses evaluating the prevalence of archaea in periodontal diseases (PROSPERO protocol: CRD42020213109). A systematic search in the literature was conducted in several databases and in grey literature, retrieving 30 reports on periodontal archaeome, published from 1980 to 2020. The methodological quality of included studies and the certainty of evidence were evaluated by using validated tools. Most studies focused on the detection of methanogens, revealing that the diversity of the periodontal archaeome is currently underestimated. Two meta-analyses concluded that individuals with periodontitis are prone to have archaeal-positive subgingival biofilms when compared to periodontally healthy individuals (OR 6.68, 95% CI 4.74-9.41 for 16S rRNA gene analysis and OR 9.42, 95% CI 2.54-34.91 for mcrA gene analysis). Despite the archaeal enrichment in sites with periodontitis, less than half of the individuals with periodontitis tested positive for archaeal DNA (general estimative of 46%; 95% CI 36-56%). Conventional treatment for periodontitis reduced the archaeal population, but systemic antibiotics used as adjunctive therapy did not increase its effectiveness. Hence, it could conceivably be hypothesised that archaea are secondary colonizers of areas with dysbiosis, probably flourishing in the inflammatory environment. Due to their lower prevalence, archaeal cells are probably underestimated by the current detection protocols. It may also be speculated that archaea do not have a single central role in the infection, with bacterial cells directly involved in that role. New studies are necessary, with different methodological approaches, to explore the underestimated diversity of the oral archaeome.

Low-Abundant Microorganisms: The Human Microbiome's Dark Matter, a Scoping Review.

Research on the human microbiome has mainly been restricted to the identification of most abundant microbiota associated with health or disease. Their abundance may reflect their capacity to exploit their niche, however, metabolic functions exerted by low-abundant microrganisms can impact the dysbiotic signature of local microbial habitats. This scoping review aims to map the literature regarding the management of low-abundant microorganisms in studies investigating human microbiome samples. A systematic literature search was performed in 5 electronic databases, as well as grey literature. We selected clinical microbiome studies targeting human participants of any age, from any body site. We also included studies with secondary data which originated from human biofilm samples. All of the papers used next-generation sequencing (NGS) techniques in their methodology. A total of 826 manuscripts were retrieved, of which 42 were included in this review and 22 reported low-abundant bacteria (LB) in samples taken from 7 body sites (breast, gut, oral cavity, skin, stomach, upper respiratory tract (URT), and vagina). Four studies reported microbes at abundance levels between 5 and 20%, 8 studies reported between 1 and 5%, and 18 studies reported below 1%. Fifteen papers mentioned fungi and/or archaea, and from those only 4 (fungi) and 2 (archaea) produced data regarding the abundance of these domains. While most studies were directed towards describing the taxonomy, diversity and abundance of the highly abundant species, low-abundant species have largely been overlooked. Indeed, most studies select a cut-off value at <1% for low-abundant organisms to be excluded in their analyses. This practice may compromise the true diversity and influence of all members of the human microbiota. Despite their low abundance and signature in biofilms, they may generate important markers contributing to dysbiosis, in a sort of 'butterfly effect'. A detailed snapshot of the physiological, biological mechanisms at play, including virulence determinants in the context of a dysbiotic community, may help better understand the health-disease transition.

Presence of Archaea in dental caries biofilms.

OBJECTIVE: Although the prevalence and functions associated with members of Bacteria are well known in dental caries, the role of Archaea in cariogenic biofilms has not been studied yet. DESIGN: To detect the presence of Archaea in dental caries, a triplicate of carious dentine samples and duplicate of supragingival biofilms were collected, total microbial DNA was extracted and the composition of the microbiota was investigated. Total DNA was submitted to 16S rRNA gene amplification using universal prokaryotic primers; amplicons were sequenced by high-throughput DNA sequencing. As a second strategy to detect Archaea, a representative sample of caries was chosen and other PCR reactions were performed using specific primers targeting the archaeal 16S rRNA gene; amplicons were cloned and sequenced. Annotation of sequences was performed using SILVA database and the relative abundance of genus level OTUs was calculated. RESULTS: The high-throughput sequencing method detected archaeal sequences in all samples (identified as group I.1c of the phylum Thaumarchaeota), although in a very low abundance (≤0.03 % of the total sequences). For the second strategy, 14 archaeal clones were detected, with an OTU affiliated to Methanocella clade, and another one affiliated to group I.1b of the phylum Thaumarchaeota. CONCLUSIONS: Archaeal sequences were detected in dental caries and biofilms from surfaces without caries lesions. DNA sequences of Thaumarchaeota were also identified, showing that overall archaeal diversity in the human oral cavity could be currently underestimated and not restricted to methanogens.