Análise metagenômica da microbiota associada à doença periodontal

Abstract

Periodontitis is an infectious disease that affects the tissues involving and supporting teeth. Although specific bacteria have been implicated with periodontitis, application of molecular-based research can bring additional information, since a significant parcel of the oral microbiota cannot be detected by traditional cultivation methods. This prospective case-control study aims to assess the taxonomic affiliation and functional processes involved in periodontitis, based on the metagenomic analysis of periodontitis carriers and healthy subjetcts. Saliva samples were collected from healthy (control group, n = 13) and periodontitis subjects (case group, n = 14) attended at the UEA Dental Clinic. Total DNA was extracted from the samples, quantified, and submitted to next-generation sequencing in Illumina Hiseq2500 platform. After data cleaning, the nucleotide sequences were subjected to pairing by the PEAR software, taxonomic prediction by Metaphlan 2.0 and MG-RAST (RefSeq data), measurement of alpha (Shannon-Wiener and Simpson) and beta diversity indexes (Whittaker), and functional annotation with KEGG and CAZy molecular database tools. The difference in the frequency of taxa and functional categories between groups was assessed by the Kruskal-Wallis test (α = 0.05). A total of 104.004.730 sequences was submitted to annotation, of which 3.267.049 (3.23%) and 49.391.119 (47.49%) were annotated by Metaphlan 2.0 and MG-RAST, respectively. The statistical analyzes were performed by STAMP and R v3.4.3 softwares. Based on the results, the most abundant phyla were Firmicutes (29.52%), Actinobacteria (27.83%), Bacteroidetes (20.43%), Proteobacteria (17.94%), Fusobacteria (2.97%), Candidatus Saccharibacteria (0.45%), Spirochaetes (0.33%) and Tenericutes (0.03%). Comparative analysis revealed variations in the abundance or presence/absence of taxa between the study groups that suggests an intricate association with periodontal disease. Morevoer, the increased frequencies of representatives of Bacteroidetes, Fusobacteria, Candidatus Saccharibacteria, Spirochaetes and Synergistes in the periodontitis group suggest potential pathogenic roles. Moreover, correlation analysis indicated that the genera Eubacterium, Leptotrichia, Corynebacterium, Treponema, Parvimonas, Fusobacterium, Filifactor, Capnocytophaga, Tannerella, Bifidobacterium, Peptostreptococcus, Dialister, Catonella, Selenomonas, Campylobacter and Porphyromonas embody a potential periodontopathogenic group. Functional analysis showed that KEGG’s “Metabolism” category was predominant in the oral cavity. Meanwhile, the category “Genetic Information Processing” was positively related to periodontal disease. Other categories that demonstrated higher abundance (p < 0.05) for the sick group were the Aminoacyl-tRNA biosynthesis, pyruvate metabolism, and carbon fixation pathways in prokaryotes. In addition, functional analysis resulted in 11 potential bioindicator genes of periodontal disease such as: rpoC, rpoB, carB, secA, uvrA, pflD, thrS, pheT, ntpA, cobQ e aspS. It can be concluded that the oral cavity presents a diverse microbial community with high inter-individual variability. However, there are select groups of bacteria that appear to be associated with health and disease conditions. This microbiota is able to live in synergism by modulating its functional activity and by complicating even more the mechanism of the disease. Finally, this study reiterates that the oral cavity houses a significant number of unknown bacteria, as well as genes potentially involved in periodontal disease, and that high throughput DNA sequencing constitutes an important tool in the study of the oral microbiome.