The prevalence of novel periodontal pathogens and bacterial complexes in Stage II generalized periodontitis based on 16S rRNA next generation sequencing

Abstract Objective: To define the subgingival microbial profile associated with Stage II generalized periodontitis using next-generation sequencing and to determine the relative abundance of novel periodontal pathogens and bacterial complexes. Methodology: Subgingival biofilm samples were collected from 80 subjects diagnosed with Stage II generalized periodontitis. Bacterial DNA was extracted, and 16S rRNA-based bacterial profiling via next-generation sequencing was carried out. The bacterial composition and diversity of microbial communities based on the age and sex of the patients were analyzed. The bacterial species were organized into groups: bacterial complexes (red, orange, purple, yellow, and green), novel periodontal pathogens, periodontal health-related species, and unclassified periodontal species. The results were analyzed and statistically evaluated. Results: The highest number of bacteria belonged to the phylum Bacteroidetes and Firmicutes. In terms of relative abundance, the orange complex represented 18.99%, novel bacterial species (Fretibacterium spp. and Saccharibacteria spp.) comprised 17.34%, periodontal health-related species accounted for 16.75% and unclassified periodontal species represented (Leptotrichia spp. and Selenomonas spp.) 15.61%. Novel periodontal pathogens had outweighed the periodontal disease-related red complex (5.3%). The one-sample z-test performed was statistically significant at p<0.05. The Beta diversity based on the unweighted UniFrac distance at the species level demonstrated a total variance of 15.77% based on age and 39.19% on sex, which was not statistically significant. Conclusion: The bacterial species corresponding to the disease-related orange complex and novel periodontal pathogens are predominant in Stage II generalized periodontitis.


Introduction
Periodontitis is a biofilm-associated inflammatory disease of the periodontium characterized by the destruction of periodontal tissue. Three significant factors are involved in the pathogenesis of periodontitis: susceptible host, presence of periodontal pathogens, and reduction or absence of beneficial bacterial species for periodontal health. Whilst, the microbial composition of subgingival biofilm differs in healthy individuals, the development of periodontitis is directly co-related to a characteristic shift in the microbiome referred to as "Dysbiosis." 1,2 The inter-relationship between dysbiosis of the oral microbiome and the periodontitis progression has been established by several studies. 3,4 These investigations postulate the role of the microbiome on the onset and progression of periodontitis.
The role of microbiome relating to periodontal health and disease was first described by Socransky, et al. 5 (1998). This path-breaking study -using whole genomic DNA probes to 40 bacterial species -has defined the potential role of the bacterial complex as opposed to individual bacterial species. The red complex is composed of three bacterial species: Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia that were associated with periodontal disease. The yellow complex composed of different Streptococcus species and green complex composed of Capnocytophaga species were firmly related to periodontal health. The orange complex -including Fusobacteria species -members of Prevotella, and Campylobacter were classified as periodontopathogens. The detection and quantitation of these bacterial complexes were described with whole genomic DNA probes, checkerboard DNA-DNA hybridization, 5 and using highly sensitive techniques such as real-time PCR (polymerase chain reaction). 6 These test may also be used to quantify species from Socransky complexes.
However, recent investigations using metagenomics and metatranscriptomics proposed that more diverse periodontitis-associated microbiota are involved in periodontal disease. According to this model, periodontitis results from polymicrobial synergy and dysbiosis that disturbs the ecologically balanced biofilm associated with periodontal tissue homeostasis. 7 In this model, the host's immune response is initially disrupted by keystone pathogens supported by accessory pathogens and it is subsequently overactivated by pathobionts resulting in homeostasis breakdown and destructive inflammation. The host's immune response is dysregulated either because it is disrupted by the microbial community or due to host immunoregulatory defect, resulting in bacterial outgrowth and overt pathogenicity. 8 Recent studies have also identified novel periodontal pathogens and additional bacterial species associated with periodontal health and disease. A study conducted by Kirst, et al. 9 (2015) has confirmed the presence of Rothia and Streptococcus species related to periodontal health. Similarly, investigations based on next-generation sequencing has revealed the presence of novel periodontal pathogens/pathobionts like 10,11,12  16SAmpliconPCRForwardPrimer=5' UniFrac distance -which represents the phylogenetic measurement of beta diversity -was estimated using QIIME. The Spearman's correlation coefficient was used to analyze the difference between the Unweighted UniFrac distances among two groups (based on the age and sex) and significance was set at p<0.05.   Elusimicrobia, and Verrucomicrobia. The species of bacteria in these groups of phyla were not considered in this study as they were not consistently present in all samples, also, they represented a frequency inferior to 1%. Levene's test for equality of variances for our analysis was F (2,78)=1.49, p=0.226. Since the significance was greater than 0.05, the Levene's Test was non-significant, and homogeneity of variance at phylum level were observed for age and sex.  Abudance of microbial complexes and novel bacterial species Table 1 reveals the data obtained for the relative abundance of bacterial species sorted into nine groups. The one-sample z-test performed for each group was statistically significant at p<0.05. The t statistics results are described in Table 1    sex for all samples were also measured using Jaccard Index. The percentage of variation was calculated to be 10.69% and the Jaccard distance was obtained as 0.89. Therefore, a Jaccard index of 0.89 stated an 89% similarity for two categories of age and sex in  The total abundance in each group was calculated by taking the sum of relative abundance of each bacterial species. † represents the highest bacterial species in each group. ‡ species that has been excluded in the remaining eight groups and their corresponding prevalence indicated greater than 0.1%.   The accompanying phyla identified in Stage II periodontitis along with the above-stated ones, were Proteobacteria, Actinobacteria, Patescibacteria, Epsilonbacteraeota, Synergistetes, and Choloroflexi.

Sequencing
In this study, we identified proteobacteria phylum, and their presence was related to periodontal health.  Synergistetes constitute a dysbiotic microbial community and these findings were consistent with the current literature. 8,12,13 A species-level of identification relating to their representative bacterial complex were described in order to recognize their prevalence in Stage II generalized periodontitis. The orange complex, healthrelated species, unclassified health/disease periodontal species, and novel periodontal pathogens represented a higher count, which was statistically significant.
This was followed by purple, yellow, red, and green complexes. The health-related species (Neisseria spp., Rothia spp., Kingella spp., and Corynebacterium spp.) and novel species (Fretibacterium spp. and The two novel bacterial species detected in our study were the Fretibacterium spp. and Saccharibacteria spp. The Fretibacterium spp. showed significantly increased levels in the periodontitis cases and were positively correlated with ≥ 4 mm periodontal pocket depth (PPD) and bleeding on probing (BOP). 33 As these bacterial species could not be cultivated, their characteristics and virulent aspects relating to the disease progression is unknown. The presence of Fretibacterium was also confirmed in the subgingival plaque of early stages of chronic periodontitis patients. 34 Saccharibacteria has dynamic interaction with its host bacteria and exhibits virulent killing of host bacterium. These bacteria can inhibit the host's growth dynamics and affect the oral microbial ecology. 35 Saccharibacteria previously known as TM7 detected in our study were also described by Kirst, et al. 9 (2015) in periodontitis cases. The role of novel periodontal pathogens has been reported. 32 These pathogens have shown a strong association with periodontal disease compared to less numerous periodontal pathogens previously implicated.
In addition to the novel species, other relevant species on our study were Neissera spp. Gemella spp., Rothia spp. Kingella spp., and Corynebacterium spp. The species belonging to Leptotrichia and Selenomonas genera -representing a higher relative abundance -were neither classified as health species nor pathogens as their role in either situation remains uncertain. Leptotrichia spp. are associated with both disease and normal health, representing distinct pathogenic potentials of bacteria in the same genera. 16 Selenomonas species have been strongly associated with periodontitis, and they play a role in the onset and progression of the disease. Higher levels of Selenomonas Sputigena were detected in the subgingival biofilm of deep periodontal pockets ≤ 6mm, that represents Stage II & III periodontitis. 38 However, their exact role in inducing periodontal destruction remains undetected, a higher prevalence of Selenomonas species identified in this study directly associates these species' presence in the periodontal pocket of Stage II generalized periodontitis.
Apart from the role of periodontal microbes, it is essential to consider the host-microbial interaction for the development and manifestation of periodontitis. The beta-diversity measurement based on Unweighted UniFrac distance indicated that the distance between two different age groups was minimal. Although a total variance of 39.19% was observed for sex, the findings were not statistically significant. While bacterial species variation occurs in males and females, a non-significant difference in this study could have resulted from the exclusion of subjects who presented systemic conditions. Systemic conditions like pregnancy would induce hormonal changes and variation in bacterial species compared to male subjects. A Jaccard Index of 89%, similar in both age and sex, suggests that the microbial profile did not vary between biofilm samples and are representative of Stage II generalized periodontitis.
The 16s rRNA based microbial profiling via NGS in periodontal conditions is the first of its kind to be studied in the United Arab Emirates. Exact quantification of bacterial species less than 0.01% has not been described in our study, which could be a limitation. Also,16-23S rRNA providing high-resolution analyses for bacterial species identification has not been employed in our study. However, 16S rRNA-based bacterial profiling via next-generation sequencing of supragingival and subgingival plaque has been employed in previous studies to determine the composition and diversity of bacterial communities. 16,25 Based on our study, it is possible to suggest that there is an association between Stage II generalized periodontitis and novel periodontal pathogens. Future studies based on proteomic and metabolomic analyses would provide information on the interbacterial relationship of these novel periodontal pathogens and improve our understanding on microbial function in periodontal pathogenesis.

Conclusion
The 16S rRNA via NGS approach in patients diagnosed with Stage II generalized periodontitis indicated that the bacterial species belonging to the orange-complex and novel periodontal pathogens are the most abundant.