Comparative evaluation of subgingival microbiome in healthy periodontium and gingivitis using next-generation sequencing technology: A case-control study.

Background: Human dental plaque is a complex microbial community containing millions of species. Gingivitis is a dysregulated immune-inflammatory response induced by dysbiotic plaque biofilm that interrupts symbiosis. The emergence of next-generation sequencing with 16S rRNA gene has greatly contributed in understanding the complexity of microbiota. However, studies focusing on microbiome in gingivitis are limited. The whole bacterial community is important in causing periodontal disease than a small number of periodontal pathogens. In this study, we attempted to profile the subgingival microbiome from individuals with healthy gingiva and in patients with gingivitis using next-generation sequencing technology. Materials and
Methods: Subgingival plaque samples from 15 healthy periodontium (Group I) and 15 gingivitis (Group II) were collected and 16s rRNA sequencing was done in Illumina Solexa Sequencer. Data analysis using 16s metagenomics tool from BaseSpace onsite operational taxonomic units was assigned to each sequence using HOMD database. Individual variation in the microbiome of the subgingival samples between the two groups was also evaluated.
Results: The comparison of top 20 species between Group I and Group II revealed no significant species group between them. Synergistetes was absent in Group I samples but found in Group II. At the genus level, HACEK group species were found in both the groups, while Dialister and Aneroglobus were found abundantly in the Group II.
Conclusion: The presence of unique genera and species seen in Group II samples could point toward a dysbiotic shift that could be taking place in the subgingival environment leading to gingivitis. Copyright:

INTRODUCTION

The human microbiota is comprised trillions of symbiotic microbes in different areas of our body, each area having their own unique microflora that mirror their microenvironment.[1] Microflora harboring the mucosa-lined surfaces have their own ecology where these bacteria flourish. In an apparently healthy individual, the microbiome establishes a symbiotic mutual interconnection with the host, where the microbes find themselves in a nourishing environment. In addition to this, the bacterium assists the host through metabolic activities, priming the immune cells and shielding from pathogenic foreign organisms.[2] A disruption in the symbiosis leads to dysbiosis which has a profound impact on systemic health leading to dire consequences. On the tooth surface, the bacterial colonies adhere and form complex biofilms which when accumulated leads to plaque-induced gingivitis. This inflammatory process triggers the host immune response and results in the destruction of the periodontal attachment apparatus.[3]

Chronic gingival inflammation results in irreversible destruction of periodontal tissue.[4] Both gingivitis and periodontitis are closely related with higher load of microbial colonies demonstrated through community richness and Shannon indices. This close association was also found to be in sociodemographic factors. A study by Shaw et al. discovered numerous taxa related to gingival inflammation. The abundant taxa that were seen were correlated with an increase in the severity of gingivitis.[5] However, their abundance alone is insufficient to explain the presence of disease. There has been a big leap in our understanding the role of plaque in gingivitis and periodontitis from the “Experimental gingivitis model” of Loe due to the rapid advancement in technologies.[6]

Research involving the microbiome of the oral cavity was identified using bacterial cultures in the early days.[7] Since 40%–60% of bacteria from oral microflora cannot be cultured due to which culture-independent methods such as polymerase chain reaction (PCR), in situ hybridization, immunologic, and enzymatic assays were introduced, however, they were found to be close ended.[8] To address this issue, next-generation sequencing (NGS) was introduced and has revolutionized the studies of oral microbiome in individuals with healthy or inflamed gingival apparatus and is an open-ended technique.[9] NGS works on the principle involving sequences of oligonucleotides that are subjected to cyclical ligation through an automated machine using repetitive cycles of nucleotide extension sequences.[10] A single NGS run provides us with millions of nucleotide sequences owing to reactionary processes occurring in a parallel manner resulting in high sequence reads per run.[11] Despite the fact that research on the subgingival microbiome has been undertaken, there is a scarcity of information on the microbial population in gingivitis. Hence, as a first of its kind, this study was done to investigate the subgingival microbiome in gingivitis and normal healthy periodontium using NGS technology among the South Indian population.

MATERIALS AND METHODS

Ethical approval was obtained from Institutional Ethical Committee and the study was carried out from August 2019 to December 2019. This case–control study was conducted among 55 subjects, of which 30 individuals who fulfilled the inclusion criteria were enrolled. The participants were further split into two groups, 15 patients with gingivitis[12] were considered as the test group (Group II) and 15 individuals with healthy periodontium with no signs of inflammation[13] were the control group (Group I).

Inclusion criteria

For Control group (Group I):

Healthy gingiva without inflammation

Absence of bleeding on probing (BOP)

Probing pocket depth (PPD) ≤3 mm

No clinical attachment loss (CAL).

For Test group (Group II):

Subjects with gingivitis who had bleeding score ≥1 according to Muhlemann and Son Bleeding Index[14]

Patients who scored ≥2 in Silness–Loe plaque index[15]

PPD ≤3 mm and no CAL.

Exclusion criteria

Patients who were under the antibiotic medication within the last 6 months

Patients who had undergone periodontal treatment within the last 12 months

Patients who used commercial mouthwash

Patients with the habit of smoking or tobacco chewing

Individuals under immunosuppressant or steroid for the past 6 months.

Sample collection

Prior to the clinical evaluation and sample collection, written informed consent was obtained along with plaque and bleeding index from all the participants after explanation of the study. In Group II, subgingival plaque samples were collected from areas with BOP. The site was first air-dried and isolated using cotton. Following this, supragingival plaque was removed with gauze and subgingival plaque was collected with sterile Gracey Curette that was inserted 2 mm of the sulcus depth. The plaque on the curette tip was retrieved with a mild pull motion toward the coronal surface of the associated tooth. The tip of the curette containing the subgingival plaque was dropped in an Eppendorf tube containing ionized molecular water and shaken until the plaque dislodged from the tip and into the tube. In Group I, subgingival areas that did not exhibit any sign of inflammation were chosen for plaque samples. The same method was followed for collecting the samples, as shown in Figure 1. The collected samples were stored at a temperature of − 20°C. To prevent degradation, the collection of samples was done in 2 days, after which it was sent for analysis.

Figure 1

(a) Collection of subgingival plaque samples with Gracey curettes; (b) Sample collected in curette's tip; (c) Sample transferred to Eppendorf tube containing ionized molecular water; (d) FastStart essential DNA kit

DNA extraction

According to the manufacturer's instructions (MP Biomedicals, Santa Ana, CA, USA), genomic DNA was extracted from 30 plaque samples using the Fast DNA kit and the FastPrep 24-5G. Silica-based spin filters were used to purify the extracted DNA. The 16S V3 (341F) forward and V4 (805R) reverse primer pairs with Illumina adapter overhang nucleotide sequences were used for amplification of the extracted DNA.

16S rRNA amplification

Amplicon synthesis was performed using thermocycling with genomic DNA (8.5 μl), amplicon PCR forward primer and reverse primer (2 μl each), and 2x KAPA HiFi HotStart Ready Mix (12.5 μl). Initial denaturation was performed for 3 min at 95°C, followed by annealing in 25 cycles of 95°C, 62.3°C, and 72°C for 30 s each, with a final elongation of 5 min at 72°C. Agencourt AMPure XP beads (Beckman Coulter Genomics) were used to clean up the reactions. Five microliter of amplicon PCR product DNA, Illumina Nextera XT Index Primer 1 (N7xx), and Illumina Nextera XT Index Primer 2 (S5xx), 25 μl of 2x KAPA HiFi HotStart Ready Mix, and 10 μl of PCR-grade water were used to attach dual indices and Illumina sequencing adapters (UltraClean DNA-free PCR water; MO BIO Laboratories, Inc., Carlsbad, CA, USA). Following this process, thermocycling was done for 3 min at 95°C, after which 8 cycles of 95°C, 55°C, and 72°C were performed for 30 s each, with final extension done for 5 min at 72°C.

Library construction and sequencing

Agencourt AMPure XP beads were used to purify 16S metagenomic libraries. The quantification of the same was done using Quant-iT PicoGreen and the KAPA Library Quantification Kit (KAPA BIOSYSTEMS). The Agilent Technologies 2100 Bioanalyzer was used to perform a library quality control. Illumina Nextseq 500 System was used to carry out sequencing. Using 2 × 150 bp paired-end sequencing, all the plaque samples were sequenced in a single lane on the NextSeq.

Statistical analysis

Data analysis was done by using 16s metagenomics tool from BaseSpace onsite operational taxonomic units which were assigned to each sequence using HOMD database. At the genus level, a circular maximum likelihood phylogenetic tree was generated using the iTOL and PhyloT tools, according to Letunic and Bork.[16] The Mann–Whitney U-test was used to compare species abundance.

RESULTS

The study population consisted of 15 individuals in Group I who are periodontally healthy and 15 patients in Group II with gingivitis, whose subgingival plaque samples were obtained and subjected to 16s rRNA sequencing using NGS technology. Bacterial phyla, genera, and species were described for each of the 30 plaque samples and their relative abundance was quantified through taxonomic assignment to the reference database. A total of 6 phyla, 21 genera, and 37 species were found in the Group I samples and 7 phyla, 32 genera, and 59 species in the Group II samples were found.

On comparing the abundance of phyla among Group I and Group II, the concentration of Firmicutes and Proteobacteria was more in Group I. Compared to Group I, the abundance of Candidatus saccharibacteria, Actinobacteria, Bacteroidetes, and Fusobacteria was found to be higher in Group II. Synergistetes have only been observed in the Group II samples. This comparison is represented in Figure 2.

Figure 2

Comparison of abundance of phylum in Group I and Group II

In Group I samples, the most abundant genus present was Veillonella (22%) followed by Neisseria (15%) and Saccharibacteria genera incertae sedis (13%). The list of most abundant top 8 genera in Group I samples is shown in Table 1. In Group II samples, Veillonella (17%) was found to be the most abundant genus, followed by Saccharibacteria genera incertae sedis (16%) and Dialister (14%). The list of most abundant top 8 genera in Group II samples is shown in Table 2.

Table 1

Evaluation of abundance of top 8 genera and their percentage among Group I samples

Genus	Relative abundance expressed in logs	Percentage of genera (%)
Veillonella	3.050	22
Neisseria	2.136	15
Saccharibacteria genera incertae sedis	1.799	13
Actinomyces	1.623	12
Haemophilus	1.591	11
Kingella	1.591	11
Eikenella	1.255	9
Prevotella	1	7

Table 2

Evaluation of abundance of top 8 genera and their percentage among Group II samples

Genus	Relative abundance expressed in logs	Percentage of genera (%)
Veillonella	2.514	17
Saccharibacteria genera incertae sedis	2.303	16
Dialister	2.086	14
Anaeroglobus	1.995	14
Kingella	1.462	10
Eikenella	1.380	10
Prevotella	1.380	10
Neisseria	1.342	9

Comparison of abundance of top 20 species in Group I versus Group II is described in Table 3 and Figure 3. The results were not statistically significant (P = 0.081), as shown in Table 4. Comparison of abundance of top 20 species in Group II versus Group I is described in Table 5 and Figure 4. The results were not statistically significant (P = 0.296), as shown in Table 6. There are about 11 species present in Group I but are absent in Group II samples and about 46 species that are present in Group II but are absent in Group I samples, as shown in Figure 5. The organisms which are present only in the Group II samples may be the causative factor for the gingivitis.

Table 3

Comparison of abundance of top 20 species in Group I versus Group II

Organism	Abundance
	Group I	Group II
Veillonella tobetsuensis	3.003	2.475
Veillonella parvula	2	0.778
Kingella oralis	1.591	1.4623
Haemophilus parainfluenzae	1.579	0.845
Actinomyces odontolyticus	1.477	0.845
Eikenella corrodens	1.255	1.380
Actinomyces meyeri	0.954	0.301
Dialister invisus	0.903	2.068
Veillonella denticariosi	0.903	0.602
Veillonella rogosae	0.778	0.845
Capnocytophaga leadbetteri	0.698	0.477
Capnocytophaga sputigena	0.602	0.004
Cardiobacterium hominis	0.602	0.477
Streptococcus sanguinis	0.602	0.698
Aggregatibacter segnis	0.477	0.301
Prevotella maculosa	0.477	0.845
Actinomyces oris	0.301	0.301
Aggregatibacter aphrophilus	0.301	0.602
Veillonella atypica	0.004	1.041
Cardiobacterium valvarum	0.004	0.954

Figure 3

Comparison of abundance of top 20 species in Group I versus Group II

Table 4

Comparison of abundance of top 20 species in Group I versus Group II

Group	n	Mean	SD	SEM	P a
Abundance log
Group I	20	0.925	0.726	0.162	0.881
Group II	20	0.865	0.601	0.134

a Statistical significance - P≤0.05; Mann-Whitney U-test. SD – Standard deviation; SEM – Standard error mean; n – Number of samples; P – Statistical significance

Table 5

Comparison of abundance of top 20 species in Group II versus Group I

Organism	Abundance
	Group II	Group I
Veillonella tobetsuensis	2.475	3.003
Dialister invisus	2.068	0.903
Eikenella corrodens	1.380	1.255
Veillonella atypica	1.041	0.004
Atopobium parvulum	0.954	0.004
Cardiobacterium valvarum	0.954	0.004
Haemophilus parainfluenzae	0.845	1.579
Prevotella maculosa	0.845	0.477
Veillonella rogosae	0.845	0.778
Veillonella parvula	0.778	2
Megasphaera micronuciformis	0.698	0.004
Streptococcus sanguinis	0.698	0.602
Veillonella denticariosi	0.602	0.903
Aggregatibacter aphrophilus	0.602	0.301
Capnocytophaga leadbetteri	0.477	0.698
Cardiobacterium hominis	0.477	0.602
Actinomyces meyeri	0.301	0.954
Aggregatibacter segnis	0.301	0.477
Prevotella oulorum	0.301	0.004
Actinomyces naeslundii	0.301	0.004

Figure 4

Comparison of abundance of top 20 species in Group II versus Group I

Table 6

Comparison of abundance of top 20 species in Group II versus Group I

Group	n	Mean	SD	SEM	P a
Abundance log
Group I	20	0.847	0.567	0.126	0.296
Group II	20	0.726	0.775	0.173

aStatistical significance - P≤0.05; Mann-Whitney U-test. SD – Standard deviation; SEM – Standard error mean; n – Number of samples; Pa – Statistical significance

Figure 5

List of species present in Group I and Group II

The comparison of subgingival microbiome in Group I versus Group II at the genus level is depicted as phylogenetic tree in circular form, as given in Figure 6. The tree has been built using phyloT software and is exhibited using iTol.[16] In Group I (orange) and Group II (blue) sites, the relative abundance of bacterial genera is reflected by the bars in the outer band (green).

Figure 6

Circular maximum likelihood of phylogenetic tree at the genus level

DISCUSSION

Gingivitis is a type of reversible inflammatory periodontal disease characterized by BOP and changes in the color and texture of the gingiva.[12] Accumulation of plaque leads to uncontrolled gingivitis which progresses to periodontitis. Literature indicates that periodontitis is associated with an increase in bacterial accumulation and shift in the subgingival microbial composition from Gram-positive microorganisms to Gram-negative anaerobes.[1718] However, most of the literature has focused on methods of microbiological research such as culture, immunodiagnostic methods, and techniques of hybridization of DNA-DNA. The relative presence, abundance, and properties of particular species associated with periodontal disease have been shown in the DNA-DNA checkerboard study as red complex organisms such as Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola.[19]

The present study was carried out using Illumina sequencing method as it allows more sequence per run and detecting diverse bacteria compared to conventional sequencing. The 16s rRNA amplicon sequencing was sufficient to determine low abundance taxa.[20] In this study, there was an overall increase in the number of colonies of bacterium in Group II as compared to Group I samples. In both Group I and Group II, Firmicutes was the most abundant phylum present, comprising approximately 30% and 24%, respectively. Proteobaceria, C. saccharibacteria, Actinobacteria, and Fusobacteria were the other phyla present at approximately identical levels in both groups, which is in accordance with other research.[2122] Firmicutes, Proteobaceria, and Actinobacteria are early colonizers of the plaque biofilm.

The only phylum presents exclusively in the Group II community was Synergistetes, which is in line with earlier studies.[2324] Synergistetes was recognized as one of the 13 distinct phyla described in the Human Oral Microbiome Database. Although information on the existence of Synergistetesis is scarce, it is not quite clear how this phylum is associated with plaque-inducing gingivitis.[25] In both Group I and Group II, Veillonella was the most abundant genus comprising 22% and 17%, respectively. Veillonella is a thin, Gram-negative cocci, obligatory anaerobes, aflagellate with spores or capsules which play a crucial role in promoting species succession in a multispecies biofilm environment.[26] In Group I, Actinomyces and Haemophilus were found to be more abundant compared to Group II samples, which is in line with the previous studies.[27] The microbiome of the subgingival plaque from Group II had more variation at the genus level than Group I, where the ecological succession is not a replacement strategy by the primary organisms, but rather viewed as the introduction of dominant ones as the biofilm matures.[9]

At the genus level, Streptococcus, a vital group of bacterium that initiates the formation of plaque biofilm, was not abundant in both the groups. This could be due to the impact on the lifestyle and dietary habits.[28] It could be hypothesized that supragingival plaque could lead to the accumulation of streptococci in the oral cavity. A mild shift in the environmental ecology was seen in our study owing to the subgingival ecology, which is different from the other parts of the oral cavity. In Group II, among the abundant genus, Aneroglobus was exclusive in the subgingival region.

Dialisters are small anaerobic coccobacilli that grow on Colombia's blood agar as small, circular and transparent colonies. In mixed cultures, they can be hard to differentiate from other species and often requires alternative molecular methods. Dialister pneumosintes and Dialister invisus are said to cause periodontal diseases.[2529] Our study findings are in resonance with the above and its very presence among the most abundant genera in the Group II samples suggests a shift in the atmosphere from a commensal to a dysbiotic one. The HACEK group (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella) are the Gram-negative, fastidious bacilli which were seen in abundance in both the groups, and these organisms were frequently isolated from the subgingival setting.[30] Their role in periodontal inflammation and systemic participation is widely documented in the literature.[29]

At the species level, the abundant ones were Veillonella tobetsuensis, Veillonella parvula, Actinomyces odontolyticus, and Actinomyces meyeri. Veillonella which achieves symbiosis with the other bacterium, thereby promoting the development of a microbial ecology.[31] When we compared the top 20 species between Group I and Group II samples, there was no statistical significance, indicating that there may be no distinct shift between periodontal health and gingivitis in the microbial communities. It could be hypothesized that at this point, the species in the shallow subgingival microenvironment could only be comprised commensal origin, and as changes in the environment became more pronounced, a more drastic shift could possibly lead to dysbiosis. The organisms which are present only in Group II may be the etiological risk factor for gingivitis. However, the limitation of the study is that it was conducted with a limited sample size of 30 patients owing to the financial constraints and complexity of the technique involved, which was similar to other studies.[3233] The results of this study should further be validated in the future with a robust sample size which are essential for further understanding the role of plaque in gingivitis.

CONCLUSION

There was a mild change in the microbiome between Group I and Group II samples. Certain species of Dialister, namely D. pneumosintes, D. Invisus, and Aneroglobus which have been known to cause periodontal disease were found in abundance in Group II. This study proves few genera and species were exclusive to gingivitis and they could facilitate dysbiosis and cause a shift in the subgingival environment, thereby causing disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.