Comparative analysis of the red-complex organisms and recently identified periodontal pathogens in the subgingival plaque of diabetic and nondiabetic patients with severe chronic periodontitis

Abstract

Aim: 

This analytical case–control study sought to evaluate the presence of the recently established putative periodontal pathogen organisms, Filifactoralocis and Fretibacteriumfastidiosum, against the levels of the already established red-complex pathogens, Porphyromonasgingivalis, Tannerellaforsythia, and Treponemadenticola, in chronic periodontitis sites of patients with and without diabetes mellitus.

Materials and Methods: 

Fifty-six subgingival plaque samples were obtained from the deepest sites of subjects diagnosed with severe chronic periodontitis with and without diabetes mellitus. These patients were categorized into two groups of 28 each. Clinical parameters were recorded and microbial analysis was done with quantitative polymerase chain reaction, and the bacterial counts of F. alocis and F. fastidiosum were determined and then compared with that of the red-complex organisms.

Results: 

The bacterial counts were found to be higher in the diabetic group than that in the nondiabetic group, which was statistically significant for T. forsythia (P < 0.037) and T. denticola (P < 0.003). The study found very less number of F. alocis, which was slightly higher in the diabetic group. When correlating the bacterial levels within the nondiabetic groups, the red complex species had a strong positive correlation both individually with F. alocis (P < 0.0001) and F. fastidiosum (P < 0.001) and also when the newer species was clubbed together as a cohort (P < 0.0001). Whereas, in the diabetic group, although there was a positive correlation, there was no statistical significance.

Conclusion: 

The results of this study highlighted the presence of a definite difference in the subgingival microbiota of both the patient groups evaluated. They also indicate that of the newly identified microorganisms, both the cohorts had higher levels of F. fastidiosum, suggesting a pathobiont-like role of this bacteria among both these periodontitis groups. F. alocis was comparatively lesser in number among the cohorts evaluated, and the cause for this decreased level of F. alocis needs to be further evaluated. The results of the present study depict a higher bacterial load in the diabetic group when compared to the nondiabetic group. Further, the study demonstrates a strong correlation between the red-complex species and the newer organisms in the nondiabetic group.

INTRODUCTION

Periodontitis is a multifactorial chronic inflammatory disease affecting the supporting structures, the initiation and progression being the result of interplay between essential bacterial factors and the host susceptibility. Our interpretation of the pivotal role of microorganisms in the etiology of periodontitis has gone through a sea change in the last 50 years.

The “polymicrobial synergy and dysbiosis” (PSD) model suggests that commensals that are part of a biofilm, once it has become dysbiotic, would in turn amplify the inflammatory state and perpetuate it, resulting in periodontal tissue destruction. A key component of a dysbiotic biofilm is the role of the pathobionts and of the keystone pathogens.[¹] In the PSD model, the established red-complex pathogens, with particular reference to Porphyromonasgingivalis, facilitate the uncontrolled growth of other species in the same biofilm enabling the biofilm to become inflammophilic and thereby promoting periodontal destruction. Among these newly identified species, Filifactoralocis and Fretibacteriumfastidiosum are bacteria that frequently seem to be associated with disease progression.[^2-4]

Diabetes mellitus that has an increasing global prevalence and incidence, has a significant role in modifying the onset and progression of periodontal disease. Zambon etal. first reported on the periodontal microflora in diabetes mellitus, indicating a significantly diverse flora, when compared to nondiabetics with chronic periodontitis.[⁴] A further study in a Hispanic population with diseased periodontal sites of patients modified with Type II diabetes showed that the most frequently isolated microorganisms are the red-complex bacteria, Aggregatibacteractinomycetemcomitans and Campylobacterrectus.[⁵] An independent study by Moon etal. assessed the composition of the bacterial communities in periodontitis patients modified by smoking, and found that F. alocis and F. fastidiosum along with other previously cultivated members were predominant in the subgingival plaque.[⁶]

The current study hypothesized a similar increase in the number of F. alocis and F. fastidiosum in patients diagnosed with diabetes mellitus over nondiabetic patients with chronic periodontitis. Hence, the study sought to assess the levels of red-complex bacteria P. gingivalis, Tannerellaforsythia, and Treponemadenticola and these new candidate periodontal pathogens – F. alocis and F. fastidiosum – in sites with severe chronic periodontitis in patient groups diagnosed with and without diabetes mellitus.

MATERIALS AND METHODS

The study proposal was placed before the Institutional Scientific and Ethical Review Board, and prior approval was obtained before the commencement of the study. All the patients participating in the study were fully explained about the nature of the study, following which written informed consent was obtained. With an α-set at 0.05 and β-set at 0.9 and an effect size of 0.91, the required sample was a total of 56 patients. Diabetic mellitus (DM) group comprised 28 diabetic patients diagnosed with severe chronic periodontitis and non-DM (NDM) group comprised 28 nondiabetic patients diagnosed with severe chronic periodontitis.

The patients recruited for the study were 30–60 years of age diagnosed with chronic periodontitis according to AAP 1999 classification with at least 20 remaining teeth and at least one site with a probing pocket depth (PPD) or Clinical attachment loss (CAL) ≥5 mm with radiological evidence of bone loss. The DM group included patients previously diagnosed and under treatment for ≥2 years with controlled type II diabetes mellitus, i.e., patients with a HbA1c value of ≤8%. Patients with known systemic conditions other than diabetes mellitus, patients with history of previous periodontal treatment and antibiotic intake in the last 3 months, patients who currently used tobacco products, and patients who were pregnant or lactating were excluded from the study. The clinical parameters such as the plaque index, gingival bleeding index, PPD, and Clinical attachment loss (CAL) were assessed by a single calibrated examiner, to rule out examiner bias prior to sample collection.[⁷⁸]

Plaque samples were collected from one of the deepest pockets based on the CAL in each patient. The selected site was isolated with cotton rolls and the supragingival plaque was removed with sterile cotton swab, then a sterile Gracey curette (Hu-Friedy) was positioned into the subgingival pocket, and the plaque was removed without blood contamination [Figure 1]. The plaque obtained was immediately transferred into Eppendorf tubes containing bacterial lysis buffer (Cat # 51104-1KT, Qiagen, Germany). The collected plaque samples were stored at 2°C–4°C until analysis.

Figure 1: Subgingival plaque sample collection

Plaque samples collected in DNA extraction buffer containing 100 mM of Tris (pH 8), 25 mM ethylenediaminetetraacetic acid, and 2% sodium dodecyl sulfate were digested with 10 mg/ml of lysozyme (Cat#L6876, Sigma-Aldrich, USA) at 37°C for 30 min. Following cell lysis, 20 mg/ml of Proteinase K was added and the lysates were incubated at 57°C for 2 h to digest all protein components present in the lysate. In order to quantitatively determine the copy numbers of each of the bacteria (relative to each other and among the samples), a standard curve was established. 2.5 ng of total DNA was subjected to polymerase chain reaction (PCR) amplification of the 16S rRNA gene hypervariable regions V1 to V9 with the following set of primers: Forward: AGTTTGATCCTGGCTCAG, and Reverse: TACCTTGTTACGACTT under standard PCR conditions. This PCR amplified products were subsequently quantified with Qubit fluorometer and were then diluted to obtain 3 nanogram concentrations in all the samples that were used as a template in the next round of PCR with species-specific primers [Table 1]. 10 mM of each of the primers was added to high-resolution melting EvaGreen RT-Master Mix (Cat# 206542, Qiagen, Germany) in 20 ml reaction, and samples were analyzed in Rotor-Gene Q real-time PCR equipment (Qiagen, Germany) under standard amplification conditions for 25 cycles. Upon completion of the real-time run, the samples were quantified with in-system software in reference to standards, and the presence of bacteria was expressed as copy numbers.

Table 1: Polymerase chain reaction primer sequences

All statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, version 17, IBM, India) for Microsoft windows. The data were expressed as mean and SD. Normality of the data was assessed by one–sample Kolmogorov–Smirnov test, and the data were not normally distributed; therefore, parametric/nonparametric tests were applied. Independent Student’s t-test was used to compare the clinical parameters between the study groups. The data obtained on the microbial quantification were nonparametric in nature, and therefore, Mann–Whitney U-test was rendered to be appropriate to evaluate the significance of the data. Intra-group correlation of the organism in the control and test groups was done using Kendall’s Tau b test. Correlation of the red-complex organisms and new organisms was done using Kendall’s Tau b test. P ≤ 0.05 was considered to be statistically significant.

RESULTS

In the present study, a total of 56 patients with severe chronic periodontitis fulfilling the selection criteria of our study as described previously were placed accordingly into DM and NDM groups. Following the clinical data documentation, plaque samples were collected and subjected to quantitative PCR. The descriptive statistics of the age, gender distribution, PPD, clinical attachment level, site-specific plaque, and gingival bleeding index are shown in Table 2.

Table 2: Descriptive statistics of the diabetic mellitus and nondiabetic mellitus group with comparative analysis

The mean quantity and the copy number of the organisms for all the samples are presented in Table 2. The results indicated that the bacterial counts were higher in the DM group for all the organisms except for the counts of F. alocis that was slightly higher (2869.93 ± 14098.252 copies) in the NDM group. On intergroup comparison, a statistically significant difference was seen for the levels of T. forsythia (P < 0.037) and T. denticola (P < 0.003). Although the levels of F. alocis were higher in the NDM group, there was no statistical significance.

The quantitative levels of the organisms were correlated with each other within the DM group. There was a positive correlation that existed between the organisms. However, a significant positive correlation was elucidated between P. gingivalis and T. forsythia (P < 0.006) and between T. forsythia and F. alocis (P < 0.007) [Table 3]. Whereas, in the NDM group, the correlation was strongly positive between the organisms. P. gingivalis, T. forsythia, and F. alocis positively correlated with all the other bacteria quantified. However, T. denticola and F. fastidiosum correlated with all the bacteria except with each other [Table 4].

Table 3: Comparison of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Filifactor alocis, and Fretibacterium fastidiosum in the diabetic mellitus group

Table 4: Comparison of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Filifactor alocis, and Fretibacterium fastidiosum in the nondiabetic mellitus group

F. alocis and F. fastidiosum were grouped together as new organisms and were correlated with the red-complex organisms in the DM and the NDM groups. There was no significant positive correlation between the red-complex and new organisms in the DM group (correlation coefficient: 0.201; P < 0.133). However, in the NDM group, there was a correlation between the red-complex organisms and new organisms, both individually and when grouped, particularly with P. gingivalis and T. forsythia indicative of some synergism [Table 5].

Table 5: Correlation of the red-complex organisms and the new organisms in the nondiabetic mellitus group

DISCUSSION

Pathobionts are low-volume members of the host biofilm in health, that are upregulated in disease, causing a pro-inflammatory priming of the host tissue as a result of the dysbiotic changes to the biofilm. It is apparent that the inflammophilic nature of the dental biofilm in periodontal disease is primarily caused by the reaction of pathobionts to keystone pathogens.[⁹¹⁰] The current analytical case–control study sought to evaluate the potential role of F. alocis and F. fastidiosum that have been identified in multiple metatranscriptomic studies, as probable pathobionts in the causation of periodontal disease.

F. alocis belonging to the phylum Firmicutes is considered an important periodontal pathogen that contributes to butyrate production that in turn induces the production of reactive oxygen species, inhibits the growth of gingival fibroblast, and induces apoptosis and autophagic cell death in gingival epithelial cells.[¹¹¹²] Further, Aruni etal. also highlighted a possible cooperative interaction between F. alocis and P. gingivalis in the diseased dysbiotic state.[¹³] F. fastidiosum that belongs to the phylum Synergistetes, has been shown to be in a cluster with other periodontopathic organisms associated and shown to be elevated in abundance in periodontitis sites. F. fastidiosum is also claimed to have co-occurrence with the red-complex species, most importantly with T. forsythia.[¹⁴¹⁵] Szafranski etal. confirmed that both F. alocis and F. fastidiosum along with the red-complex species are not only abundant, but that they are also an active part in the microbial community of periodontitis.[¹⁶]

Multifold studies indicate a significantly altered microbial community in diabetes mellitus patients with periodontitis.[^5,14,17-20] Casarin etal. suggested that there is a diversity between the diabetic and nondiabetic microbial communities.[²⁰] A similar diversity was reported by Ganesan etal., who indicated that diabetic cohorts had two distinct subgingival microbial signatures, that differentiated the diabetic individual with periodontitis, from the nondiabetic patient with periodontitis.[²¹] Among these two distinct microbial communities was one which had predominant levels of Fretibacterium species and F. alocis.

The elevated levels of T. denticola in the DM group of our study is in agreement with a study done by Li etal.[²²] Similarly, P. gingivalis titers did not show any significant difference in diabetics and nondiabetics associated with chronic periodontitis as recognized by multiple studies possibly indicating that P. gingivalis could be modulating the overgrowth of other bacteria that might be pathobionts, within this systemically compromised environment.[^22-25]

This study found a stronger positive correlation of F. fastidiosum with the red-complex species in both the groups. This aggregation might be indicative of F. fastidiosum’s role as a pathobiont. The overgrowth of this organism along with P. gingivalis certainly indicates a possible symbiotic relationship between these two organisms, and this relationship needs to be investigated further.

Barnes etal.[²⁶] in their metabolomic analysis of human saliva and plasma from healthy and diabetic subjects with and without periodontal disease suggest a reduced diversity of the oral microbiome in a diabetic state which was more recently highlighted by Sabharwal etal.[²⁷] Our study also observed a similar trend as with the levels of F. alocis. Fine etal. in their longitudinal study on periodontal destruction highlighted that F. alocis levels increased prior to bone loss, and decreased thereafter.[²⁸] This should also be taken into consideration in the understanding of the role of F. alocis in periodontal disease. However, Ganesan etal.[²¹] show significantly greater levels of F. alocis and F. fastidiosum in a diabetic cohort, suggesting the co-occurrence of these species, playing an important role in increasing the susceptibility and progression of periodontal disease.[²⁹]

The observation of low levels of F. alocis in our study could be indicative of a geographical variation in the Indian subcontinent or may be due to the primer-binding region of F. alocis genome being polymorphic. This possible polymorphism needs to be carefully evaluated through cultural studies and further sequencing of F. alocis primer-binding site. If, however, the levels indicated in this study are accurate, it would suggest that the microbiome across the world could have diverse bacteria that synergize periodontal destruction along with the internationally acknowledged red-complex organisms.

Our study further highlights a coactive relationship between P. gingivalis, a putative keystone pathogen, and F. alocis in the oxidatively stressed periodontium of patients with type II diabetes mellitus. Earlier studies quote that F. alocis is relatively resistant to oxidative stress, and that the levels of P. gingivalis are increased in an environment rich in oxidative stress, especially in the presence of F. alocis.[¹³³⁰] Several previous studies also pinpoint F. alocis to have a symbiotic association with P. gingivalis.[¹³³¹³²] The current study demonstrates a possible nexus of F. alocis with T. forsythia for which further strategized elucidation is warranted. Further, when comparing the entire red-complex species to the new organisms, i.e., F. alocis and F. fastidiosum as a cohort in the DM group, there was no significant positive correlation between them. However, when comparing the levels of organisms in the NDM group, the organisms P. gingivalis, T. forsythia, and F. alocis, all positively correlated with every other bacteria quantified. Further, T. denticola and F. alocis correlated with all the bacteria tested except with each other. This observation reiterates the fact that the diabetic cohort is systemically conditioned to elaborate greater host-driven inflammatory cytokines, that are expressed due to the higher oxidative stress secondary to the hyperglycemic state, that obviates the need for specific microbe driven periodontal breakdown. The current study highlights that clusters apart from red-complex pathogens need to be evaluated based on microbiome studies, to have a better understanding of the role of the diverse bacteria in the subgingival flora of periodontitis patients with varied risk profiles.

CONCLUSION

The results of this study have indicated a definite difference in the microflora of patients diagnosed with severe chronic periodontitis with and without diabetes mellitus and underline the need for extensive molecular and culture-based studies on the periodontal flora. The study highlights a synergy between F. fastidiosum and the red-complex species, which further energizes the call for F. fastidiosum to be identified as a pathobiont in chronic periodontitis. However, the role of F. alocis in chronic periodontitis is confusing based on its titer levels in our study. Future studies that address potential geographical variations/polymorphisms related to F. alocis will need to be vigorously investigated. In addition, studies that evaluate the role of newly identified clusters need to be carried out in periodontitis patients with varied risk profiles with longitudinal monitoring of these clusters, to declare with certainty the role of these clusters in periodontal disease progression.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.