|Year : 2021 | Volume
| Issue : 4 | Page : 433-438
The varied proportion of filifactor alocis in periodontal health and disease in the South Indian subpopulation
Anila Neelakandan1, Ravishankar Potluri1, Pradeep Kumar Yadalam1, Priyankar Chakraborty1, AV Saravanan1, Rex Arunraj2
1 Department of Periodontology and Implantology, SRM Dental College, SRM Institute of Science and Technology, Chennai, Tamil Nadu, India
2 Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, Chennai, Tamil Nadu, India
|Date of Submission||17-Sep-2020|
|Date of Decision||29-Sep-2020|
|Date of Acceptance||31-Oct-2020|
|Date of Web Publication||21-Dec-2021|
Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, Kattankulathur, Chennai - 603 203, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Aim: The periodontal microbiome being complex, this study was aimed to detect and quantify the prevalence of Filifactor alocis in various stages of periodontitis and to evaluate its prospect as a diagnostic marker for periodontal disease. Settings and Design: Sixty subjects were selected (20 healthy controls, 20 with chronic periodontitis, and 20 with aggressive periodontitis) for the study. Materials and Methods: Clinical parameters probing depth and the level of clinical attachment was recorded, subgingival plaque samples were collected. The F. alocis 16srDNA was cloned, sequenced, and used as the standard for real-time quantification of bacterial load using SYBR green chemistry. Statistical Analysis: Clinical, microbiological, and quantitative polymerase chain reaction (PCR) data were analyzed using ANOVA and Pearson's coefficient correlation. Results: (a) Real-time PCR analysis showed the highest average F. alocis count in chronic periodontitis subjects (32,409.85), which was followed by count in healthy controls (3046.15) and the least count in aggressive periodontitis subjects (939.84). The bacterial count was statistically significant at P = 0.005. (b) An intra-group comparison reveals that there was a statistically significant increase in the bacterial count with age and mean probing pocket depth at P = 0.0005. Conclusion: F. alocis population in aggressive periodontitis was lower compared to chronic periodontitis and healthy controls. The F. alocis population surge in healthy controls may be due to geographical variations and the ethnicity of the subjects. A higher population of F. alocis in chronic periodontitis proves its high pathogenic potential to invade the host tissues to aid in further periodontal destruction.
Keywords: Chronic periodontitis, Filifactor alocis, microbial load, real-time quantitative polymerase chain reaction
|How to cite this article:|
Neelakandan A, Potluri R, Yadalam PK, Chakraborty P, Saravanan A V, Arunraj R. The varied proportion of filifactor alocis in periodontal health and disease in the South Indian subpopulation. Contemp Clin Dent 2021;12:433-8
|How to cite this URL:|
Neelakandan A, Potluri R, Yadalam PK, Chakraborty P, Saravanan A V, Arunraj R. The varied proportion of filifactor alocis in periodontal health and disease in the South Indian subpopulation. Contemp Clin Dent [serial online] 2021 [cited 2022 Jan 28];12:433-8. Available from: https://www.contempclindent.org/text.asp?2021/12/4/433/333151
| Introduction|| |
Periodontitis is an intricate interplay of bacteria-induced infection and host counteraction, modified by systemic factors that prompt the obliteration of the encompassing periodontal structures. The three keystone pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola initiate the disease.,,, P. gingivalis is the most extensively studied organism, but P. gingivalis alone does not suffice to cause infection. Pathobiont is a critical factor in infection and disease establishment; it exist in the normal oral microbiota and act opportunistically during inflammation. Pathobionts ameliorate the microenvironment for other periodontal pathogens and aid in host immunity manipulation. F. alocis, a periodontal pathogen identified through novel culture-independent techniques such as polymerase chain reaction (PCR), Next-Gen Sequencing, DNA hybridization is emerging as a critical pathobiont due to its increased presence in periodontally diseased patients. F. alocis is obligately anaerobic, fastidious, Gram-positive asaccharolytic bacilli that are slow-growing and difficult to culture.
F. alocis is found along with P. gingivalis qualifying as a microbial diagnostic marker.
Technological advances such as immunoassays, biochemical tests, and nucleic acid hybridization have been used for microbial examination. Since bacterial culture is a prerequisite, the above techniques are mostly laborious and time-consuming. Advanced molecular biology techniques have largely evolved over the past few years to engage in culture-independent methods; PCR is a robust, rapid, and sensitive technique for the identification of periodontal pathogens. The 16S RNA (16SrDNA) gene of the small ribosomal subunit is often used as a target for PCR for its highly conserved regions through evolution and species-specific variable sequences. The conserved sequences can offer PCR primers for amplification of 16SrDNA from all the bacterial species and species-specific regions for the identification of unique bacterial species.
F. alocis is emerging as a pathobiont, gaining more relevance in the world of periodontal microbiology. There is very little done to report F. alocis prevalence in periodontitis in the Indian ethnic population. This study aims to detect the presence and estimate the absolute count of F. alocis in periodontal health and disease in the South Indian ethnic population.
| Materials and Methods|| |
Sixty systemically healthy individuals visiting the Department of Periodontics underwent a comprehensive periodontal examination that included probing depth (PD) and level of clinical attachment (CAL). The subjects were assigned into three groups: periodontally healthy (Group A), aggressive periodontitis (Group B), and chronic periodontitis (Group C) [AAP 1999] based on the following inclusion criteria: group A-Clinically healthy gingiva without bleeding on probing, CAL and PD ≤ 3 mm [Figure 1]a, Group B-aggressive periodontitis, at least four sites showing ≥3 mm CAL and PD ≥5 mm including rapid attachment loss and bone destruction, radiographically visible vertical or arc-shaped bone loss, particularly in central incisors and first molars, amount of destruction does not commensurate with local factors [Figure 1]c and Group C-Chronic periodontitis, at least four sites showing ≥3 mm CAL and probing pocket depth (PPD) ≥5 mm, amount of destruction consistent with local factors, radiographically visible bone loss in the horizontal direction, frequent detection of subgingival plaque and calculus [Figure 1]b. The patients who had any systemic diseases like diabetes mellitus, HSV or HIV infection, patients with a history of either smoking or chewing tobacco, and patients who underwent periodontal treatment or antimicrobial therapy in the duration of the pst 6 months were excluded from the study.
|Figure 1: Representation of the various clinical specimens involved in the study. (a) Healthy periodontium, (b) chronic periodontitis, (c) aggressive periodontitis, and (d) sample collection using paper points|
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The plaque samples were collected the next day following clinical examination to avoid contamination of the samples by bleeding induced by probing. The samples were collected from four deep pockets using sterile paper points of size 35, pooled and transferred immediately to Eppendorf tubes containing 1 mL of Tris-EDTA buffer [Figure 1]d. This ensures high sensitivity and accurate representation of the subject. The samples were freeze stored at −80°C until the bacterial count.
16srDNA cloning and sequencing
From the total sample collected, 1 μl of the sample was used to amplify 594 bp of 16srDNA region of F. alocis by PCR using the primer pair FP-CTAATACCGCATACGTCCTAAG/RP-CTACTAAGCAATCAAGTTGCCC. The PCR conditions used were 95°C for 3 min (initial denaturation) followed by 35 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s and a final extension at 72°C for 3 min. The PCR product was gel purified, cloned into the pGEM-T easy vector, transformed into Top10 cells and confirmed by DNA sequencing. The plasmid was used as a standard for the absolute quantification of F. alocis.
Absolute quantification by quantitative polymerase chain reaction
A 20 μl reaction was set using 2.5 pm each of primers, with known concentrations of standard plasmid DNA. Absolute quantification was performed using the following program: 95°C, 7 min, 35 cycles of 95°C for 20 s, 55°C for 20 s, and 72°C for 20 s, melt analysis consisting of 95°C for 1 min, −65°C for 1 min, −95°C continuous acquisition. Fluorescence data was captured during the extension stage of each cycle. The plasmid containing the 16srDNA fragment was used as a standard [Figure 2]a. A standard graph was computed using log of the copy number of the plasmid against Cq value.
|Figure 2: Absolute quantification of Filifactor alocis in healthy, chronic and aggressive subjects of periodontitis. The 16srDNA cloned plasmid was used as a standard to quantify Filifactor alocis in the subjects (a). The average absolute count of the bacteria in the various subjects were estimated by pooling of paper points of individual subjects and absolute count quantified in triplicates using quantitative polymerase chain reaction (b)|
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The data were analyzed with IBM. SPSS statistics software 23.0 Version (SPSS statistics software produced by SPSS Inc , Chicago, Illinois USA). Descriptive statistics were performed using mean and standard deviation, while multivariate analysis was computed using one-way ANOVA with Tukey's Post-Hoc test at P = 0.05. To assess the relationship between the variables, Pearson's Correlation was used.
| Results|| |
PPD and CAL correspond to the severity of periodontitis
The mean age of Group A, Group B, and Group C was 36.85 ± 8.55, 26.20 ± 3.32, and 43.05 ± 9.29, respectively [Supplementary Table 1]; Group B subjects were younger than Group C and Group A subjects. In addition, there was no significant difference in gender-wise distribution among the three groups. Periodontal examination revealed that PPD and CAL were high in chronic subjects, but was maximum in aggressive subjects. The mean PPD for Group A, Group B, and Group C was 2.45 ± 0.65, 6.75 ± 0.75, and 5.71 ± 0.98, respectively [Supplementary Table 1]. The mean CAL for Group A, Group B, and Group C was 0, 7.28 ± 1.47 and 6.23 ± 1.23, respectively [Supplementary Table 1]. The mean PPD and mean CAL were statistically significant at P ≤ 0.05 (0.0005) [Supplementary Table 2].
Cloning and absolute quantification of Filifactor alocis
The 16srDNA of F. alocis was PCR amplified, cloned, and sequence confirmed (MT573543) for use as a standard to quantify the pathogen in healthy, aggressive, and chronic patient groups. The standard curve was prepared with varying copy numbers of the 16srDNA containing plasmid (r2 = 0.9989), and the bacteria were quantified in all three groups [Figure 2]a.
Filifactor alocis colonization and growth depends on the periodontal microbiome
Absolute count of F. alocis was determined using real-time quantitative PCR (qPCR). The average bacterial count in Group A (healthy) was 3046.15; 40% of the samples showed more than the average bacterial count, whereas the average bacterial count in Group B (aggressive) was only 939.84; 40% of the samples showed more than the average. The mean bacterial count in Group C (chronic) was 32,409.85, with 15% of the samples having an absolute count more than the group's average count [Figure 2]b.
Inter- and intra-group comparison for clinical parameters and absolute count
Analysis of variance showed a significant difference between age, PPD, CAL, and the count of F. alocis in healthy, aggressive, and chronic patients [Supplementary Table 1]. The correlation within the groups for the absolute bacterial count with age, mean PPD, and mean CAL was studied using intra-group Pearson's coefficient relation [Supplementary Table 2], [Supplementary Table 3], [Supplementary Table 4], [Supplementary Table 5] and [Figure 3]. An intra-group comparison across the three groups for the bacterial count showed a significant increase with age, mean PPD, and mean CAL increased in healthy, aggressive, and chronic periodontitis groups (P ≤ 0.01).
|Figure 3: The correlation between age and bacterial count in Group A, Group B, and Group C shows a linear increase in the count as age increases with a positive linear association of 0.695 (a), 0.441 (b), and 0.337 (c) respectively. The correlation between mean probing pocket depth and bacterial count in Group A, Group B, and Group C shows a linear increase in the count as mean probing pocket depth increases with a positive linear association of 0.538 (d), 0.568 (e), and 0.396 (f). The correlation between mean clinical attachment and bacterial count in Group B and Group C shows a linear increase in the count as mean clinical attachment increases with a positive linear association of 0.581 (g), and 0.465 (h) respectively|
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| Discussion|| |
Periodontitis is characterized by chronic inflammation of the surrounding periodontal structures leading to alveolar bone loss and destruction of gingival and periodontal ligament attachments to the teeth.,,
Advances in technology have redefined the oral microbiome into a synergistic and dysbiotic microbial community rather than by select “periopathogens,” such as the “redcomplex.” Certain newer organisms, such as F. alocis, TM7, Selenomonas, Desulfobulbus, and Synergistes, have been implicated in periodontal pathogenesis. Culturing F. alocis, the third-most prevalent pathogen in aggressive periodontitis, the second most prevalent in chronic periodontitis is difficult owing to its fastidious and anaerobic nature, prevalent at mild to moderate infections in healthy controls.,,
Here, we have used qPCR to quantify F. alocis in clinically categorized healthy, aggressive and chronic periodontal subjects; the pathogen was detected in all the samples analyzed. The prevalence of F. alocis is high in periodontitis compared to gingivitis and healthy; more common in sites with periodontitis than in healthy sites., We observed the highest average bacterial count in the chronic subjects. The prevalence of F. alocis vary from14%, 20%, 30%, 44% and up to 60% in chronic periodontitis. In some other parts of the world, it varies from 30% in Sweden, 66.7% in Germany to 83% and 36%, respectively, in deep and shallow sites of periodontal region in Korea.
Periodontal therapy reduced F. alocis occurrence in patients with chronic periodontitis conditions in the Thai population; however, in another study, non-surgical periodontal treatment failed to reduce F. alocis. Therefore, we believe that the microbiome is complex and might be region or population specific with more factors implicated in the constitution of the microbiome. Furthermore, the prevalence and virulence of one bacteria influence other members of the microbiota. F. alocis and P. gingivalis co-exist together and have a symbiotic relationship; P. gingivalis is more dominant in chronic periodontitis, it can enhance the multiplication and virulence potential of F. alocis. Coexistence and resistance to oxidative stress might be the critical cause for the increased incidence of F. alocis in chronic periodontitis.
The F. alocis prevail at low to moderate levels in healthy controls.,,,,, In our study, about 40% of the healthy controls showed more than the average bacterial count of 3046. Although the incidence of F. alocis varied among healthy controls, a maximum of 29% has been detected in healthy controls. The variation in the incidence of F. alocis may be due to strain variability and difference in PD; less virulent strain of F. alocis ATCC 35896 was shown to be less invasive than the isolate D-62D. Since only PCR was used to detect F. alocis, the magnitude of prevalence was unknown, our report is the first to report real-time detection and quantification of F. alocis in various subjects.
In our study, the average estimate of F. alocis in the aggressive subjects was low (939.84), the tissue invasiveness and lack of subgingival plaque might be a compelling factor for the lower incidence. Furthermore, F. alocis prevail in consortium with Aggregatibacter actinomycetemcomitans and Streptococcus parasanguinis before the bone loss in localized aggressive periodontitis patients. The incidence and prevalence of F. alocis are complicated by the age of the patient, personal habits, population type, and country of origin., Biologically, the population of F. alocis is dependent on the periodontal microbiome; the microbiota varies significantly between chronic and aggressive patients. In an in vitro community model study, Streptococcus gordonii, Gram-negative bacteria of aggressive periodontitis microbiota was antagonistic to F. alocis, and A. actinomycetemcomitans interacted with F. alocis in a strain-specific manner.
The present study observed a statistically significant association between age, clinical periodontal parameters such as PPD and CAL, and the presence of the organism. Intra-group correlation of the age with bacterial count was substantial in all the three groups (P ≤ 0.01). Similarly, intra-group correlation reveals that there was a positive correlation between PPD and CAL with the bacterial population in all three groups (P ≤ 0.01). Geographical locations, ethnicity, food habits, smoking habits influence the distribution, prevalence, and virulence of the oral microbiome; variations prevail in the subgingival microbiota worldwide.
| Conclusion|| |
F. alocis is a novel microorganism gaining more relevance in periodontal pathogenesis. It has been implicated to be the second- and third-most prevalent organism in chronic and aggressive periodontitis, respectively. The highest bacterial count was observed in chronic periodontitis patients (32,409.85), followed by healthy controls (3046.15) and least in aggressive periodontitis patients (939.84). There was a statistically significant difference between the bacterial count of three groups with P value at 0.005 (P ≤ 0.05). On intra-group comparison, there was a statistically significant increase in the bacterial count as the age and mean PPD increases with P value at 0.0005 (P ≤ 0.05). The higher prevalence of F. alocis in chronic periodontitis patients in the Indian population indicates that it can be a candidate marker for chronic periodontitis; however, to qualify it as a marker, validation with more longitudinal studies in a larger study group is warranted.
We would like to acknowledge the facilities provided by SRM IST to carry out the work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]