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 ~  Materials and Me...
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 1  |  Page : 54-59
 

Sequencing of Porphyromonas gingivalis from saliva in patients with periodontitis and type 2 diabetes mellitus


1 Department of Microbiology, Diabetes and Metabolism, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Department of Endocrinology, Diabetes and Metabolism, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India

Date of Web Publication16-Aug-2019

Correspondence Address:
Dr. Padma Srikanth
Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research Institute, Porur, Chennai - 600 116, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_409

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 ~ Abstract 


Background: Porphyromonas gingivalis is a major periodontal pathogen. Saliva is the most easy, non-invasive microbiological sample for detection of periodontal pathogens. Aim and Objectives: A prospective study on 37 diabetic patients was grouped into well-controlled diabetes with/without periodontitis and uncontrolled diabetic with periodontitis. PCR and sequencing of P. gingivalis was performed in saliva samples. Materials and Methods: DNA was extracted from saliva using Triton X-100 and 16s rRNA gene (404 bp) was amplified by polymerase chain reaction. DNA sequencing was performed for two samples. Results:P. gingivalis was detected in 27.03% (n = 10), of which 30% (n = 9) were diabetic with periodontal disease and 14.3% (n = 1) were diabetic without periodontal disease. The percentage of poor oral hygiene was 50% and 20% in uncontrolled and controlled glycaemic patients, respectively. DNA sequencing of two samples showed 100% identity with the sequences in the GenBank database (Gen Bank accession no: KX640913-KX640914). Conclusion: Type 2 diabetes mellitus and periodontitis are interlinked. Early detection of P. gingivalis and appropriate treatment with doxycycline will also assist in controlling the glycaemic status.


Keywords: Glycaemic control, polymerase chain reaction, Porphyromonas gingivalis, saliva


How to cite this article:
Radhakrishnan P, Anbalagan R, Barani R, Mani M, Seshadri KG, Srikanth P. Sequencing of Porphyromonas gingivalis from saliva in patients with periodontitis and type 2 diabetes mellitus. Indian J Med Microbiol 2019;37:54-9

How to cite this URL:
Radhakrishnan P, Anbalagan R, Barani R, Mani M, Seshadri KG, Srikanth P. Sequencing of Porphyromonas gingivalis from saliva in patients with periodontitis and type 2 diabetes mellitus. Indian J Med Microbiol [serial online] 2019 [cited 2019 Oct 21];37:54-9. Available from: http://www.ijmm.org/text.asp?2019/37/1/54/264482





 ~ Introduction Top


Diabetes mellitus (DM) is a clinical syndrome of chronic hyperglycaemia due to relative insulin deficiency, resistance or both. The WHO estimates that diabetes deaths will double between 2005 and 2030 and more than 346 million people worldwide will have diabetes and around 470 million people are projected to be pre-diabetic by 2030.[1] In India, 32 million individuals were affected by diabetes in the year 2003, and it is also estimated that it would go up to 80 million by the year 2030.[2]

An epidemiological survey on the prevalence of periodontitis and DM has estimated the overall prevalence of diabetes to be 9.3% and in patients with periodontitis (17.7%) and was found to be significantly higher than the non-periodontitis group (4.5%). In India, the incidence of periodontal disease is higher (70%) in the adult population,[3] and among diabetic patients, this is estimated to increase up to 82.7%.[4] Diabetes is often associated with severe periodontal disease and is a risk factor for periodontal disease progression.[5] There is a bidirectional relationship between diabetes and periodontal disease[6] systemic disease predisposes to oral infections, and after infection is established, it exacerbates systemic disease.[7]

Periodontal disease is primarily a bacterial infection caused by a diverse group of microorganisms. Porphyromonas gingivalis is considered the most common etiological agent of periodontal disease[8] and major periodontal pathogen causing adult periodontitis.[9] In addition to periodontitis, P. gingivalis has been reported to cause certain systemic diseases, such as lung and brain abscess,[10],[11],[12] pulmonary infections and development of atheromas in cardiovascular disease.[12],[13],[14],[15] Periodontal diseases have been associated with an increased incidence of pre-term delivery of low-birth-weight infants.[16]

P. gingivalis is black-pigmented, Gram-negative, non-sporing, non-motile, obligate anaerobe, coccobacillus which belongs to the family Porphyromonadaceae, genus Porphyromonas formerly known as Bacteroides gingivalis.[17]

P. gingivalis have been isolated from saliva and dental plaque samples from gingival crevices.[18] Saliva may act as a source of transmission for periodontal pathogens.[19] Initial entries of P. gingivalis into the oral cavity could occur through transmission from infected individuals.

The collection of saliva is easier and also the recovery of P. gingivalis is easier in saliva than from periodontal pockets in patients with severe periodontal disease.[20],[21],[22],[23],[24] Anaerobic culture methods have been traditionally used for the isolation of P. gingivalis. Molecular methods such as conventional polymerase chain reaction (PCR) and real-time PCR allow for rapid detection, high sensitivity and specificity than conventional culture method from saliva.[24],[25],[26]

Since anaerobic culture is labour intensive and time-consuming, we decided to undertake to perform conventional PCR by in-house method using simple DNA extraction method. This study was carried out to detect a major periodontal pathogen P. gingivalis from saliva of diabetic patients with and without periodontal disease, and the results were clinically correlated with the glycaemic status.


 ~ Materials and Methods Top


Study design

The study was conducted on diabetic patients attending the outpatient clinic in a tertiary care hospital. Institution's ethics committee were obtained to carry out the study and only diabetic patients who consented to participate in the study voluntarily were enrolled. A pro forma was maintained with details of general health conditions, oral hygiene behaviours, glycaemic status and periodontal evaluation.

Inclusion and exclusion criteria

Adult diabetic patients (≥18 years) who had not received any kind of dental treatment in the past 6 months were included in the study. Patients with systemic conditions such as hypertension, cardiovascular disorders, abnormal hepatic function, haemoglobinopathies, renal failure, retinopathy, pregnancy, smoking, pan chewing and alcohol consumption, antibiotic treatment or prophylaxis that may influence or altered the presence of periodontal microbiota.[11],[12]

Assessment of glycaemic status

At the time of enrolment, glycated haemoglobin (HbA1c) values were measured and determined by the HbA1c value. Patients with HbA1c <7% were considered to have well-controlled diabetes and poorly controlled/uncontrolled diabetes if HbA1c ≥7%.[13] Fasting blood sugar level, post-pyramidal blood sugar level and mean plasma glucose and lipid profile were also recorded.

Oral health assessment

Periodontal status was assessed by oral examinations which included Simplified Oral Hygiene Index, evaluation of bleeding on probing (BOP), periodontal probing depths (PPD) and clinical attachment loss (CAL). Presence of BOP, PPD >5 mm and CAL >3 mm were considered as diagnostic criteria for periodontal disease.[11]

Categorisation of the study population

Diabetic patients (n = 37) were categorised into three groups based on their glycaemic control and periodontal status. Diabetic patients with good glycaemic control (HbA1c <7%) and showed no signs of periodontal disease (absence of BOP, PPD <5 mm and CAL <3 mm) were categorised as Group I (n = 7). Group II (n = 15) consisted of well-controlled diabetic patients (HbA1c <7%) with evidence of periodontal disease (presence of BOP, PPD >5 mm and CAL <3 mm) and Group III (n = 15) consisted of diabetic patients with poor glycaemic control (HbA1c ≥7%) and showed evidence of periodontal disease.

Collection of saliva

Expectorated whole saliva was collected from the diabetic patients (n = 37). The patients were asked to hold their saliva in their mouth without swallowing for 5–10 min. At the end of 5 min, they were asked to spit it out in a 10 ml sterile collection container and immediately transported to molecular biology laboratory where the samples were aliquoted in multiple storage vials and stored at −70°C until used for PCR.

Quality control

All the reagents were placed into aliquots based on the number of samples to be tested. Dedicated micropipette and hand care (gloves) were used for sample processing, reagent preparation, DNA extraction, amplification and detection of DNA in order to prevent cross-contamination. Quality assurance was performed by incorporation of nuclease-free water as a negative control in-between three samples and the positive control (P. gingivalis ATCC 33277, Lot no: 912-49-4, Ref no: 0912P). Samples were interpreted according to the base pair size of the sample DNA with positive control DNA.

DNA extraction

The DNA was extracted by simple boiling method,[24] in which equal volume (200 μl) of lysis buffer (10 mmol/L Tris, 1.0 mmol/L EDTA, 1% Triton X-100, pH 7.8) and samples were incubated at 56°C for 30 min. Following incubation, it was boiled for 10 min and centrifuged at 8000 rpm for 5 min, and 5 μl of the supernatant was used as a template for PCR or immediately stored at −20°C until used for the assay.

Polymerase chain reaction amplification

Amplification were performed to amplify a 404-bp fragment of the 16S rRNA gene[25] in a total volume of 25 μl consisting of 15.6 μl of sterile Milli - Q water, 2.5 μl of Taq Buffer, 1.0 μl of equal molar of four dinucleotide phosphates, 0.2 μl of each forward - 5'-AGG CAG CTT GCC ATA CTG CG-3'and reverse primer - 5'-ACT GTT AGC AAC TAC CGA TGT-3', 0.5 μl of Taq polymerase enzyme and with the addition of 5 μl of template. PCR amplification was performed in Applied Biosystems Veriti 96-well thermo cycler (Applied Biosystems, Foster City, CA). The cyclic conditions include initial denaturation of 95°C for 1 min and 36 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 1 min and extension for 1 min, followed by the final extension at 72°C for 2 min.[27]

Amplicon detection

The amplified PCR products were determined using 2% agarose gel electrophoresis in which the agarose is stained with 0.2 μl of ethidium bromide (EtBr-0.5 μg/ml) and subsequently the products were visualised on 260 nm wavelength of ultraviolet transilluminator in the gel documentation system (Bio-Rad Hercules, CA, USA). Appearance of the band at 404 bp was considered as positive for the presence of P. gingivalis. All the 37 samples were tested twice by two different investigators and only those who were repeatedly positive were considered as positive.

DNA sequencing

The amplified PCR products were purified by Multiscreen HTS PCR plate according to the manufacturer's instruction (Millipore, Billerica, USA). Sequencing reactions were carried out with a set of primer sequences (Forward Primer - 5'– AGG CAG CTT GCC ATA CTG CG-3', Reverse primer – 5'-ACT GTT AGC AAC TAC CGA TGT-3') targeting 16s r RNA gene using ABI Prism BigDye terminator kit, 2 μL of ready reaction buffer, 0.5 μL of ready reaction mix, 1.6 picomole primer, 4.9 μL Milli-Q water and 1 μL purified DNA. The cycling conditions were 40 repeats of 96°C for 15 s, 50°C for 20 s and 60°C for 4 min. After purification according to the manufacturer's instruction (Millipore, Billerica, USA) with Montage SEQ 96 filtration plate, the samples were loaded in ABI Sequence detection system GA 3730. Obtained bidirectional sequences were analysed using BLAST. The sequences were submitted to GenBank public domain and accession numbers were obtained KX640913-KX640914.


 ~ Results Top


A simple sample processing PCR technique was standardised for the detection of P. gingivalis in saliva. The PCR was performed as previously reported for detecting P. gingivalis in saliva.[27] However, the DNA extraction step was modified where lysis buffer[28] was used instead of Chelex 100. All the 37 salivary samples collected from the diabetic patients were tested for P. gingivalis.

The DNA band showing 404 bp in agarose gel electrophoresis was considered positive for [Figure 1] P. gingivalis ATCC 33277 was used as positive control and RNase-free water as negative control. The assay was performed repeatedly to insure reproducibility.
Figure 1: Image of gel electrophoresis showing the presence of Porphyromonas gingivalis at 404 bp

Click here to view


P. gingivalis was detected in 27.03% (n = 10) of the 37 diabetic patients. It was found in 14. 28% (n = 1) in Group I, 13.51% (n = 5) in Group II and 10.81% (n = 4) in Group III. The percentage of P. gingivalis detected in diabetic patients (Group I) without periodontal disease was 14.28% (n = 7) and in diabetic patients (Group II and Group III) with periodontal disease, it was found to be 30% (n = 30). The BLAST analysis of the two representative sequences showed as 16s rRNA of P. gingivalis.

Analysis of glycated haemoglobin and the age of the subject are positive for Porphyromonas gingivalis

The samples which are positive for P. gingivalis by PCR were analysed for age, HbA1c values, periodontal condition and oral hygiene status [Table 1]. The mean age of diabetic patients with periodontal disease (Group II and Group III) positive for the presence of P. gingivalis was 53.33 years and only one patient was <40 years of age.
Table 1: Glycated haemoglobin, oral hygiene status and age of the study participants with diabetic; periodontal disease and positive for Porphyromonas gingivalis

Click here to view


Oral hygiene scores of the subject's positive for Porphyromonas gingivalis

Oral hygiene scores of the patient's positive for P. gingivalis were either poor or fair, except for one individual with good oral hygiene status. Furthermore, the percentage of poor oral hygiene in Group III (HbA1c ≥7%) was 50%, whereas in Group II (HbA1c <7%), it was 20%. P. gingivalis was detected in only one individual with good glycaemic control and no signs of periodontal disease (group I). However, his oral hygiene score was found to be poor.

Transmission of Porphyromonas gingivalis between spouses

On analysing the data of the positive patients, we found a couple enrolled in the study. P. gingivalis was detected in both of them. The husband was 55 years old with the HbA1c of 7.9% and the presence of periodontal disease (Group III). His oral hygiene score was good. The wife was 50 years old with good glycaemic control (HbA1c = 6.5%) and with the presence of periodontal disease (Group II). Her oral hygiene score was fair.


 ~ Discussion Top


Periodontal disease (periodontitis) is considered one of the complications of DM and is characterised by progressive destruction of the tissues supporting the tooth. Periodontitis is usually caused by a periodontal pathogen such as P. gingivalis, Tannerella forsythi a and Treponema denticola. This mixed infection forms a red complex in periodontal sites and strongly correlates with the severity of adult periodontitis.[29],[30] Other periodontal pathogens such as Prevotella intermedia, Capnocytophaga spp., Actinobacillus actinomycetemcomitans and Eikenella corrodens may be detected in subgingival plaque samples in various forms of periodontal disease.[31] These microorganisms induce inflammatory changes through pro-inflammatory cytokines, chemokines and various chemical mediators which reach systemic levels and contribute to development of complications in diabetic patients and to the pathogenesis of various other systemic diseases such as hypertension and cardiovascular diseases.[32]

P. gingivalis is strongly associated with peridontitis. The detection rate of P. gingivalis in saliva is 35.4%[8] and the rate of detection is increased by 63.3% in adults over the age of 30 years[33] The detection of P. gingivalis is useful for diagnosis, monitoring of treatment and epidemiologic investigations of destructive periodontal disease. Since P. gingivalis is a fastidious anaerobic organism, anaerobic culture methods are time-consuming, difficult to maintain, have low detection levels and are labour intensive, especially from samples such as saliva or periodontal pockets,[34] and it may be detected more reliably with use of non-culture than with culture methods.

Several studies have detected the presence of P. gingivalis by molecular methods in subgingival plaque samples, oral rinses, oral swab and saliva.[27],[35],[36] Therefore, we undertook the study to determine the presence of P. gingivalis in saliva. Collection of dental plaque is highly subjective, recovery of the organism from plaque samples depend on the site of collection, technique used and the skill of the examiner. Saliva is considered as a nutrient medium to maintain the viability of the organism[23] is easy to collect and is non-invasive, and the recovery of P. gingivalis from saliva is easier in patients with severe periodontal disease.[20],[21],[22],[23],[24]

PCR is reproducible, specific and sensitive. The cost of anaerobic culture may be comparable to that of conventional PCR. The “time to result” for anaerobic culture takes at least 48–72 h, in the case of conventional PCR the “time to result” is 4–6 h. Although the cost of anaerobic culture may be lesser than conventional PCR, it is time-consuming. Culture will also allow the simultaneous growth of several bacterial species, some of which may be pathogens and some commensals, the use of PCR will enable specific detection of the pathogen under study.

Real-time PCR is a useful method for the quantitative detection of periodontal bacteria.[37],[38] The sensitivity of assay is higher in real-time PCR; however, the cost is comparatively higher than conventional PCR.[24],[26],[39] The cost per sample for one target site could be approximately INR1200 for conventional PCR and INR 3000 for real-time PCR. Since the real-time PCR is more expensive, we undertook to determine the presence of P. gingivalis using simple sample processing DNA extraction method by in-house conventional PCR which was found to be cost-effective that indicated the presence or absence of P. gingivalis in saliva.

Recovery of periodontal pathogen by manual extraction methods such as Chelax 100, SDS, boiling method, phenol-chloroform and Triton X-100 have been extensively used. Chelax-100 is more efficient than boiling extraction method;[27] however, it is more expensive than other methods. Total cost of the extraction methods using Chelax 100 (Sigma Aldrich: C7901) is INR 4,222 and using Triton X-100 (Sigma Aldrich: T8787) is INR 1452. Using this method of extraction, the cost of in-house conventional PCR was only INR 800 per sample. PCR target was also specific since specific target site primers were used.[33]

In our study, P. gingivalis was isolated from one patient from a group of good glycaemic control without periodontal disease which could be due to his poor oral hygiene. The detection rate of P. gingivalis was 30% among diabetic patients with periodontal disease (Group II and Group III) and 14.28% in patients with controlled diabetes and non-periodontitis (Group I). Studies described the association between P. gingivalis and periodontitis among type 2 DM patients.[40],[41] The occurrence of P. gingivalis was documented in 53% following periodontal treatment. In our study, the PCR results when correlated clinically were suggestive of active disease. Two representative samples of our study participants who were positive for P. gingivalis by conventional PCR were sequenced and confirmed as P. gingivalis.

We also found that the oral hygiene score for most of the study participants who were positive for P. gingivalis was either poor or fair. Poor oral hygiene was 50% in uncontrolled diabetics and 20% in well-controlled diabetic patients. Thus, poor oral hygiene may also contribute to poor glycaemic control.[42] Since P. gingivalis was recovered from 30% of the diabetic patients with periodontal disease, this can be used for targeting specific treatment. The use of doxycycline improves the periodontal prognosis and metabolic control in diabetic patients indicating that it has an equal effect on haemoglobin glycosylation.[43],[44]

Ultrasonic root debridement with systemic doxycycline, 100 mg/day for 2 weeks, decreased P. gingivalis to non-detectable levels in all patients who tested positive at baseline after 3 months of treatment and resulted in 1% decrease in the level of HbA1c.[44] Thus, therapeutic intervention may be undertaken in those positive for P. gingivalis to prevent progression of disease not only the periodontitis but also the worsening of diabetic mellitus since both diseases are interlinked.

To the best of our knowledge, there are no data on the recovery of P. gingivalis from salivary samples from India using a simple cost-effective conventional in-house PCR. The findings of this study can be used to undertake larger studies and also plan for therapeutic intervention.


 ~ Conclusion Top


We standardised an in-house conventional PCR technique to detect the presence of P gingivalis in the salivary samples of the diabetic patients. Saliva is considered the most easily, non-invasively collected microbiological sample and also represents all the sites of the oral cavity. We adopted a new hybrid technique for DNA extraction, which was not only a simple sample processing procedure but also a cost-effective method. This modified PCR protocol will be advantageous and economical in resource-limited settings. The detection rate was 30% in salivary samples of diabetic patients with periodontal disease, which is comparable with previous studies. We found that poor oral hygiene contributes to the presence of P. gingivalis, and poor glycaemic control was associated with poor oral hygiene.

Financial support and sponsorship

This study is a self-funded project.

Conflicts of interest

There are no conflicts of interest.



 
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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04