Journal of Interdisciplinary Dentistry

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 10  |  Issue : 1  |  Page : 17--23

Identification of Dialister pneumosintes in healthy and chronic periodontitis patients with Type 2 diabetes mellitus and its correlation with the red complex bacteria


Pratima Oswal, Sandeep Katti, Vinayak Joshi, Hawaabi Shaikh 
 Maratha Mandal's N.G.H., Institute of Dental Sciences, Belagavi, Karnataka, India

Correspondence Address:
Dr. Pratima Oswal
Senior Lecturer, Tatyasaheb Kore Dental College and Research Centre, New Pargaon, Kolhapur, Maharashtra
India

Abstract

Aim: The aim of the study was to assess the presence of Dialister pneumosintes in healthy and chronic periodontitis (CP) patients with Type 2 diabetes mellitus and its association with the red complex bacteria. Materials and Methods: Depending on the inclusion and exclusion criteria, a total of 36 patients, aged 35–70 years, were allotted to two groups: periodontally healthy diabetic patients (18) and CP diabetic patients (18). Subgingival plaque samples were collected from the deepest site. Microbial assessment was done by subjecting the plaque samples to DNA isolation, and conventional polymerase chain reaction was performed for the identification of D. pneumosintes with specific primers. Results and Conclusion: D. pneumosintes was found greater in number in CP patients (50%) than in healthy controls (27.78%), but it was statistically insignificant (P = 0.17). Association of the presence of D. pneumosintes with Tannerella forsythia was 83.33%. It was found that there was a statistically significant association between D. pneumosintes and T. forsythia, with P = 0.047.



How to cite this article:
Oswal P, Katti S, Joshi V, Shaikh H. Identification of Dialister pneumosintes in healthy and chronic periodontitis patients with Type 2 diabetes mellitus and its correlation with the red complex bacteria.J Interdiscip Dentistry 2020;10:17-23


How to cite this URL:
Oswal P, Katti S, Joshi V, Shaikh H. Identification of Dialister pneumosintes in healthy and chronic periodontitis patients with Type 2 diabetes mellitus and its correlation with the red complex bacteria. J Interdiscip Dentistry [serial online] 2020 [cited 2020 Jun 2 ];10:17-23
Available from: http://www.jidonline.com/text.asp?2020/10/1/17/283536


Full Text



 Clinical Relevance to Interdisciplinary Dentistry



Recent findings indicate that oral health may influence systemic health, and this may be a bidirectional relationship for some conditions. This is particularly true for the relationship between periodontal disease and diabetes mellitus, which encourages more research.

 Introduction



Periodontitis is caused by bacteria colonizing the subgingival margin in dental plaque, where multiple bacterial species act in a cooperative manner to induce infection and tissue destruction. Several Gram-negative anaerobic bacteria such as Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum have frequently been isolated from periodontal lesions, which are thought to contribute to the development of periodontal diseases.[1] One of the previously unrecognized bacteria found to be associated with chronic periodontitis (CP) is Dialister pneumosintes and has been noticed in laboratories. It was initially named as Bacterium pneumosintes by Olitsky and Gates.[2]

D. pneumosintes is nonfermentative, anaerobic, Gram-negative rods that produce colonies on blood agar.[3] Little is known about the relationship between the organism and periodontal disease, and also recent findings indicate that oral health may influence systemic health, and this may be a bi-directional relationship for some conditions. This is particularly true for the relationship between periodontal disease and diabetes mellitus.[4]

Thus, the aim of the present study is to evaluate the presence of D. pneumosintes and also to evaluate the coexistence and relationship between D. pneumosintes and red complex bacteria (P. gingivalis, T. forsythia, and Treponema denticola) in subgingival plaque samples of periodontally healthy and CP patients with Type II diabetes mellitus.

 Materials and Methods



Patient selection

Study subjects

This study was performed in accordance with the guidelines and after approval by the Ethical Committee of Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences (MMNGHIDS) and Research Centre, Belagavi. Subgingival plaque samples were collected from the outpatient Department of Periodontology, MMNGHIDS and Research Centre.

A total of 54 patients with Type II diabetes mellitus (according to the criteria of American Diabetes Association, 2011)[5] of age ranging from 35 to 70 years were screened for the study and 36 patients (23 male and 13 female) were included in the study based on the inclusion and exclusion criteria. The participants were divided into two groups – individuals with healthy periodontium (H group) and CP patients (CP group) with Type II diabetes mellitus. The diagnosis of CP was done based on the classification system of the American Academy of Periodontology, 1999, at the International Workshop for Classification of Periodontal Diseases and Conditions.[6]

Individuals who had a minimum of twenty natural teeth were selected for the study with the following inclusion criteria for both the groups:

For healthy group:

Type II diabetes mellitus patients withNo signs of gingival inflammationAbsence of bleeding on probing (BOP)Probing depth ≤3 mm andNo clinical attachment loss (CAL).

For chronic periodontitis group:

Type II diabetes mellitus patients withPresence of gingival inflammationPresence of BOPProbing depth ≥5 mm and CAL ≥3 mm were selected.

Exclusion criteria

Individuals who had received periodontal therapy or antimicrobial therapy within the last 3 months before sampling

Patients with a history of any systemic diseases/conditions except Type 2 diabetes mellitusPatients with a habit of smoke/smokeless tobacco andPregnant women and lactating mothers.

Periodontal examination was carried out, and measurements of clinical parameters such as Gingival Index (GI) (Loe and Silness in 1963),[7] Plaque Index (PI) (Silness and Loe in 1964),[7] Bleeding Index,[8] and probing depth and CAL were carefully recorded. Glycated hemoglobin (HbA1c) level for each individual was also recorded before taking samples. All the recordings were done by a single examiner.

Subgingival plaque sample collection

In the CP group, plaque samples were collected from three deepest sites with probing depth of ≥5 mm[9] and in the H group, the plaque samples were collected from normal gingival sulcus. Clinical samples were collected using strict asepsis. The selected sites were isolated with sterile cotton rolls and air dried. Supragingival plaque and calculus were removed. The subgingival plaque samples were collected from each participant, from the most apical portion of the accessible probing depth and alongside the root of the tooth at all possible sites using a sterile universal curette, which were then transferred into a microcentrifuge tube containing transport medium (Tris-EDTA buffer) and sent to the laboratory for analysis using polymerase chain reaction (PCR) technique. In the laboratory, DNA extraction procedure and PCR followed by gel electrophoresis were performed.

DNA extraction procedure

This was done using modified proteinase-K method.[10] In PCR technique, it amplifies specific DNA sequence by simultaneous primer extension of complementary strands of DNA. A conventional PCR method was used in the present study, and the following set of PCR primers were used which were specific to respective organisms.

Amplification of Dialister pneumosintes

PCR cycling conditions for amplification using primer specific for D. pneumosintes included initial denaturation step at 95°C for 5 min, followed by 36 cycles of a denaturation step at 94°C for 30 s and a primer annealing step at 55°C for 1 min; then, an extension step was performed at 72°C for 2 min, and a final step of 72°C for 10 min was done. The PCR samples were then kept at 4°C.[11]

Amplification of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia

This encompassed initial denaturation step at 95°C for 5 min which was followed by 40 cycles of a denaturation step at 94°C for 1 min; then, primer annealing step at 60°C for 1 min was done, an extension step was performed at 72°C for 1 min, and a final step of 72°C was performed for 10 min. The PCR samples were then kept at 4°C.[12]

Agarose gel electrophoresis procedure

Gel electrophoresis is a procedure for separating a mixture of molecule (DNA) through stationary material (gel) in an electric field.[11] Twenty microliter of this amplified product was taken and added carefully into the wells. A molecular weight marker was loaded in the last well. Electrodes were fixed. The power supply was turned on and the current was adjusted (16 A). The gel was run for 2 h. The photo of gel under ultraviolet light transilluminator was taken and the bands were recorded using Gel documentation system. The amplified products of size 1105, 404, 316, and 641 base pairs were identified with the help of a DNA ladder [Figure 1] and [Figure 2] which was run simultaneously with the samples in each run. The amplified products of size 1105 base pairs are indicative of D. pneumosintes, whereas 404, 316, and 641 base pairs in dicated P. gingivalis, T. denticola, and T. forsythia, respectively [Table 1].{Figure 1}{Figure 2}{Table 1}

Statistical analysis

Comparison of the two study groups with respect to presence status of different bacteria was done with Chi-square test. Association of the presence of D. pneumosintes bacteria with red complex bacteria was evaluated using Chi-square test with Yates's correction. Comparison of the presence status of D. pneumosintes bacteria with respect to plaque and GI scores was done by Mann–Whitney U-test. Comparison of presence status of D. pneumosintes bacteria with respect to PD, CAL, BOP, and HbA1c scores was done by using independent t-test.

 Results



[Table 2] shows the presence of D. pneumosintes, P. gingivalis, T. denticola, and T. forsythia in subgingival plaque samples from periodontally healthy controls and patients with CP with Type II diabetes mellitus.{Table 2}

[Table 3] describes the association between the presence of D. pneumosintes bacteria with the presence of P. gingivalis, T. denticola, and T. forsythia individually in all the 36 individuals. Association of the presence of D. pneumosintes in the present study with the presence of T. denticola was 46.15%, with P. gingivalis at 30% and with T. forsythia at 83.33%.{Table 3}

[Table 4], [Table 5], [Table 6] describe the comparison of the presence status of D. pneumosintes bacteria with respect to clinical parameters such as PI, GI, probing depth, CAL, BOP, and HbA1c. For comparison of the presence status of D. pneumosintes bacteria with respect to PI and GI scores, Mann–Whitney U-test was used. When the presence and absence status of D. pneumosintes was compared with the clinical parameters, in D. pneumosintes present group, the mean PI and GI scores were 1.33 and 1.31, respectively, whereas in D. pneumosintes absent group, the mean PI and GI scores were 0.86 and 0.82, respectively. There was a statistically significant positive correlation between the presence of D. pneumosintes with respect to PI with P = 0.05. No significant correlation was seen between GI and presence of D. pneumosintes. With respect to PD, CAL, and BOP scores, independent t-test was performed to assess the correlation with D. pneumosintes. As shown in [Table 5], there was no statistically significant correlation. In our study, a total of 14 patients were positive for D. pneumosintes, out of which 7 belonged to the category of moderately controlled diabetes, 7 belonged to the category of poorly controlled diabetes, and zero belonged to the good controlled diabetes mellitus. There was no statistically significant association between HbA1c level and presence of D. pneumosintes.{Table 4}{Table 5}{Table 6}

 Discussion



Periodontal disease is considered one of the major oral health problems and is the most common cause of tooth loss in adults. Periodontal disease is a term used to describe several pathological conditions such as the presence of CAL and alveolar bone defects.[13]

Periodontal infections are polymicrobial, and a limited number of (mainly) Gram-negative anaerobic species are frequently considered as major periodontal pathogens.[14] However, recent studies using molecular methods for microbial identification have indicated that several other organisms, including noncultivable and/or not yet identified species, may play a role in human periodontitis.[15]

Diabetes mellitus is emerging as a global epidemic, and its complications impact significantly on quality of life, longevity, and health-care costs. The escalating human and economic burden across both the developed and developing world necessitates a multidisciplinary approach, including adjunctive measures to managing diabetes and its complications.[16] The onset of diabetes is preceded by inflammation, which leads to pancreatic beta-cell dysfunction and apoptosis, as well as impacting on the development of insulin resistance and ultimately diabetes.[17] Inflammatory periodontal diseases are the most common chronic inflammatory conditions of humans worldwide. Periodontal disease is a microbial initiated chronic inflammatory disease, in which dysregulated immune-inflammatory processes are responsible for the majority of host tissue destruction, and ultimately tooth loss. Evidence has shown that systemic inflammation results from oral microbial agents and their virulence factors entering into the circulation.

Several studies have investigated the composition of plaque in patients with diabetes compared with controls. Increased number of periodontal pathogens have been isolated from periodontal pockets of diabetic patients,[18],[19] although the specific differences in the microbiota of diabetics compared with nondiabetics are not clear.

Thorstensson et al. observed significantly greater number of P. gingivalis in patients with diabetes compared to controls, although no differences were seen with Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Capnocytophaga spp., Eikenella corrodens, F. nucleatum, and P. intermedia.[20] Other studies, including longitudinal studies, found no such association.[21],[22] Using the checkerboard DNA-DNA hybridization methodology, Ebersole et al. showed significantly increased frequency of P. gingivalis, Campylobacter spp., and A. actinomycetemcomitans in the subgingival plaque of patients with diabetes compared with nondiabetics.[23]

In the present investigation, we determined the prevalence of a suspected periodontal pathogen, D. pneumosintes, in subgingival plaque samples from periodontally healthy and CP individuals with Type II diabetes mellitus using PCR technique with specific primers.

A total of 36 individuals were included in the study and were divided into two groups of 18 participants each. It was found that among 18 samples collected from periodontally healthy individuals with Type II diabetes mellitus, 5 had D. pneumosintes, whereas among 18 samples from CP individuals, 9 had D. pneumosintes, though the presence of D. pneumosintes in subgingival plaque samples was higher in patients with CP than that of the periodontally healthy controls; there was no statistically significant difference between both the groups with P = 0.17 [Table 2].

Previously, a study conducted by Ferraro et al.[15] showed that CP patients presented a significantly greater mean prevalence of D. pneumosintes, which is 62.17±6.4% compared to periodontally health individuals which is 29.47±7.9%. Another investigation done by Contreras et al.,[24] observed that among paper-point samples from periodontal pockets of 105 periodontitis and 30 gingivitis patients, D. pneumosintes was detected in 83% of patients having severe periodontitis and in 17% of patients having gingivitis. According to these investigations, it can be concluded that in systemically healthy individuals, a significantly high frequency of D. pneumosintes species was observed in CP individuals compared to periodontally healthy individuals, but when compared in Type II diabetes mellitus individuals, the results were not statistically significant. D. pneumosintes being a periodontopathic organism, its presence in healthy controls may pose a risk of transforming periodontal health to disease in diabetic individuals.

To our knowledge, this is the first study of its kind, in which the presence of D. pneumosintes was assessed in subgingival plaque samples of periodontal healthy individuals with Type II diabetes mellitus compared with CP patients. The present study also depicts the association of D. pneumosintes bacteria with red complex bacteria (P. gingivalis, T. denticola, and T. forsythia) in both H and CP groups.

In the present study, when the association between the status of the presence of D. pneumosintes bacteria with the presence of T. forsythia was compared, there was 83.33% association of T. forsythia with D. pneumosintes, as shown in [Table 3], which is statistically significant. A study conducted by Ghayoumi et al.[25] showed significant association of D. pneumosintes and T. forsythia. In their study, D. pneumosintes was detected in 44/149 (29.5%) samples and T. forsythia in 96/149 (64.4%) samples. D. pneumosintes occurred together with T. forsythia in 36/149 (24.2%) samples with CP, which shows the positive association of T. forsythia with D. pneumosintes bacteria. In our study, the finding that T. forsythia is strongly associated with D. pneumosintes in diabetic individuals is of interest. Previously, T. forsythia has been found to frequently cohabit with F. nucleatum,[26] probably because of the need for T. forsythia to acquire exogenous N-acetylmuramic acid or other products produced by F. nucleatum.[27] According to Ghayoumi et al.,[25]D. pneumosintes may provide growth factor for T. forsythia or, vice versa, T. forsythia may provide growth factor for D. pneumosintes, or a third organism may provide growth factor for both.

The association between the presence of D. pneumosintes bacteria with the presence of P. gingivalis in CP group was also compared, which was statistically not significant [Table 3]. This finding is supported by a study done by Ghayoumi et al.,[25] where D. pneumosintes and P. gingivalis occurred together in only few samples, which was statistically not significant with P = 0.13. According to Slots and Ting,[28] while P. gingivalis is generally considered to constitute a prototype of an anaerobic periodontal pathogen, it is also assumed that the virulence of other organisms can play an important role in the polymicrobial infections of periodontal disease. In addition, no association was detected between D. pneumosintes and P. gingivalis, which does not necessarily imply a clinically inconsequential interrelationship between these two bacteria. On the contrary, a study done by Kamma et al.[29] showed that subgingival co-infection by D. pneumosintes and P. gingivalis was more closely related to progressive periodontitis than any of the two species alone.

 Conclusion



The first line of evidence to define a microorganism as a true pathogen is to determine if the organism is present in a higher prevalence and/or levels and/or proportions/abundance in disease condition than in healthy condition (association studies). The objectives of the present study were to evaluate the occurrence of D. pneumosintes in periodontally healthy patients and CP patients and to compare the occurrence of D. pneumosintes in both the groups and its association with clinical parameters and red complex bacteria.

The PCR analysis of the subgingival plaque samples obtained from periodontally healthy and CP individuals with Type II diabetes mellitus indicated no statistically significant differences in the presence of D. pneumosintes in both the groups. D. pneumosintes was present in both the healthy and CP individuals with Type II diabetes mellitus. D. pneumosintes being a periodontopathic organism, its presence in healthy controls may pose a risk of transforming periodontal health to disease in diabetic individuals. However, it is hard to draw any general conclusions depending on this finding as the sample size is smaller, therefore further investigations are needed with larger sample size.

The important finding is that there is a positive association between T. forsythia and D. pneumosintes in Type II diabetic individuals. Furthermore, research is needed to delineate the association between these two periodontopathogens.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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