|Year : 2012 | Volume
| Issue : 3 | Page : 398-403
Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture
Sangita Agarwal1, KD Jithendra2
1 Department of Periodontology, People's Dental Academy, Bhopal, Madhya Pradesh, India
2 Department of Periodontology, K D Dental College and Hospital, Mathura, Uttar Pradesh, India
|Date of Submission||08-May-2010|
|Date of Acceptance||11-Apr-2012|
|Date of Web Publication||12-Sep-2012|
Department of Periodontics, People's Dental Academy, Bhanpur, Bhopal, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Helicobacter pylori is an important gastrointestinal pathogen that is strongly associated with gastritis as well as peptic ulcer disease. Antimicrobial therapy frequently fails to cure H. pylori infection, which suggests there may be sanctuary sites where the organism resides. This study was aimed to assess the role of oral cavity as a reservoir of H. pylori by evaluating the occurrence of the organism in subgingival plaque of dyspeptic patients by polymerase chain reaction as well as culture. Materials and Methods: Thirty chronic periodontitis patients whose biopsy specimens were found to be H. pylori positive with rapid urease test and histopathologic examination were considered as cases and 20 chronic periodontitis patients who never had any symptoms of gastritis or peptic ulcer were taken as controls. Subgingival plaque samples were collected and sent to microbiological laboratory for detection of H. pylori by 16S rRNA based polymerase chain reaction as well as culture. Results: 60% of the samples were found to be positive with polymerase chain reaction in the case group when compared to 15% in the controls. Also, 30% of the cases were found to be positive with culture compared to none in controls. Conclusion: A higher frequency of detection of H. pylori in those patients with positive antral biopsy report was seen. Also, polymerase chain reaction was found to be more sensitive than culture for detection. Thus, we conclude that detection of H. pylori in dental plaque of dyspeptic patients cannot be neglected and might represent a risk factor for recolonization of stomach after systemic eradication therapy.
Keywords: Culture, gastritis, Helicobacter pylori, polymerase chain reaction, subgingival plaque
|How to cite this article:|
Agarwal S, Jithendra K D. Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture. J Indian Soc Periodontol 2012;16:398-403
|How to cite this URL:|
Agarwal S, Jithendra K D. Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture. J Indian Soc Periodontol [serial online] 2012 [cited 2019 Oct 21];16:398-403. Available from: http://www.jisponline.com/text.asp?2012/16/3/398/100919
| Introduction|| |
Helicobacter pylori is a gram-negative, curved microaerophilic organism that has been implicated in the etiology of gastritis, in the process of gastric and duodenal ulcer formation, and in gastric carcinoma. , Approximately 10% of individuals are affected by gastritis and/or gastric ulcer during their lifetime and over 50% of the world's population carries this infection. 
The exact mode of transmission of H. pylori is not yet fully understood. As human infection by this pathogen appears to involve an oral route, it seems biologically plausible that oral health status directly or indirectly influences the process of H. pylori infection or reinfection. 
H. pylori has been observed in saliva, in the microbiota from the dorsum of the tongue, in dental plaque, on the surface of oral ulceration, and in oral neoplasias. ,,,, While H. pylori could be isolated from the oral cavity in some cases, most attempts to culture the organism have failed. This is attributed to factitious nature of H. pylori, which requires a microaerophilic environment, supplemented media, and up to 7 days incubation for growth. Under these conditions, overgrowth by other species is likely, and direct growth inhibition of H. pylori by oral species in vitro has also been reported.  With the advances in the molecular technology, the potential difficulties with culture have been circumvented by the use of polymerase chain reaction (PCR), which allows for the detection of even small numbers of specific bacteria within a sample and obviates the need for viable organisms. 
H. pylori gastric infection can be successfully treated with systemic antibiotic therapy. Despite the current treatment regimens that lead to successful management of H. pylori positive chronic gastritis, the reinfection rate is high.  Therefore, studies have been carried out to evaluate sites other than stomach that could be implicated in the eventual transmission of the bacteria and in the reinfection process. Although H. pylori may be transmitted through the oral cavity, it is unclear whether it is a permanent or transient reservoir and its specific niche has not yet been identified.
Thus, the present study aimed at evaluating the occurrence of H. pylori detected by PCR as well as culture in dental plaque samples of periodontitis patients testing positive for this bacterium in the stomach.
| Materials and Methods|| |
Source of data
Patients from the gastroenterology division of KLES's Hospital, Belgaum, with alteration of superior digestive tract were evaluated. Chronic periodontitis patients who had never suffered from gastritis/peptic ulcers were randomly selected from the patients reporting to Department of Periodontics, KLES's Institute of Dental Sciences, Belgaum. The criteria for selection of study subjects were as follows:
- Chronic generalized periodontitis patients with symptoms of gastritis and peptic ulcer, whose antral biopsy specimens tested urease positive, were included in the test group.
- Chronic generalized periodontitis patients who never had symptoms of gastritis/peptic ulcer were taken as controls.
- Patients above 30 years of age.
- Patients with minimum of three posterior teeth in each quadrant.
The following subjects were excluded:
- Patients with systemic diseases/conditions other than peptic ulcer/gastritis.
- Patients who had taken antibiotics, bismuth containing compounds, or omeprazole within the previous 2 months.
A total of 50 patients (28 males and 22 females) in the age range of 30-65 years were selected. It included 30 cases (mean age=46.2±11.44 years) and 20 controls (mean age=44.5±11.36 years). Informed consent was obtained from all the subjects for their willingness to participate in the study. An ethical clearance was obtained from ethical committee, KLES's Institute of Dental Sciences, Belgaum, before conducting the study.
Antral biopsy samples were taken and subjected to rapid urease test [Figure 1] and histopathologic examination for detection of H. pylori. Periodontal status was evaluated for the presence of periodontitis.
|Figure 1: Rapid urease test for detection of H. pylori in antral biopsy specimens|
Click here to view
Subgingival plaque samples were collected with the help of sterile curette after careful removal of supragingival plaque [Figure 2]. In each subject, samples were collected from all posterior teeth and pooled to be suspended in phosphate-buffered saline (pH 7.5) for transport. Then, the samples were carried to microbiology laboratory for culture and PCR.
- Culture: Samples were centrifuged and inoculated in H. pylori selective media (Columbia blood agar ±5% defibrinated sheep blood along with antibiotic supplements, i.e. Vancomycin, Trimethoprim, Cefsoludin, and Amphotericin-B) [Figure 3]. The agar plates [Figure 4] were incubated in a microaerophilic atmosphere at 37°C for 5 days. A Gram stain was then performed and H. pylori were identified by characteristic curved gram-negative rod appearance.
- PCR: PCR is a molecular biology technique for enzymatically replicating DNA without using a living organism. It allows a small amount of DNA to be amplified exponentially.
The basic PCR technique involves 30-50 repetitive cycles, with each cycle comprising three sequential reactions:-
Step 1: Denaturation of target nucleic acid at 92-96°C
Step 2: Primer annealing
Primer pair [Figure 5] mixes with denatured target DNA.
One primer anneals to a specific site at one end of the target sequence of one target strand (against 16S rRNA) while the other primer anneals to a specific site at the opposite end of the other, complementary target strand.
Duplexes are formed.
Step 3: Extension of primer target duplex [Figure 6].
In this study, Hot start PCR with variation in basic 16S rRNA gene based technique was used. It was performed manually by heating the reaction components to the denaturation temperature (e.g. 95°C) before adding the polymerase.
The PCR amplification product was then gel electrophoresed and analyzed under UV transilluminator [Figure 7] and [Figure 8]. Finally, PCR amplification products are compared with standard molecular markers and analyzed.
|Figure 7: Gel electrophoresis results of PCR showing samples 2, 3, 5, 7, 8, 12, 14, 15 positive for H. pylori in cases|
Click here to view
|Figure 8: Gel electrophoresis results of PCR showing samples 2, 3, 12 positive for H. pylori in controls|
Click here to view
The results were expressed as being either positive or negative. The data obtained were subjected to statistical analysis using chi-square test. The level of significance was set at 95% confidence interval.
| Results|| |
Culture and PCR detection of H. pylori in subgingival plaque samples have shown a high prevalence of the bacterium in subjects with alteration of superior digestive tract. Prevalence of H. pylori in subgingival plaque samples of gastritis/peptic ulcer patients was found to be significantly more when compared to controls (P=0.0198) [Table 1], [Figure 9]. Again with PCR, 60% of samples was found to be positive in cases compared to 15% in the control group, which was found to be very significant (P=0.0042) [Table 2], [Figure 10]. When comparing the results of PCR and culture, it was found that out of 50 samples, 21 (42%) detected positive with PCR and 9 (18%) with culture, which is again significant (P=0.0164) [Table 3], [Figure 11]. When considering totally 50 samples, PCR gave 100% sensitivity and 70.73% specificity. False-positive rate was 29.26% and false-negative rate was 0%, which is encouraging [Table 4].
| Discussion|| |
The significant role of H. pylori in the etiology of gastric disease is now undisputed. Despite the current treatment regimens that lead to successful management of H. pylori positive chronic gastritis, relatively high infection rate is still a major problem.  Detection of H. pylori within dental plaque or saliva would provide a foundation for a role of the oral cavity in H. pylori transmission and the literature addressing this issue is considerable. 
The oral cavity is colonized by various micro-organisms and interspecies coaggregation is thought to be important for bacterial colonization. Fusobacterium nucleatum and Porphyromonas. gingivalis were found to coaggregate with H. pylori. Since F. nucleatum and P. gingivalis are potential periodontal pathogens, a positive correlation between the oral existence of H. pylori and periodontitis cannot be ruled out. 
In addition, some oral bacteria, i.e. Streptococcus and Actinomyces species produce bacteriocin-like inhibitory proteins against H. pylori. The fact that good oral hygiene patients harbor less H. pylori in their mouth could also be explained by this inhibitory activity of the early colonizers of the mouth.  Also, it is reasonable to hypothesize that H. pylori survives in moderate to advanced periodontal pockets because the architecture and the microcosm of these periodontal conditions promote a viable habitat for micro-aerophilic and anaerobic microorganisms. However, few authors had found no association of H. pylori infection with periodontal status or oral hygiene status. ,
Many of the earlier studies ,, reported a high prevalence of H. pylori in dental plaque, whereas some ,, reported a very low prevalence or absence of H. pylori in dental plaque. Possible reasons for discrepancies in results include the small number of patients studied, differences in population prevalence, and the difficulty of culturing this fastidious micro-organism from the oral cavity with its complex and varied normal flora. ,
The reported prevalence of H. pylori in the developing countries is higher than in developed countries.  Most of the studies done on Indian population , have shown high prevalence of H. pylori in oral cavity, and so has our study. The reason for this may be related to the poor oral hygiene and socioeconomic status of the patient population studied.
Since, H. pylori is the only urease-positive bacterium known to reside in the stomach, urease test is a suitable detection method for use on gastric samples. However, as the oral cavity is residence to several urease-producing species, including Streptococcus spp., Haemophilus spp., and Actinomyces spp., it is inappropriate to conclude that high urease activity in dental plaque is indicative of the presence of H. pylori. Similarly, diagnosis of H. pylori based on microscopic appearance (gram negative, curved, or spiral-shaped rods) , is likely to provide low specificity in case or oral samples, where spirochetes including Treponema spp. are routinely found.
The culture medium used in the present study was selective for H. pylori. Also, the primer used for the molecular method (PCR) was specific for H. pylori. This eradicates the possibility of giving false-positive results because of other urease-producing species in the oral cavity.
In the present study, 16S rRNA gene based PCR was used for H. pylori DNA amplification, which is highly sensitive. rRNA is a common but distinctive cellular component, and 16S rRNA gene based sequencing methods have been shown to be highly suitable for demonstrating phylogenetic diversity of bacteria.  Although sensitivity is tenfold more than other PCR methods, this nonspecifically amplifies human DNA. ,
Hot start PCR methods provide a solution to this lack of specificity by reducing or eliminating nonspecific product formation before high temperature cycling. The term "Hot start" is used to describe the inactivation of a DNA polymerase until the initial denaturation step of PCR cycling. It is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature. 
Thus, Hot start PCR significantly reduces nonspecific priming and the formation of primer dimmers, and often increases product yields.
Considering the 50 samples examined in this study, only 18% of the samples were found to be H. pylori positive in culture, whereas 44% of the samples were detected to be H. pylori positive with PCR (P=0.0164) [Table 3], [Figure 11]. The lack of culture growth compared to PCR detection might be due to insufficient number of cells for detection, the presence of inhibitors, or the presence of unculturable but viable coccoid forms in polymicrobial oral specimens. 
One may question the importance of PCR in detecting oral H. pylori as PCR allows for detection of low numbers of bacteria, which may be too few to influence gastric health. Moreover, PCR can permit detection of non-viable H. pylori, which is, by definition, non-infectious. 
There are differing points of view in the literature concerning the contention that the oral cavity is a reservoir of H. pylori. Some authors believe that H. pylori has only a transient presence in the oral cavity as they detected low percentage of H. pylori in the mouths of their patients. ,, Occasional recovery of viable microorganisms in the mouth might be associated with gastroesophageal reflux.  On the other hand, authors who find this bacterium in almost all of their studied population consider it as part of the normal microbiota of the oral cavity. ,
H. pylori has a characteristic distribution pattern within the oral cavity. The prevalence of H. pylori in dental plaque has been shown to decrease from molar to premolar to the incisor region.  If H. pylori has a preferred oral niche, sample collection from a single site could potentially give rise to false-negative results. Therefore, to exclude this possibility, cumulative dental plaque samples were collected from all posterior teeth and pooled on an individual basis in the present study.
As oral carriage is suggested to be population dependent, appropriate population comparison studies and longitudinal investigations should be carried out to prove oral cavity as the potential reservoir of H. pylori. Also, more successful culturing techniques for oral H. pylori are needed to confirm its viability and thus infectious status.
| Conclusion|| |
The subgingival environment of dyspeptic patients with chronic periodontitis is a potential reservoir for H. pylori, enhancing the risk for re-infection of the stomach. However, very low detection of H. pylori in subgingival plaque of healthy subjects suggests that periodontal pocket does not constitute a natural reservoir of H. pylori. Analyzing the diagnostic tests used, it was found that PCR was highly sensitive with adequate specificity for detection of oral H. pylori, when compared to culture.
The ability to detect H. pylori in dental plaque samples offers the potential for a non-invasive test for infection and would lend support for the oral spread as the principal mode of transmission.
| Acknowledgment|| |
We would like to acknowledge Dr. Kishore Bhat MD (Microbiology) for his valuable help in carrying out the study.
| References|| |
|1.||Martinez-Gomis J, Diouf A, Lakhssassi N, Sixou M. Absence of helicobacter pylori in the oral cavity of 10 non-dyspeptic subjects demonstrated by real-time polymerase chain reaction. Oral Microbiol Immunol 2006;21:407-10. |
|2.||Kilmartin CM. Dental implications of helicobacter pylori. J Can Dent Assoc 2002;68:489-93. |
|3.||Dowsett SA, Kowolik MJ. Oral helicobacter pylori: Can we stomach it? Crit Rev Oral Biol Med 2003;14:226-33. |
|4.||Dye BA, Kruszon-Moran D, Mcquillan G. The relationship between periodontal disease attributes and helicobacter pylori infection among adults in the United states. Am J Public Health 2002;92:1809-15. |
|5.||Gebara EC, Faria CM, Pannuti C, Chehter L, Mayer MP, Lapa L. Persistence of helicobacter pylori in the oral cavity after systemic eradication therapy. J Clin Periodontol 2006;33:329-33. |
|6.||Kignel S, Pina FA, Andre EA, Mayer MP, Birman EG. Occurrence of helicobacter pylori in dental plaque and saliva of dyspeptic patients. Oral Dis 2005;11:17-21. |
|7.||Krajden S, Fuksa M, Anderson J, Kempston J, Aoccia A, Petrea C, et al. Examination of human stomach biopsies, saliva and dental plaque for campylobacter pylori. J Clin Microbiol 1989;27:1397-8. |
|8.||Ferguson DA, Jrchuanfu LI, Patel NR, Mayberry WR, Chi DS, Thomas E. Isolation of helicobacter pylori from saliva. J Clin Microbiol 1993;31:2802-4. |
|9.||Gebara E, Pannuti C, Faria CM, Chehter L, Mayer MP, Lapa L. Prevalence of helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients. Oral Microbiol Immunol 2004;19:277-80. |
|10.||Ishihara K, Miura T, Kinizuka R, Ebihara Y, Mizuno Y, Okuda K. Oral bacteria inhibit helicobacter pylori growth. Fems Microbiol Lett 1997;152:355-61. |
|11.||Miyabayashi H, Furihata K, Shimizu T, Ueno I, Akamatsu T. Influence of helicobacter pylori on the success of eradication therapy against gastric helicobacter pylori. Helicobacter 2000;5:30-7. |
|12.||Anderson RN, Ganeshkumar N, Kolen-Brander PE. Helicobacter pylori adheres selectively to fusobacterium species. Oral Microbiol Immunol 1998;13:51-4. |
|13.||Anand PS, Nandakumar K, Shenoy KT. Are dental plaque, poor oral hygiene and periodontal disease associated with helicobacter pylori infection? J Periodontol 2006;77:692-8. |
|14.||Namiot DB, Namiot Z, Kemona A, Bucki R, Gotebiewska M. Oral health status and oral hygiene practices of patients with peptic ulcer and how these affect helicobacter pylori eradication from the stomach. Helicobacter 2007;12:63-7. |
|15.||Umeda M, Kobayashi H, Takeuchi Y, Hayashi J, Morotome-Hayeshi Y, Yano K. High prevalence of helicobacter pylori detected by PCR in the oral cavities of periodontitis patients. J Periodontol 2003;74:129-34. |
|16.||Majumdar P, Shah SM, Dhunjibhoy KR, Desai HG. Isolation of helicobacter pylori from dental plaques in healthy volunteers. Indian J Gastroenterol 1990;9:271-2. |
|17.||Desai HG, Gill HH, Shankaran K, Mehta PR, Prabhu SR. Dental plaque: A permanent reservoir of Helicobacter pylori. Scand J Gastroenterol 1991;26:1205-8. |
|18.||Asikainen S, Chen C, Slots J. Absence of helicobacter pylori in subgingival samples determined by polymerase chain reaction. Oral Microbiol Immunol 1994;9:318-20. |
|19.||Berroteran A, Perrone M, Correnti M, Cavazza ME, Tombazzi C, Goncalvez R, et al. Detection of helicobacter pylori DNA in the oral cavity and gastroduodenal system of a Venezuelan population. J Med Microbiol 2002;51:764-70. |
|20.||Oshowo A, Tunio M, Gillam D, Botha AJ, Holton J, Boulos P, et al. Oral colonization is unlikely to play an important role in helicobacter pylori infection. Br J Surg 1998;85:850-2. |
|21.||Young KA, Allaker RP, Hardie JM. Morphological analysis of Helicobacter pylori from gastric biopsies and dental plaque by SEM. Oral Microbiol Immunol 2001;16:178-81. |
|22.||Butt AK, Khan AA, Suleman BA, Bedi R. Randomized clinical trial of helicobacter pylori from dental plaque. Br J Surg 2001;88:206. |
|23.||Paster BJ, Dewhirst FE. Phylogeny of campylobacters, wolinellas, Bacteroides gracillis and Bacteroides ureolyticus by 16S ribosomal ribonucleic acid sequencing. Int J Syst Bacteriol 1998;38:56-62. |
|24.||Lu JJ, Perng CL, Shyu RY, Chen CH, Lou Q, Chong KF, et al. Comparison of five PCR methods for detection of Helicobacter pylori DNA in gastric tissues. J Clin Microbiol 1999;37:772-4. |
|25.||Chong SK, Lou Q, Fitzgerald JF, Lee CH. Evaluation of the 16S rRNA gene PCR with primers Hp1 and Hp2 for detection of Helicobacter pylori. J Clin Microbiol 1996;34:2728-30. |
|26.||Variations in basic PCR technique. Available from: http://en.wikipedia.org/wiki/polymerase_ chain_ reaction. [Last accessed on 2010 Apr 24]. |
|27.||Madinier IM, Fosse TM, Monteil RA. Oral carriage of helicobacter pylori: A review. J Periodontol 1997;68:2-6. |
|28.||Nguyen, Engstrand L, Genta RM, Graham DY, el-Zaatari FA. Detection of helicobacter pylori in dental plaque by reverse transcription - polymerase chain reaction. J Clin Microbiol 1993;31:783-7. |
|29.||Song Q, Lange T, Spahr A, Adler G, Bode G. Characteristic distribution pattern of Helicobacter pylori in dental plaque & saliva detected with nested PCR. J Med Microbiol 2000;49:349-53. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2], [Table 3], [Table 4]
|This article has been cited by|
||Oral Cavity as an Extragastric Reservoir of Helicobacter pylori
| ||Arwa Al Sayed,Pradeep S. Anand,Kavitha P. Kamath,Shankargouda Patil,R. S. Preethanath,Sukumaran Anil |
| ||ISRN Gastroenterology. 2014; 2014: 1 |
|[Pubmed] | [DOI]|
||Can eradication rate of gastric Helicobacter pylori be improved by killing oral Helicobacter pylori?
| ||Song, H.-Y., Li, Y. |
| ||World Journal of Gastroenterology. 2013; 19(39): 6645-6650 |
||Extragastric Diseases andHelicobacter pylori
| ||Claire Roubaud Baudron,Francesco Franceschi,Nathalie Salles,Antonio Gasbarrini |
| ||Helicobacter. 2013; 18: 44 |
|[Pubmed] | [DOI]|