Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture

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.[12] 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.[3]

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.[4]

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.[5–9] 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.[10] 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.[2]

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.[5] 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.
• Smokers/alcoholics.
• 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

Procedure

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.

Figure 2

Subgingival plaque collection

• 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.

Figure 3

Selective media for H. pylori

Figure 4

Culture plate showing H. pylori colonies

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.

Figure 5

PCR detection kit for H. pylori

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].

Figure 6

Thermocycler

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 {Figures 7 and 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

Figure 8

Gel electrophoresis results of PCR showing samples 2, 3, 12 positive for H. pylori in controls

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].

Table 1

Culture results

Figure 9

Culture results comparing cases and controls

Table 2

PCR results

Figure 10

PCR results comparing cases and controls

Table 3

Comparison of PCR with culture

Figure 11

Comparison of PCR with culture

Table 4

Assessment of diagnostic tests used

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.[5] 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.[11]

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.[10]

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.[12] 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.[1314]

Many of the earlier studies[15–17] reported a high prevalence of H. pylori in dental plaque, whereas some[11819] 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.[220]

The reported prevalence of H. pylori in the developing countries is higher than in developed countries.[2] Most of the studies done on Indian population[1617] 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)[2122] 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.[23] Although sensitivity is tenfold more than other PCR methods, this nonspecifically amplifies human DNA.[2425]

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.[26]

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.[27]

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.[2]

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.[11819] Occasional recovery of viable microorganisms in the mouth might be associated with gastroesophageal reflux.[28] 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.[1617]

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.[29] 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.