Is Helicobacter pylori Linked to Halitosis in Non-Dyspeptic Chronic Periodontitis Patients? – A Case-Control Study

ABSTRACT

Objective:  To quantify H. pylori in the subgingival plaque of non-dyspeptic chronic periodontitis patients with halitosis and healthy controls.

Material and Methods:  Sixty participants, distributed into subjects with chronic periodontitis and halitosis who were systemically healthy (Group A, cases, n=30) and subjects with healthy gingiva (Group B, control, n=30) were included in the study. The clinical periodontal status was evaluated, and various clinical parameters, including the gingival index, gingival bleeding index, pocket probing depth, clinical attachment levels, and tongue coating scores. Quantitative Real-time PCR was performed with the subgingival plaque of both cases and control by using highly specific primers that amplify the ureA gene of Helicobacter pylori.

Results:  The H. pylori ureA gene was not identified in either the cases or the control group. The results strongly support the fact that H. pylori is a transient member of the oral microflora.

Conclusion:  The finding of the present study reveals a negative correlation of H. pylori with non-dyspeptic chronic periodontitis patients with halitosis.

Keywords:
Chronic Periodontitis; Helicobacter pylori ; Real-Time Polymerase Chain Reaction

Introduction

Halitosis, also termed as fetor ex ore or fetor oris, is derived from two Latin words: Halitus (breath) and osis (disease) [¹]. It is defined as an unpleasant or offensive odor emanating from the breath formed by volatile molecules, which are caused by pathological or non-pathological reasons, and originates from an oral or a non-oral source.

The basic pathophysiology process in halitosis is microbial degradation of sulfur-containing amino acids substrates. Bacterial metabolism of these amino acid leads to metabolites such as volatile sulfur compounds (VSCs), hydrogen sulfide, methyl mercaptan, dimethyl sulfide, skatole, and indole [²]. Although halitosis has multiple sources, the primary cause is attributed to the oral cavity, specifically poor oral hygiene, periodontal disease, tongue coating, food impaction, unhygienic dentures, defective restorations, oral carcinomas, and throat infections. Halitosis hampers daily life activities and the morale of an individual, and determining its origin and effective management proves to be a challenge.

H. pylori is a spiral, microaerophilic, Gram-negative bacterium that colonizes the human gastrointestinal tract, primarily the stomach. It is believed to be responsible for gastritis, peptic ulcers, and is a risk factor for gastric cancer [³,⁴,⁵].

The mode of transport for Helicobacter pylori is poorly understood. Several studies have detected Helicobacter pylori in the human oral cavity, particularly in patients with gingivitis or periodontitis, suggesting that the oral cavity may serve as a primary extragastric reservoir for H. pylori. However, it remains to be established whether H. pylori is a transient resident of the oral cavity or whether the oral cavity serves as the original reservoir, where this bacterium can multiply before entering and infecting the stomach [⁶].

Although treatment regimens lead to the successful management of H. pylori chronic gastritis, the reinfection rate is relatively high, indicating that other pathways of infection are involved [⁷]. Helicobacter pylori has been reported as a biological indicator for halitosis [⁸]. In addition, a Canadian study has reported the absence of halitosis in patients who underwent therapy for destruction of H. pylori infection [⁹]. Hence, the present study aims to assess the prevalence of oral H. pylori in the dental subgingival plaque of non-dyspeptic subjects with chronic periodontitis and halitosis, and to correlate the findings with levels of oral volatile sulfur compounds (VSCs).

Material and Methods

Ethical Clearance

This study was cleared by the Institutional Ethics Committee of Sree Balaji Dental College & Hospital, Chennai. (Approval No: SBDCH/IEC/01/2018/14). Informed written consent was obtained from all the subjects.

Study Design and Sample

This Case-Control Study population included thirty subjects with non-dyspeptic, chronic periodontitis with halitosis who were systemically healthy (Group A) and thirty subjects with healthy gingiva (Group B).

Systemically healthy patients, age between 25yrs to 65yrs, presence of no less than 20 permanent teeth, > 4mm periodontal probing depth, > 2mm clinical attachment loss in at least 30% of the teeth was the inclusion criteria for Group A. Age and sex matched individuals with no mucosal lesions and no history of deleterious habits were recruited under Group B. Subjects with the habit of smoking, diabetes, antibiotic or steroidal therapy in the past three months, lactating and pregnant women, denture wearers, dyspeptic patients, periodontal treatment in the past three months were excluded.

Clinical Data

The clinical periodontal status was evaluated, and various clinical parameters, including the gingival index, gingival bleeding index, pocket probing depth, clinical attachment levels, and tongue coating scores, were recorded. Volatile sulfur compound assessment was performed using a portable volatile sulfide monitor (FitScan Breath Checker, Tanita Corp., Tokyo, Japan). After carefully removing supragingival plaque with a sterile cotton roll, subgingival plaque was collected from deep sites in each quadrant using a Gracy curette. The subgingival plaque was pooled in a sterile Eppendorf tube containing 1ml of phosphate-buffered saline. The samples were stored at -20 °C until assayed.

DNA Extraction

The subgingival plaque present in PBS was centrifuged at 7500 rpm for 10 min and the supernatant was discarded. DNA was extracted from all samples using the DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA was quantified by Qubit 4.0 Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Real-Time PCR Protocol

Quantitative Polymerase Chain Reaction (qPCR) was performed by using primers specific for H. pylori ureA, coding for the small subunit of urease – forward, 5’-GTATTGAAGCGATGTTTCCT -3’; reverse, 5’-GCTTTTTTGCCTTCGTTGAT-3’. For quantitation, quantity and purity were assessed by Qubit fluorometer, and a series of dilutions with H. pylori positive control (10² to 10¹⁰ dilutions) were prepared. Each dilution of positive control was amplified with the H. pylori ureA primer set. A standard graph was constructed with the C_T (threshold cycle) values obtained from serially diluted H. pylori positive control (Figure 1).

Figure 1
The standard curve of the quantitative real-time PCR assay. Serial dilutions of H. pylori positive control from 10² to 10¹⁰ dilutions.

The CT values from clinical samples were plotted on the standard curve, and the copy number was calculated automatically by Sequence Detector (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA), a software package for data analysis. Each sample was tested in duplicate, and the mean of the two values was shown as the copy number of the sample. The estimation of H. pylori DNA in each sample was expressed as the number of H. pylori DNA copies per 50ng of purified DNA. QuantiNova SYBR Green PCR Kit (Qiagen, Hilden, Germany) was used for the study. PCR Thermal cycling conditions were: Hold stage – 95oC for 2 minutes, PCR stage – 40 cycles of 95oC for 5 seconds, 60oC for 20 seconds, melt curve stage – 95oC for 0.05 seconds, 60oC for 0.20 seconds, and 95oC for 0.01 seconds.

Each 10-μl reaction contained: 5μl of QuantiNova SYBR Green PCR Master Mix, 0.1 μlof QN ROX Reference Dye, 0.5μl of 30ρmol each of the forward and reverse primers, 2 μl of DNA template. To check for amplicon contamination, every run contained at least two “no template” controls in which nuclease-free H₂O was substituted for template.

Melting Curve Analysis

Reaction specificities were verified by melting curve analysis, which involved a progressive temperature increase from 60°C to 95°C at a 0.1°C/s transition rate, accompanied by continuous fluorescence acquisition. Successive runs with the same primers can be modified to remove the contribution of primer dimer formation to the product signal by collecting data in an additional cycling step, where the temperature is set between the already determined dimer and product melting temperatures.

Results

The number of cycles required to amplify the full range of serially diluted DNA from the H. pylori positive control ranged from 4 to 25 for the H. pylori ureA gene (10² to 10¹⁰) (Figure 2).

Figure 2
Amplification plot of Helicobacter pylori standards from 10² to 10¹⁰ dilution. The number of cycles required to amplify the 10² to 10¹⁰ diluted DNA from the H. pylori positive control ranged from 4 to 25.

No evidence of nonspecific or cross-reaction products was observed for species-specific markers. Dissociation profiles of standard DNA amplicons revealed well-depicted peaks with the absence of primer-dimers (Figure 3).

Figure 3
Melt curve analysis. The melting curves for genomic DNA extracted from the H. pylori positive control. The SYBR Green quantitative PCR assays with the Positive control yielded specific products, and the dissociation curve peaks were unequivocal.

The subgingival plaque of subjects with non-dyspeptic, chronic periodontitis with halitosis who were systemically healthy (Group A) did not harbor H. pylori. Thus, a complete absence of the H. pylori ureA gene was observed among the samples of Group A. The majority of the subgingival plaque of subjects with healthy gingiva also did not harbour the H. pylori ureA gene, except for two samples. Each of the two samples was amplified at the 32nd cycle and the 33rd cycle, respectively. Amplification of DNA above the 30th cycle in real-time PCR may not be considered a significant positive. The H. pylori ureA gene was not detected in Group A and Group B subjects.

Discussion

Halitosis is a concern for millions of people, affecting interpersonal social communication with ensuing personal discomfort and social embarrassment [¹⁰]. Although the existence of halitosis has been documented in the literature for thousands of years, it has been a long-neglected quandary until recently. It was found that 80% to 90% of patients with halitosis had oral conditions. The causative organisms from the halitogenic biofilm on the posterior dorsal tongue, and/or within gingival crevices/periodontal pockets are habitually gram-negative anaerobic bacteria [¹¹,¹²]. The basic pathophysiological process involves the microbial degradation of sulfur-containing amino acid substrates, such as methionine and cysteine [¹³]. H. pylori has been reported to produce hydrogen sulfide and methyl mercaptan, which suggests that this microorganism can be a potential risk factor for the development of halitosis [¹⁴,¹⁵].

Currently, portable VSC monitors boast high sensitivity, consistency, accuracy, and ease of use, measuring the cumulative amount of various VSCs present to provide diagnostic value [¹⁶]. One such example of a compact sulfide monitor is the Tanita FitScan Breath Checker (Tanita Asia Pacific, Tokyo, Japan). In our study, we used the Fit Scan breath checker to estimate volatile sulfur scores and found a statistically significantly higher value (mean 1.63) in halitosis cases compared to controls.

The likely link between halitosis and H. pylori infection was first reported by Tiomny et al. [¹⁷]. Few earlier studies have suggested that suppression of H. pylori, either in the oral cavity or in the stomach, would help improve halitosis [¹⁸,¹⁹]. 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 [²⁰]. 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 [²¹]. While H. pylori can be isolated from the oral cavity in some cases, most attempts to culture the organism have been unsuccessful. This is attributed to the fictitious nature of H. pylori, which requires a microaerophilic environment, supplemented media, and up to seven 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 [²²]. With advances in molecular technology, the potential difficulties associated with culture have been circumvented by the use of polymerase chain reaction (PCR), which enables the detection of even small numbers of specific bacteria within a sample and obviates the need for viable organisms [²³]. In the present study, real-time PCR was performed to quantify the H. pylori ureA gene. Primers that amplify the H. pylori ureA gene were selected due to their high specificity [²⁴,²⁵].

There are different perspectives on the contention that the oral cavity serves as a reservoir for H. pylori. Studies that detected low percentages of H. pylori in the oral cavity consider that it is not a significant reservoir for this bacterium, which might have only a transient presence in the mouth [²⁶,²⁷,²⁸]. On the other hand, authors who found high amounts of H. pylori in their studied population consider it a normal component of the oral cavity microbiota [²¹,²⁹,³⁰,³¹].

Real-time PCR has proven to be the most accurate method for detecting H. pylori in gastric biopsies due to its high specificity, short working time, low risk of contamination, and the ability to yield quantitative analysis [³²]. Martinez-Gomis et al. [³³] used real-time PCR to detect H. pylori in the oral cavity of ten non-dyspeptic patients and reported 0% prevalence. On the contrary, we aimed to quantify the presence of H. pylori by real-time PCR. The results of our study failed to detect the organism in both cases and controls, indicating that H. pylori exhibits a negative correlation with halitosis and is not a permanent resident of the oral microflora. Thus, the findings of the present study align well with those of Martinez-Gomis et al. [³³], particularly for subjects with chronic periodontitis and halitosis. The absence of H. pylori in the oral samples of non-dyspeptic halitosis patients is consistent with the findings of earlier studies by conventional PCR [³⁴,³⁵,³⁶]. The results of the present study do not show a positive correlation between H. pylori and halitosis. The current finding further supports the statement that H. pylori is more stable in the presence of sporadic gastric reflux than as a permanent colonizer in the oral cavity; further longitudinal analysis might detect its presence if it is a transient resident.

Conclusion

H. pylori is a transient member of the oral microflora of specific populations. H. pylori is not linked to halitosis. Longitudinal studies with a large sample size will shed more light on the association between H. pylori and halitosis in patients with chronic periodontitis.

Acknowledgements

The authors wish to thank for utilizing the research equipment facilities of Sree Balaji Dental College and Hospital, Pallikaranai, Chennai,Tamil Nadu, India.

• Financial Support
  The Indian Department of Science & Technology's (DST) Fund for Improvement of S&T Infrastructure (FIST) Program (Grant Ref.No.SR/FST/College-23, 2017).

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

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Edited by

• Academic Editor:
  Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  28 Nov 2025
• Date of issue
  2026

History

• Received
  13 May 2023
• Reviewed
  28 Aug 2024
• Accepted
  14 Feb 2025