Is There a Real Relationship between the Presence of Helicobacter pylori in Dental Plaque and Gastric Infection? A Genotyping and Restriction Fragment Length Polymorphism Study on Patient Specimens with Dyspepsia in Southwest Iran

Background The oral cavity can act as an extra gastric reservoir for H. pylori, and the presence of the bacteria in the oral cavity is associated with a higher risk of dental caries development. This study aimed to determine the genotype and evaluate the association between the presence of H. pylori in dental plaque and gastric biopsy specimens in dyspeptic patients in Ahvaz, Southwest Iran. Methods In this study, 106 patients with recruited dyspeptic complaints were selected, and from each patient, two gastric antral biopsy specimens and two dental plagues were examined. The presence of H. pylori was identified by the rapid urease test (RUT) and the amplification of ureAB and 16S rRNA genes. Also, to verify a hypothetical mouth-to-stomach infection route, the enzymatic digestions of three genes of cagA, vacA, and ureAB in H. pylori strains isolated from dental plaques and stomach samples were compared for each same case. Results H. pylori was found in the stomach of 52.8% (56/106) and the dental plaques of 17.9% (19/106) of the studied cases. On the other hand, H. pylori was recognized in the stomach of all 19 cases with oral colonization. Following a combination of restriction fragment lengths 21 polymorphism (RFLP) patterns of these three known genes on stomach and dental plague samples, 14 and 11 unique patterns were seen, respectively. However, for all H. pylori-positive cases (19), the comparison of RLFP patterns of these genes in dental plaque and gastric biopsy specimens was different for the same case. Conclusions In this study, it seems that there is no significant association between the presence of H. pylori in dental plaque and the stomach of the same case.


Introduction
Helicobacter (H.) pylori has been known as one of the most common agents of human bacterial infections.Tis bacterium is located under the gastric mucous layer, adjacent to the gastric epithelial cells.Infection caused by this bacterium is now recognized as a serious transmissible infectious disease [1].H. pylori infections are common in areas with low levels of social-economic health [2].Despite many investigations, the exact route of infection transmission of this microorganism is still unknown.However, several diferent modes are suggested: gastro-oral, oral-oral, and fecal-oral transmission [3].H. pylori is the only known pathogenic bacterium that survives in the highly acidic environment of the human stomach.Tis bacterium by producing a urease enzyme that converts urea into carbon dioxide and ammonia increases the pH and neutralizes the acid in the stomach environment.Reports also show that for colonization in the gastrointestinal mucosa, strains of H. pylori produce more urease enzymes than other pathogens [4,5].
In addition to the stomach, H. pylori has also been isolated from the saliva and feces of the human as a likely reservoir of it [6].Since the oral cavity can act as an additional gastric reservoir for H. pylori, it may lead to gastric reinfection and is, therefore, a possible factor for eradication failure and reinfection [7].On the other hand, Moseeva et al. found a relationship between the colonization of this bacterium in the oral cavity and the risk of dental caries development [8].In contrast, some reports did not show the simultaneous presence of H. pylori in the mouths of patients with gastric infection, because oral and stomach H. pylori had diferent genotypes [9,10].
Tese controversial results may be due to several factors including the variations in the study population, sampling procedures, and methodologies used for the detection of H. pylori in dental plaques.Tere are several diagnostic tests for H. pylori in dental plaques, including rapid urease test (RUT), polymerase chain reaction (PCR), immunoassay, cytology, and bacterial culture.In general, the detection rate of H. pylori reported in studies was higher using RUT than other tests.Te lowest detection rate was when microbial culture was used to detect the presence of this bacterium in dental plaque [11].
Several virulence factors have been identifed in H. pylori that can develop the severity of pathogenicity.Among them, cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) have been mentioned as the two main virulence factors of H. pylori that are involved in the pathogenesis of diferent strains.Te cagA gene is located within a 40-kilobase DNA fragment and is known as the cag pathogenicity island (cag PAI).Te cag PAI encodes nearly 27 proteins, one of the most important of which is oncoprotein.Twenty of these proteins have been designed as cag type IV secretion system (T4SS), which can induce the production of interleukin 8, mucosal infammation, and an increased risk of the peptic ulcer [12,13].Some reports showed that 48% and 80% of H. pylori strains isolated from Tai and Korean patients, respectively, had an intact cag PAI gene [14].Te vacA is a housekeeping gene that can induce vacuolation and apoptotic processes in epithelial cells, as well as immunosuppressive efects on some immunological cells [12].
In this study, to investigate the hypothesis of oral-tostomach transmission in H. pylori infection, it was necessary to compare the RFLP patterns of several H. pylori genes between the mouth and stomach for the same case.Tis method was also able to analyze DNA extracted from H. pylori digested by high-frequency restriction endonucleases and showed distinct digestion patterns between strains [15].In our study, three genes of cagA, vacA, and ureAB were selected for these analyses.To our knowledge, no sufcient research has been conducted on the detection of H. pylori in dental plaque and gastric biopsy specimens isolated from patients with dyspepsia in Ahvaz, Southwest Iran.
Terefore, the purpose of this study with registration number OG-93129 was to determine the genotype and investigation of Helicobacter pylori in dental plaque and gastric biopsy samples, as well as evaluate the relationship between Helicobacter pylori colonization in dental plaque and stomach

Study Population and Sampling.
In this study, specimens were collected from 106 patients with dyspeptic complaints who were referred to the teaching hospital of Imam Khomeini, Ahvaz, Iran.Initially, two biopsy specimens from each patient were taken by a gastroenterologist from the antral, within 5 cm of the pylorus.Te exclusion criteria were as follows: treatment with any antibiotics, proton pump inhibitors, H2 blockers, or bismuth compounds during one month preceding this study, recent use of nonsteroidal antiinfammatory drugs, periodontal therapy within the past year, and signs of severe periodontal infections within the preceding 6 months [16].
We retained one gastric biopsy specimen for RUT and stored the other at −70 °C for DNA extraction.To perform RUT, samples were inoculated into the urea broth medium (Sigma Co.).Te required time for a positive test depends on the concentration of H. pylori and temperature.Although most RUTs will change to positive within 120-180 minutes, for negative tests and increased sensitivity, it is best to read the test for 24 hours, so in this study, the results were interpreted after one overnight.
On the other hand, two dental plaque samples were taken from each patient before endoscopic examination.To remove the remaining foods, each patient was requested to wash his/her mouth with normal saline before sampling.Dental foss was used to completely clean the interdental spaces.Te samples were transported in the tubes containing digestion bufer (100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 250 mM ethylene diamine tetra acetic acid (EDTA) (pH 8.0), and 1% sodium lauryl sarcosine) on the day of sampling [6].One of these samples was used for screening H. pylori by RUT, and the other was stored at −70 °C for DNA extraction and PCR.

DNA Extraction.
Samples were crushed and mixed by a vortex.Ten, the mixtures were centrifuged at 12000 × g for 3 min.A volume of 200 μl of suspended pellets in normal saline was transferred to a 1.5 ml microtube.Te genomic DNA was extracted using a High Pure PCR Template Preparation Kit (Roche Diagnosis, Mannheim, Germany) according to the protocol of DNA extraction from mammalian tissues.Te concentration of the extracted DNA was measured at 260 nm, using a Nano-Drop instrument (Termo Scientifc, USA) and gel electrophoresis.DNA purity was acceptable when the light absorption ratio at 260 nm to 280 nm was between 1.5 and 2.

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Identifcation of H. pylori Using PCR. Helicobacter pylori
was identifed by PCR amplifcation of ureAB (encoding urease enzyme conserved in all Helicobacter isolates), as well as vacA, cagA, and 16S rRNA genes [17,18].Te sequence of used primers is shown in Table 1.For each gene, a uniplex PCR reaction was prepared in a volume of 25 μL.Te master mixture consisted of 0.02 units of Taq DNA polymerase, 1.5 mM MgCl2, 0.4 μM of each primer, 1xPCR bufer, 0.2 mM dNTP mix, 25 ng DNA template, and distilled water up to a fnal volume of 25 μl.
After electrophoresis of PCR products and staining of 2% gel agarose with ethidium bromide, the amplicons were observed and photographed using an ultraviolet light gel documentation system (Viber, Germany).

Restriction Fragment Length Polymorphism (RFLP)
Analysis.Te vacA, cagA, and ureAB gene fragments were amplifed by PCR and then were digested with HaeIII and Sau3AI for ureAB, HhaI and HphI for vacA, and HinfI for cagA for 3 hours at 37 °C in the appropriate bufers suggested by the supplier (Fermentas, US).Te digested products of each gene target were electrophoresed on 2% agarose gel stained with ethidium bromide [20].

Statistical Analysis.
Te descriptive statistics and chisquare test were performed in SPSS version 16.00, and the signifcance level of p < 0.05 was used in this study.

Results
A total of 106 gastric biopsy and dental plaque specimens from patients with dyspeptic complaints were evaluated.Of these patients, the number of male cases (88) was signifcantly higher (p < 0.05) compared with women (18) cases.
In this study, the prevalence of H. pylori in the stomach specimens (56/106, 52.8%) compared with the dental plaques (19/106, 17.9%) specimens was signifcantly higher (p < 0.05).On the other hand, H. pylori was found in the stomach of all 19 cases with dental plaque colonization.All patients with H. pylori colonization in dental plaque and gastric biopsy had positive results in both molecular identifcations of ureAB and 16S rRNA genes and RUT (Figure 1).
On the other hand, among 56 gastric biopsy specimens containing H. pylori, 35 cases (62.5%) had the cagA gene (Figure 1).According to our results, the highest prevalence of the cagA gene belonged to patients with gastritis (20 of 28, p < 0.05), compared to patients with peptic ulcer (12 of 21) and duodenum ulcer (3 of 7).Also, the frequency of the cagA gene in dental plaque specimens was 63.1% (12 of 19).
Following enzymatic digestion of the vacA gene via HhaI and HphI (Figure 2 2. On the other hand, due to the enzymatic digestion of the ureAB gene (Figure 2(c)) by HaeIII, four RFLP patterns of 3, 4, 5, and 6 bands were visually recognized.Te RFLP patterns of the ureAB, vacA, and cagA genes in the stomach samples are shown in Table 3.According to the data, we recognized 14 unique patterns.
According to Table 4, by combining the RFLP patterns of these three genes in the stomach and dental plaque specimens for the same case, 14 and 11 unique patterns were seen, respectively.
However, for all cases with a positive H. pylori diagnosis, a combined analysis of the RFLP patterns of these three genes was diferent in the dental plaque with the gastric biopsy samples for the same case.According to the data, there was no association between H. pylori colonization in the oral cavity and stomach for the same case (p > 0.05) because the comparison of fngerprint patterns due to enzymatic digestion of three genes (cagA, vacA, and ureAB) showed that these patterns were diferent.

Discussion
In the past, the human stomach seemed to be the only reservoir of Helicobacter pylori colonization, until the identifcation of this bacterium was reported from some dental plaques, oral cavity, and saliva.However, the recovery of H. pylori from the oral cavity is a controversial issue.In addition, most attempts to isolate the bacterium from the oral cavity by culture failed [3].Because H. pylori has a viable, noncultivable coccoid form, several investigators have proposed that H. pylori in this form may survive in the oral and is only detectable by nonculture methods [21,22].
As an indirect test that can detect the presence of H. pylori, RUT has advantages over serology because it can only detect active H. pylori infection.To obtain a positive RUT result, there must be approximately 105 bacteria in the sample.Since, in our study, all patients with H. pylori colonization in the stomach and dental plaque (19 cases) had the same positive results for both RUT and PCR, therefore, it is concluded that the sensitivity of both tests is the same.In agreement with our study, some researchers also demonstrated that RUT sensitivity is higher or similar to PCR.Khalifehgholi et al. and Souod et al. have shown that the sensitivity of RUT and PCR was 95.6% and 93.5%, respectively [23,24].   4

International Journal of Microbiology
International Journal of Microbiology However, Chamanrokh et al. showed PCR as a more sensitive method rather than RUTfor the detection of H. pylori [25].Moreover, RUT is a specifc method for the detection of H. pylori in gastric biopsy specimens, and in some cases, sensitivity and accuracy of 89.7% and 86.7% were shown, respectively, for this test in detecting H. pylori in dental plaque, but some researchers have questioned the validity of RUT for the diagnosis of H. pylori in oral specimens.Chamanrokh et al. believed that a negative RUT result does not necessarily exclude H. pylori infection and a positive-result RUT also does not confrm infection with this bacterium, because these results may be due to infection with other positive-urease bacteria [25].Moreover, some studies indicate that false positive or negative results may occur.For example, the presence of other urease-positive organisms in the specimen can cause a false-positive result.Also, the use of antimicrobial drugs and proton pump inhibitors as well as the presence of intestinal metaplasia may result in false-negative results [26].
Tese controversial results can be explained by the fact that although H. pylori is the only urease-positive bacterium known in the gastric cavity, other urease-positive bacterial species, such as Streptococcus species, Haemophilus species, and Actinomyces species, also reside as part of the normal oral fora.However, according to some reports, only H. pylori can produce large amounts of urease for a short time, such as 20 min, while delayed urease-producing bacteria do not have positive results within one hour [11].
In the present study, we identifed H. pylori by PCR method through the amplifcation of both urease and 16S rRNA genes in gastric biopsy and dental plaque specimens.H. pylori was found in the stomach of 52.8% (56/106) of the studied cases.
According to some reports, the frequency of H. pylori in dyspeptic patients in other regions of Iran using PCR was high and ranged from 48 to 85% [27][28][29].Tis diference in rates can be explained according to the socio-economic status of population density in diferent regions of Iran, diferent methods in sample collection and processing, and primers used in PCR [6].
On the other hand, we found H. pylori simultaneously in dental plaques and gastric biopsies of 19 (17.9%) cases.Similar to our study, Eskandari et al. found H. pylori in 17.4% of cases in both the stomach and dental plaque samples simultaneously, in Iran [30].Cellini et al. also showed that all the saliva and esophagus samples of 19 patients examined were positive by molecular analysis and suggested that these two sites may be considered reservoirs of H. pylori in humans, although they emphasize the need to use more sensitive techniques for H. pylori detection, especially in over-crowded sites [31].Also, a simultaneous diagnosis of the same strain of H. pylori in both plaque and gastric mucosa was performed by Bharath et al. [32].
In contrast to our study, Momtaz et al. did not fnd any H. pylori in the dental plaques of patients with H. pylori gastritis, while they detected this bacterium in 77.6% of gastric biopsies, 10.7% of saliva, and 71.6% of stool samples [6].Tese diferences in the prevalence of H. pylori may be due to variations in sampling techniques, diferent methodologies, sets used in PCR, or some diferences in the study population.In general, H. pylori colonization in the oral cavity is less than in the stomach.Te low rate can be explained by two reasons: (1) suppressor efects of oral microbiota producing bacteriocin-like inhibitory proteins against H. pylori strains and (2) hiding H. pylori in yeast such as Candida spp. in the oral cavity [8].Furthermore, unstable oral temperature due to eating and saliva as mechanical fushing may be a barrier to the long-term oral survival of H. pylori [5].Although the performed investigations by many epidemiologists have shown a positive relationship between H. pylori in gastric infection and periodontal diseases [5,33], many studies, however, could not confrm this association [34,35].Also, the study of Rossi-Aguiar et al. by evaluating the frequency of cagA and vacA genotypes of oral H. pylori in patients with functional dyspepsia showed that the oral cavity is not a reservoir for H. pylori in these patients [36].
In our study, the frequency of the cagA gene in the dental plaque and gastric biopsy specimens with H. pylori-positive results was 63.1% and 62.5%, respectively.Te prevalence of cagA-positive H. pylori strains was diferent from one geographic region to another.A study in Austria showed that the prevalence of cagA in patients with gastric cancer and duodenal ulcers was 86% and 78%, respectively [37].Also, the prevalence of this gene in Morocco, 42.3%, and in Egypt, 57.4%, has been reported [38,39].In addition, the prevalence of this gene was diferent in other regions of Iran, including 69% in Tehran [40], 92% in Shahrekord [24], and 69.7% in Tabriz [41].
In the present study, the number of cagA-positive H. pylori strains was higher in patients with gastritis than in patients with peptic ulcer and duodenum ulcer.However, Boukhris et al. and Eskandari et al. demonstrated a higher prevalence of this gene among patients with peptic ulcer (50%) and gastric cancer (85%), respectively [30,38].
In this study, we found 14 and 11 unique patterns of RFLP in the stomach and dental plaque samples, respectively.In addition, to fnd the correlation between H. pylori colonization in dental plaque and stomach for the same case, the RFLP analyses of vacA, cagA, and ureAB genes were performed on the DNA extracted from the dental plaque and gastric biopsy samples.According to these  International Journal of Microbiology analyses, diferent RFLP patterns were seen for each case; in other words, in each case, there were no identical RFLP patterns for both samples (dental plaque and gastric biopsy).In summary, although fnding the same strain in the mouth and stomach would support the role of the oral cavity as a reservoir for gastric H. pylori, however, more extensive studies are required to confrm the identity of closely related strains in the mouth and stomach [15].

Conclusions
It seems that in our study, there is no correlation between H. pylori colonization in dental plaque and the stomach.In other words, in this study, we could not consider dental plaque as a likely reservoir for reinfection of the gastric with H. pylori.
(a)) and the cagA gene via HinfI (Figure 2(b)) on DNA extracted from gastric biopsy and dental plaque specimens, two RFLP patterns of 3 and 4 bands for the vacA gene and two RFLP patterns of 1 and 3 bands for the cagA gene were visually detected.Te RFLP patterns of the vacA and cagA genes are shown in Table

Table 1 :
Te sets of the used primers in this study.

Table 2 :
Te enzymatic RFLP patterns for vacA and cagA genes.

Table 3 :
Te RFLP pattern of ureAB, cagA, and vacA genes on stomach specimens.

Table 4 :
Te combination of RFLP patterns of ureAB, cagA, and vacA genes on stomach and dental plaque specimens.