Genotyping and Association of the Presence of Helicobacter Pylori in Dental Plaque and Gastric Biopsy Specimens in Dyspeptic Patients by PCR-RFLP Technique in Southwest of Iran

The oral cavity can act as an extra gastric reservoir for H pylori, and also the presence of the bacteria in the oral cavity is associated with a higher risk of dental caries development. The aim of this study was to determine the genotype and evaluate the association of the presence of H. pylori in dental plaque and gastric biopsy specimens in dyspeptic patients in Ahvaz, Southwest of Iran. 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 identied by the Rapid Urease Test (RUT) and the amplication 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. 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 length 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 the dental plaque and gastric biopsy specimens was different for the same case. This study showed, no signicant association was observed between the presence of H. pylori in dental plaque and the stomach of the same case.


Introduction
Helicobacter pylori have been known as one of the most common agents of human bacterial infections. This 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 (Veiga et al. 2015).
H. pylori infections are common in areas with low levels of social-economic health (Goh et al. 2011). Despite many investigations, the exact route of infection transmission of this microorganism is still unknown. However, two different modes are suggested: oral-oral and fecal-oral transmission (Umeda et al. 2003).
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 (Momtaz et al. 2012). Since the oral cavity can act as an extra gastric reservoir for H pylori, it may lead to recurrent gastric infection (Al Asqah et al. 2009). On the other hand, Moseeva et al. (2010) found a relationship between the colonization of this bacterium in the oral cavity and the risk of dental caries development. In contrast, Olivier et al. (Olivier et al. 2006) and Chitsaz et al. (Chitsazi et al. 2006) failed to nd H. pylori in the dental plaque of patients with gastric infections. These 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. There 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, using RUT was higher than other tests. The lowest detection rate was when the microbial culture was used to detect the presence of this bacterium in dental plaque (Anand et al. 2014).
Several virulence factors have been identi ed 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 different strains. The cagA gene is located within a 40-kilobases DNA fragment known as the cag pathogenicity island (cag PAI). Approximately, more than 60% of H. pylori strains carry this gene. This gene is associated with increased production of interleukin 8, mucosal in ammation, and an increased risk of peptic ulcer (Palframan et al. 2012;Erzin et al. 2006). The vacA is a housekeeping gene that can induce vacuolation and apoptotic processes in epithelial cells, as well as immunosuppressive effects on some immunological cells (Palframan et al. 2012).
In this study, to investigate the hypothesis of oral to-stomach transmission in H. pylori infections, it is necessary to compare the RFLP patterns of several H. pylori genes between the mouth and stomach for the same case.
In our study, three genes of cagA, vacA, and ureAB were selected for these analyses. To our knowledge, no su cient research has been done on the detection of H. pylori in dental plaque and gastric biopsy specimens isolated from patients with dyspepsia in Ahvaz, Southwest of Iran. Hence, the present study aimed to determine the genotype and investigate H. pylori in dental plaque and gastric biopsy specimens, as well as the evaluation of an association between the colonization of H. pylori in dental plaque specimens and gastric biopsy.

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 antral, within 5 cm of the pylorus. The 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 non-steroidal anti-in ammatory drugs, periodontal therapy within the past year, and signs of severe periodontal infections within the preceding 6 months (Liu et al. 2009).
We retained one gastric biopsy specimen for RUT and the other was stored at -70℃ for DNA extraction.
For performing RUT, the samples were inoculated in the urea broth medium (Sigma Co.). The needed time for a positive test depends on the concentration of H. pylori and temperature. Although most RUT will be change positive within 120-180 minutes, for the negative tests and increased sensitivity, it is best to read the test at 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 the endoscopic examination. To remove the remaining foods, each patient was requested to wash his/her mouth with normal saline before sampling. Dental oss was used to completely clean the interdental spaces. The samples were transported in the tubes containing digestion buffer [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 (Momtaz et al. 2012). One of these samples was used for screening H. pylori by RUT and the other was stored at -70℃ for DNA extraction and PCR.

DNA extraction
The gastric biopsy and dental plaque samples were crushed and mixed by a vortex. Then, the mixtures were centrifuged at 12000 x g for 3 min. A volume of 200 µl of suspended pellets in normal saline was transferred to a 1.5 ml microtube. The 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. The concentration of the extracted DNA was measured at 260 nm, using a Nanodrop instrument (Thermo Scienti c, 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.
Identi cation of H. pylori using PCR Ampli cation of ureAB (encoding urease enzyme that is conserved in all H. pylori isolates) and 16S rRNA genes were performed for identi cation of H. pylori (Rasmussen et al. 2012;Han et al. 1998). The sequence of primers used is shown in Table 1. For each gene, a uniplex PCR reaction was prepared in a volume of 25 µL. The master mixture consisted of 0.02 Units of Taq DNA polymerase, 1.5 mM MgCl 2 , 0.4 µM of each primer, 1x PCR buffer, 25 ng DNA template, and distilled water up to a nal 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
The vacA, cagA, and ureAB genes fragments were ampli ed 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 buffers suggested by the supplier (Fermentas, US). The digested products of each gene target were electrophoresed on 2% agarose gel stained with ethidium bromide (Han et al. 1998).

Statistical analysis
The descriptive statistics and chi-square test were performed in SPSS version 16.00 and a signi cance 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, 88 cases were men and 18 cases were women. The patient's mean age was 33.9 ± 2.07 years. The patients were classi ed into four main groups based on clinical diagnosis including 58 cases with gastritis (54.7%), 35 cases with peptic ulcer (33%), 11 cases with duodenum ulcer (10.4%), and 2 cases of asymptomatic.
In this study, H. pylori was recognized in the stomach of 52.8% (56/106) and the dental plaques of 17 Table 2 The enzymatic RFLP patterns for vacA and cagA genes On the other hand, due to the enzymatic digestion of the ureAB gene ( Figure 2c) by HaeIII, four RFLP patterns of 3, 4, 5, and 6 bands were visually recognized. The 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 stomach and dental plaque specimens for the same case, 14 and 11 unique patterns were seen, respectively. Table 3 The RFLP pattern of ureAB, cagA, and vacA genes on stomach specimens Table 4 The combination of RFLP patterns of ureAB, cagA, and vacA genes on stomach and dental plaque specimens However, for all cases with positive H. pylori diagnosis, a combined analysis of the RFLP patterns of these three genes was different 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, because the comparison of ngerprint patterns due to enzymatic digestion three genes (cagA, vacA, and ureAB) showed that these patterns were different.  PCR, or some differences in the study population. In general, there is less H. pylori colonization in the oral cavity than in the stomach. The low rate can be explained by the two reasons: 1) suppressor effects 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 (Moseeva et al. 2010).
In our study, the frequency of the CagA gene in the dental plaque and gastric biopsy specimens with H. pylori-positive results were 52.6% and 63%, respectively. The prevalence of cagA-positive H. pylori strains was different from one geographic region to another, including 94% in Malaysia ( In this study, we found 14 and 11 unique patterns of RFLP in the stomach and dental plaque samples, respectively. In addition, to nd a correlation between the colonization with H. pylori in the dental plaque and the 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 analyses, the different RFLP patterns were seen for the same case. Because for each similar case, a combination of these RFLP patterns was not present in both samples (dental plaque and gastric biopsy), we concluded that there are no correlations between H. pylori colonization in dental plaque and stomach. In other words, in our study, the dental plaque was not considered as a likely reservoir for re-infection of the gastric with H. pylori.
However, in contrast to our study, Oshowo et al. (1998) demonstrated that the RFLP patterns of dental plaque and stomach samples of 13 out of 15 patients were identical for the same case. With regard that, these researchers used only one restriction endonuclease, it is di cult to prove a correlation between H. pylori colonization in dental plaque and the stomach for the same case.
One of the main limitations of our study was the lack of isolation of H. pylori by bacterial culture. Therefore, we could not determine the genotypes of the vacA and cagA genes.

Conclusion
We found a high prevalence of H. pylori in the dental plaque and gastric specimens. RUT was a sensitive method as same as PCR for the detection of H. pylori. According to the RFLP analyses on the dental plaque and gastric specimens, we showed that there was no signi cant correlation between the colonization of H. pylori in the dental plaque and stomach. However, because insu cient of sample size, our results could not con rm the dental plaque as a likely reservoir of H. pylori infection in the human stomach.

Study Limitation
In Fig. 1 and Fig. 2a