Prevalence of Helicobacer pylori in saliva and dental plaque related to periodontal disease and gastritis

1 Facultad de Odontología, Universiad Autónoma De Nuevo León, Monterrey, México. 2 Centro de Investigaciones y Desarrollo en Ciencias de la Salud, Universidad Autónoma de Nuevo Leon, Monterrey, México. 3 División de Ciencias de la Salud, Universidad de Monterrey, Monterrey, México. 4 Departamento de Biología Celular, Facultad de Biología Universidad Autónoma de Nuevo León, Monterrey, México. 5 University of Texas Health Science Center at Houston, Houston, USA. 6 División de Biología Celular y Molecular, Centro de Investigaciones Biomédicas del Noreste, Instituto Mexicano del Seguro Social. Monterrey, México.


INTRODUCTION
Helicobacter pylori is now recognized as a major etiologic factor in the development of chronic superficial gastritis and peptic ulcer disease in adults and children (NIH Consensus Conference, 1994).In 1994, H. pylori was classified as a group 1 carcinogen by the International Agency for Research on Cancer because of its association with gastric cancer (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 1994).H. pylori acquisition occurs predominantly during early childhood, and its incidence and prevalence is higher in developing countries (Thomas et al., 1999;Mitchell and Megraud, 2002;Malaty et al., 2002).Several risk factors have been associated with acquisition and transmission of H. pylori infection and they are mainly correlated with poor sanitary conditions and low socioeconomic status (Malaty et al., 2002;Chong et al., 2003;Malaty et al., 1996a).
In some studies, the prevalence rate of H. pylori infection was 49.7% in healthy children and it was 88% in adults (Muhsen et al., 2006;Malaty et al., 1996b).The oral cavity has been proposed as a reservoir for H. pylori (Watts, 2002), however, some authors have failed to isolate this bacterium in the oral cavity (De Sousa et al., 2006;Olivier et al., 2006).Also, researchers have isolated H. pylori in samples from the oral cavity without any gastrointestinal disease association (Chitsazi et al., 2006).This has created controversy about the reservoir role of the oral cavity in the progression of gastrointestinal disease.
Polymerase chain reaction (PCR) has been used to isolate H. pylori in previous studies (Olivier et al., 2006;Chitsazi et al., 2006;Umeda et al., 2003).The prevalence of H. pylori isolation using PCR was 42 and 35.1% in patients with and without periodontal disease, respectively (Umeda et al., 2003).The aim of this study was to determine the prevalence of H. pylori in the saliva and dental plaque of adult patients with periodontal disease, and determine its association with gastrointestinal symptoms of gastritis.

Subject population
A total of 60 clinical samples were obtained from 30 untreated patients.Patients were over 35 years old and were recruited from the Clinic of Periodontics, Faculty of Dentistry at the Autonomic University from Nuevo Leon, Mexico.Salivary and dental plaque samples were obtained and analyzed from each patient.Informed consent was obtained from each individual.Clinical measurements and sample collection.Periodontal disease was determined by clinical and radiographic examination.We measured the level of attachment loss and probed the pocket depth in all teeth.If the pocket depth was more than 4 mm with a conventional North Caroline periodontal probe (Hu-Friedy Chicago, IL, USA) in a least six sites, the patient was considered to have periodontal disease.
Patients that had used systemic antibiotics in the previous six months or had any systemic disease were excluded.Information about gastrointestinal symptoms was obtained from patients by direct interview.All oral specimens were inoculated onto brain heart infusion agar plates (Merck, Darmstadt, Germany) supplemented with 5% sheep blood and cultured in anaerobic conditions using Gaspack ® (Becton Dickinson Franklin Lakes, NJ, USA).All patients with symptoms of gastritis underwent upper endoscopy to confirm the diagnosis and underwent a rapid urease test for the presence of H. pylori in the gastric chamber.The presumptive positive culture isolates of H. pylori were identified in vitro by urease and catalase activities.The control strain was H. pylori ATCC 43504, 43649, which was donated by Dr. Stanley Holt.

DNA extraction
Suspensions of the bacterial strain were prepared in 100 μl of sterile double-distilled water and they were obtained by the inocula-tion of a single bacterial colony from the infusion of agar plates with a standard loop.A loop full of cells was transferred to water and the mixture was boiled for 10 min at 94°C to lyse the cells.The resulting cell lysate was centrifuged briefly (30 s at 10,000 rpm; Eppendorf model 5415C centrifuge (Eppendorf, Hamburg, Germany) and 19 l of the supernatant was used as the DNA sample for the PCR mixture.

Oligonucleotide PCR primer design
Suitable primers were designed using 16s rRNA gene sequences of H. pylori available in the GenBank database.Multiple comparisons were made with related organisms using the FASTA and Clustall Wallis programs.Oligonucleotides were synthesized using a synthesizer of DNA (Microsyn 1450A; Systec Inc.) with the reagents and conditions specified by the manufacturer.We designed two primers; the first was named LH-F (5´-TAGATTATGTGCCTCTTAGTT-3´; position 123 to 146, GenBank accession AE000511:0) and the next was named LH-R (5´-AGGAGGTGATCCAACCGC-3´; position 2431 to 2446 GenBank accession AE000511:0).A Blast search was performed to check the specificity of the primer sequences.To test the sensitivity, we used dilutions of the DNA extracted from ATCC43504, 43629 H. pylori strains.The specificity of the primers and PCR conditions were evaluated by testing ATCC43504, 43629 H. pylori strains as positive controls, as well as related bucal flora Porphyromonas gingivalis ATCC 33277T, Streptococcus intermedius ATCC 27335T, Prevotella intermedia ATCC 25611T, and more distantly Escherichia coli, Proteus vulgaris, Bacillus thuringiensis and Bacillus subtilis as negative bacteria.

PCR amplification
The template DNA was added to 50 μl of a reaction mixture containing 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.3], 1.5% [vol/vol] Triton X-100), 1.5 mM MgCl2, 200 μM concentrations of each deoxynucleoside triphosphate and 50 pmol of each primer.Amplification was performed in a Perkin-Elmer Thermocycler 2400 (Perkin-Elmer, Applied Biosystems, and Foster City, Calif.)As the first step of a denaturing process begins with 5 min exposure to Master Amp Taq PCR core kit, (Epicentre® Biotechnologies, Madison, Wisconsin, USA), were subsequently added.Then, 30 cycles of the next cycle program were performed: denaturation at 94°C for 1 min, annealing at 36°C for 1 min, extension at 70°C for 1 min, and a final extension step at 70°C for 10 min.The PCR products were analyzed with 1.5% (wt/vol) agarose gels in Tris acetic acid-EDTA pH8 buffer (TAE) and the sizes of the amplicons were estimated by comparison with 100-bp DNA size markers (Promega Corporation, Madison, Wisconsin, USA).

RESULTS
We analyzed the samples from 30 patients (17 males [56.7%] and 13 females [43.3%], mean age 50.1+ 11.03 years [ Table 1]).Sixty samples were obtained; that is, two from each patient.One sample was dental plaque and the other was a saliva sample.None of the 30 Salivary samples were positive for H. pylori in the PCR analysis, while 4 of the dental plaque samples (13.3%) were positive.All patients with positive PCR isolation of H. pylori in the dental plaque had gastritis by endoscopy and 75% had a urease rapid test in the gastric biopsy (Table 1).Of all the analyzed patients, 66.7% were asymptomatic and 33.3% showed gastrointestinal symptoms confirmed by endoscopy as gastritis.Both groups that is, asymptomatic and symptomatic patients, were compared.None of the 17 asymptomatic patients (without gastritis) were positive for the presence of H. pylori in the PCR dental plaque test, while 4/13 (30.7%) of the symptomatic gastritis patients showed PCR positivity (Table 1).A Fischer exact test showed an association between the PCR positivity for H. pylori and symptomatic patients (p = 0.02).Another important finding was related to the depth of the pockets.4.7% of the samples from patients with 4-5 mm pockets were positive for H. pylori compared to 33.3% of the samples from patients with 6-7 mm pockets (p < 0.02; Table 2).When determining the association of the isolates by PCR according to age and gender, no significant differences were found using a Fisher's exact test.Subsequently, by grouping patients with and without gastritis, the proportion of males was 61.5 and 52.9%, respectively.In the group of patients with gastritis, all patients with isolates were male 4/8 (50%), but a Fisher's exact test showed this was not significant (p=0.07).The mean age of patients with gastritis was 49.46 + 10.3 y and the mean age for asymptomatic patients was 50.58 + 11.5 y (p=0.51).The mean age of patients with H. pylori isolation was 49.75 + 7.8 y and it was 50.15 + 11.5 y for the patients without H. pylori (p=0.41).

DISCUSSION
As reported in previous studies (De Flora and Bonanni, 2011;Dai et al., 2011), H. pylori is related to gastrointestinal disease that is, mainly to gastritis and gastric cancer (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 1994).We performed this study to associate H. pylori in dental plaque with gastritis because some researchers have suggested that plaque may act as a reservoir for the bacteria (Watts, 2002).Also, there is controversy over the possibility that infected people could develop gastric infection and gastrointestinal symptoms (Navabi et al., 2011).We found a lower percentage (30%) of H. pylori in symptomatic gastritis patients than reported in previous studies using PCR isolation (Umeda et al., 2003).This may be due to PCR technique variations.Umeda et al. (2003) performd nested PCR, while we used standard PCR.We found important differences compared to previous studies because none of the Salivary samples were positive for H. pylori (Sugimoto et.al., 2009;De Sousa et al., 2006;Olivier et al., 2006;Chitsazi et al., 2006;Umeda et al., 2003;Tiwari et al., 2005).This may be due to a lower concentration of the bacteria in salivary samples.Some authors have suggested that other oral bacteria may cause false positive results.We feel that our PCR technique should be improved in order to detect lower counts of bacteria in salivary samples.
No asymptomatic patient showed the presence of H. pylori in plaque or salivary samples, which is in contrast with the findings of previous studies (Fernández-Tilapa et.al., 2011).We think that these people are really free of H. pylori, however, another possibility is that the prevalence of H. pylori in this population is low.By increasing the sample size, we might be able to find individuals with positive isolations.Finally, we consider the possibility that bacterial counts were actually low and, thus, the PCR technique should be improved to detect low counts of bacteria in plaque samples.
When gender associations were analyzed, we found that males were the major proportion of cases with H. pylori isolation compared to females.In other studies, the proportion of males was the same as females for isolation in the oral cavity and females were the majority showing isolation in the gastric cavity (Berroteran et al., 2002).A strong association between H. pylori isolation and the presence of gastric symptoms was demonstrated in our study.We found that more than 30% of the patients with gastritis have H. pylori in their dental plaque.H. pylori isolation was dependent on the depth of the pocket and this was dependent on the severity of the periodontal disease.The greater the depth of the pocket, the greater the likelihood of H. pylori isolation by PCR.We demonstrated that 75% of our patients with H. pylori positive isolation had periodontal disease, while only 3.3% of the patients without periodontitis showed H. pylori isolation by PCR.Patients without gastrointestinal symptoms did not show a positive PCR result for isolation of H. pylori.We conclude that H. pylori isolation by PCR in patients with periodontal disease and gastritis was demonstrated.In our study, we were not able to detect H. pylori in Salivary samples by PCR.

Table 1 .
Patient symptoms and H. pylori isolation by PCR.

Table 2 .
Deep pocket depth and H. pylori isolation by PCR.