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On-line version ISSN 1678-9199
J. Venom. Anim. Toxins incl. Trop. Dis vol.18 no.2 Botucatu 2012
Rasmussen LTI; de Labio RWII; Neto ACII; Silva LCIII; Queiroz VFIV; Smith MACV; Payão SLMI,II
IPostgraduate Program in Oral Biology, Sacred Heart University, Bauru, São Paulo State, Brazil
IIDepartment of Genetics, FAMEMA Blood Center, Marília Medical School (FAMEMA), Marília, São Paulo State, Brazil
IIIDepartment of Anatomic Pathology, Marília Medical School (FAMEMA), Marília, São Paulo State, Brazil
IVDepartment of Digestive System Surgery, Marília Medical School (FAMEMA), Marília, São Paulo State, Brazil
VDepartment of Morphology, Federal University of São Paulo (UNIFESP), São Paulo, São Paulo State, Brazil
Helicobacter pylori, a gram-negative bacterium, possesses two important virulence factors: the vacuolating toxin (vacA), and the cytotoxin-associated gene product (cagA). The aim of the present study was to evaluate the presence of H. pylori in the stomach and oral cavity of humans and compare the cagA and vacA genotypes of H. pylori found in different samples (stomach, saliva and dental plaque) from the same patient. Gastric biopsies, saliva and dental plaques were obtained from 62 dyspeptic adults. DNA was extracted and evaluated for the presence of H. pylori and the alleles cagA and vacA. Persons with gastritis had a higher frequency of H. pylori -positive samples in the stomach while positive samples from gastric biopsies were significantly correlated with those from the oral cavity. There was a high H. pylori frequency in patients while the cagA gene was associated with vacA s1 alleles in gastric biopsies. Our results suggest a reservoir of the species in the oral cavity and that, in one patient, more than one H. pylori strain may exist in the saliva, dental plaque and stomach. We found a relationship between gastric infection and the bacterium in the oral cavity, with the cytotoxin genotype varying between saliva and dental plaque.
Key words: Helicobacter pylori, cagA, vacA, dental plaque, dyspeptic patient, saliva.
Helicobacter pylori is a spiral-shaped gramnegative flagellate bacterium that has been implicated as a major human gastric pathogen responsible for gastritis and peptic ulcer disease (1). It is estimated that in developing countries like Brazil, the prevalence of H. pylori infection approaches 80% in adults versus 30 to 70% in developed countries (2-7).
The high prevalence of the disease has propelled an extensive search for H. pylori virulence factors. This research has led to the characterization of a vacuolating cytotoxin A (vacA) and an associated cytotoxin A (cagA). The vacA gene is present in all H. pylori strains, and comprises two main regions that show significant sequence variability between strains: the signal region (s1 or s2) and the middle region (m1 or m2). These two parts of vacA gene determine cytotoxin production and are associated with pathogenicity of the bacterium. The vacA s1/m1 allelic combination exhibits the highest activity, while s2/m2 and the rare s2/m1 combinations may be less toxic (8). The vacA protein induces vacuolation and apoptotic processes in epithelial cells, as well as immunosuppressive effects in immunological cells (9).
The cagA gene, present in approximately 60 to 70% of H. pylori strains, is located within a 40-kilobase DNA fragment known as the cag pathogenicity island (cag PAI). Several epidemiological studies have revealed that the presence of cagA-positive strains is correlated with a higher risk of developing peptic ulceration, gastric atrophy and gastric cancer (10). In particular, most strains of H. pylori carrying the vacA alleles s1/m1 and cagA have been isolated from patients with severe gastric diseases, including duodenal and gastric ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma (11-13).
In addition to the genetic aspect of H. pylori, Nahar et al. (14) suggested that low socioeconomic status, crowded living condition, unfavorable dietary habits and poor personal hygiene may be associated with H. pylori infection. Although many aspects of the epidemiology of H. pylori infection are known, the modes of acquisition and transmission remain unclear; however, fecaloral, oral-oral, and gastro-oral routes have been most often considered (15).
Some authors have reported the presence of H. pylori in the oral cavity, potentially representing a source of H. pylori for gastric reinfection after therapy (16). This possibility was supported by Miyabayashi et al. (17) who confirmed the relationship between gastritis induced by H. pylori infection and oral colonization of the bacterium, besides trying to clarify the resistance mechanisms of oral H. pylori to typically triple anti-H. pylori therapy employed to eradicate the pathogen from the stomach. They reported that patients with oral H. pylori presented a significantly elevated gastric reinfection risk following successful therapy.
Rasmussen et al. (18) suggested a relationship between gastric infection and the presence of this bacterium in the oral cavity. Despite this, H. pylori was present in the oral cavity with variable distribution between saliva and dental plaques, suggesting the existence of a reservoir for the species and a potential association with gastric reinfection.
Dental plaque and saliva constitute a potential reservoir for infectious microorganisms and have been shown to harbor at least 400 different types of bacteria, including H. pylori (19). However, the detection and comparison of the presence of the cagA gene and vacA alelles of H. pylori in dental plaque, saliva and the stomach has not been thoroughly evaluated.
The purpose of this study was evaluate the presence of H. pylori in the stomach and oral cavity of patients with gastritis and to compare cagA and vacA genotypes of H. pylori among dental plaque, saliva and the stomach in the same patients, in an attempt to elucidate the infection mechanisms of H. pylori and verify its possible transmission route.
MATERIALS AND METHODS
Patients and Collection of Biological Samples
Sixty-two patients (27 males and 35 females with a mean age 52.56 years) presenting with recurrent abdominal pain participated in the study. All subjects were recruited from the Ambulatory Endoscopy Unit of Marília Medical School, São Paulo, Brazil.
All individuals signed an informed consent in order to be included in the study and the local ethics committee approved the study.
Three biopsies of the gastric antrum were collected during gastroscopy. One of the specimens was used for the rapid urease test, the second for histological examination and the other for the molecular analysis. The endoscopic forceps were sterilized in 2% glutaraldehyde solution for a minimum of 20 minutes between each pair of experimental procedures. Saliva and plaque samples were collected prior to the endoscopic examination of each subject. Salivary flow was selfstimulated by each patient and 3 mL of saliva was collected in test tubes. Multiple samples of dental plaque from different sites throughout the oral cavity (incisors, canines, premolars and molars) were taken from each subject with a sterile curette and transferred to a test tube containing 15 mL of PBS (phosphate-buffered saline) pH 7.4.
It is worth mentioning that our group recently published a study suggesting a possible relationship between the presence of H. pylori in the oral cavity and the stomach (18). The authors would like to emphasize that the present study is a more detailed analysis than the previous one and aims to elucidate the genetic diversity of H. pylori in humans using the same samples.
DNA Isolation and Preparation of DNA Probe
DNA from the gastric biopsies was extracted using the QIAamp® tissue kit (Qiagen, Germany), according to the manufacturer's instructions. DNA extraction from the dental plaques and saliva were performed by the method of Rasmussen et al. (18) as previously described (16, 20). The probe was synthesized using the Gene Images AlkPhos® Direct Labelling kit (GE Healthcare, UK), according to the manufacturer's instructions and the description of Rasmussen et al. (18).
Identification of Helicobacter pylori by Urease Test, Histological and Molecular Analysis
An antral biopsy from each patient was incubated in premade broth (TUPF; Laborclin, Brazil) for the urease test (RUT) immediately after collection. The test was considered H. pyloripositive when the color of the solution changed from yellow to orange, pink, or purple within four hours of incubation at 25ºC.
The biopsies for the histological examination were fixed in formalin and stained with HE (hematoxylin and eosin) and Giemsa. The histological parameters were graded using the criteria described in the Sydney system for analysis of chronic inflammation, polymorphonuclear activity and intestinal metaplasia (21).
PCR assays were performed with approximately 100 ng of total DNA using one set of oligonucleotides (Hpx1/Hpx2) (5'-CTGGAGARACTAAGYCCTCC-3' and 5'-GAGGAATACTCATTGCGAAGGCGA-3') that amplifies a 150-bp fragment corresponding to 16S-rRNA from H. pylori. The cycling program was initiated with a denaturation step of 94ºC for five minutes followed by 40 cycles at 94ºC for one minute, 59ºC for one minute, 72ºC for one minute, followed by a final incubation at 72ºC for seven minutes (22). In each experiment, positive (strain 26695) and negative (water) controls were included. After separation with electrophoresis in 2% agarose gels, PCR products were blotted to a Hybond N+ membrane and hybridized with the specific PCR fragments labeled by chemiluminescence (GE Healthcare, UK). The assay was considered positive when the PCR product was present.
Detection of vacA and cagA Gene
The vacA gene, "s" and "m" region genotyping and cagA gene detection were performed by PCR and Southern blotting, using one set of oligonucleotides for each gene fragment. For cagA detection, the previously described primers Cag1/ Cag2 (5'-ATGACTAACGAAACTATTGATC-3' and 5'-CAGGATTTTTGATCGCTTTATT-3') amplified 232 fragments (23). The vacA "s" and "m" regions were genotyped with the previously described primer sets SA/SC (5'-ATGGAAATACAACAAACACAC-3' and 5'-CCTGARACCGTTCCTACAGC-3'), and MA/ MB (5'-CACAGCCACTTTYAATAACGA-3' and 5'-CGTCAAAATAATTCCAAGGG-3'), respectively (23-25). The SA/SC primers amplified "s1" fragments of 176 bp and "s2" fragments of 203 bp. The "m1" fragments were 400 bp and "m2" fragments 475 bp (23-25). The same amplification condition was used for every gene. The cycling program was initiated with a denaturation step of five minutes at 94ºC, followed by 40 cycles of 94ºC for one minute, 53ºC for one minute, 72ºC for one minute, followed by a final incubation at 72ºC for seven minutes.
The chi-square, Fisher's exact and Kappa tests were used to analyze differences in the prevalence and cytotoxin genotypes of H. pylori in gastric biopsies, saliva and dental plaque samples. The significance level was set at p < 0.05.
Statistical analyses were performed using the statistical package SPSS 11.5.1 (USA).
Detection of Helicobacter pylori in Gastric Biopsies, Saliva and Dental Plaque by Urease, Histological and Molecular Analysis
H. pylori was detected in antral biopsies in 50/62 (80.6%) patients via PCR and hybridization by Southern blotting. The histological analysis revealed the presence of H. pylori in only 19 subjects (30.6%) whereas the urease test detected H. pylori infection in 27 patients (43.5%). All the samples in which the histology and urease test demonstrated the presence of H. pylori were also positive by PCR and Southern blot hybridization. The detection of H. pylori in gastric antrum samples was significantly higher when the Southern blotting technique was used as compared to the histological or urease tests (chi-square - p = 0.0001).
The histology of 45 (72.6%) patients revealed chronic gastritis while 17 (27.4%) had normal gastric mucosa. Of the former, 41 (91%) were found H. pylori-positive by PCR and Southern blot hybridization.
H. pylori was found in the saliva of 26 (41.9%) and in the dental plaque of 29 (46.7%) patients (Table 1). The oral cavity was operationally defined as positive when the saliva or dental plaque was positive for H. pylori. Sixteen patients presented with H. pylori confined to the stomach, while H. pylori DNA was concurrently detected in the gastric biopsy, saliva and dental plaques of ten patients. Twenty-four subjects harbored H. pylori in gastric biopsies and the oral cavity. H. pylori DNA was not detected in 12 gastric biopsy samples, but six of these 12 cases were found to have H. pylori in the oral cavity.
No statistically significant difference was observed between strains in the saliva and dental plaque (chi-square = p < 0.58). Despite this, a statistically significant difference was observed between gastric biopsies and the oral cavity (chisquare = p < 0.0001).
Detection of cagA and vacA Alleles of Helicobacter pylori Isolates from Gastric Biopsies, Saliva and Dental Plaque
We isolated 25 strains of the common vacA genotype s1/m1 and only 12 strains of vacA s2/ m2. Twenty percent (ten strains) of the H. pylori bacterium were found to have vacA genotype s1/m2, and three strains showed vacA genotype s1/m1/m2, suggesting a coinfection with two different H. pylori strains. The cagA gene was detected in 36 patients infected with H. pylori. Of these 36, 32 were associated with the toxinproducing vacA s1 and can be associated with chronic gastritis while only four cagA-positive strains were vacA s2 (p < 0.0001).
The cagA gene was detected in 13 saliva samples. Of the 13 patients who were cagA positive, seven were associated with vacA s1, two were found to be vacA s2 and four were s1/s2. The middle region (m1 or m2) of the vacA gene was genotyped only in H. pylori gastric isolates. The middle region of vacA was not detected in H. pylori isolates from these samples, which is likely related to the heterogeneity in the vacA gene as previously described (8, 10).
From patients with positive dental plaque, only seven of 29 strains had the cagA gene and three were associated with the toxin-producing vacA s1. However, 11 of the patients with cagA-negative samples demonstrated the vacA genotypes s1.
When associated with positivity for H. pylori, the presence of cagA and vacA genes and their alleles, there was approximately 85% disagreement among the three materials, just five patients showed the same genetic profile, according to the genes analyzed, in the oral cavity and biopsies, highlighting a wide variety of strains, a mixed colonization in the same host with an unequal distribution, independent of gastric biopsy, saliva or dental plaque. No statistically significant relationship was found between the presence of cagA and vacA alelles in saliva or dental plaques. However, there was a statistically significant relationship between the presence of cagA and vacA genotypes s1/m1 (Fisher = p < 0.001). The association between the presence of cagA and vacA alleles in all strains is described in Table 1.
Our methodologies of using PCR in concordance with Southern blotting are supported by Li et al. (26) and Song et al. (27) who found a significant increase in sensitivity after incorporating the Southern blotting technique. The high sensitivity and specificity of the PCR test with Southern blotting and hybridization in our study did not reveal false positives, false negatives or contamination. This is attributed to the fact that the probe was synthesized from genomic DNA of H. pylori culture by PCR, which excludes false results (28, 29). In addition, we used more amplification cycles, optimized buffers and conditions while all H. pylori-positive samples were genotyped to confirm the presence of H. pylori.
Out of 62 samples, H. pylori was detected in 50 of the gastric biopsies in which the most virulent vacA genotype s1/m1 was most common. The cagA gene was detected in 72% of our patients, a level comparable to two other studies of Brazilian patients where, respectively, 70% and 79% of samples were found to be cagA-positive (3, 30). Several studies have described the associations between s2/m2 and cagA-negative strains and between s1/m1 and cagA-positive strains, both supported by the results of the present study.
Twenty-three and 31 biopsies that were positive as determined by the Southern blotting method were negative when analyzed with the RUT and histological analysis, respectively. This is expected because the sensitivity of the Southern blotting technique is much greater for the detection of H. pylori when compared to the RUT and histological analyses. In addition to reduced sensitivity, the histological analysis may be biased by the subjectivity of the assessment, inter-observer variation or human error (21). Researchers and clinicians should be aware of these potentially serious limitations when using histological analysis. The observation of de Francesco et al. (31) of H. pylori in 20% of histology-negative biopsies by PCR is similar to our findings. This increased the prevalence of the pathogen from 43 to 58% in their patient samples.
In the oral cavity, H. pylori had been demonstrated earlier, although the exact time course of the infection is not fully elucidated. It is not clear whether the oral cavity is a permanent or a temporary reservoir for this microorganism. In our study 26 (41.9%) and 29 (46.7%) of the patients had H. pylori in saliva and dental plaque, respectively.
Li et al. (26) verified 75% positivity among saliva specimens, similar to the findings of Wang et al. (32), who found the bacterium in 71% of the saliva samples in their study. Al Asqah et al. (33) also found a significant association between the presence of H. pylori in the stomach and in dental plaque. In a study of 120 dyspeptic patients, H. pylori was detected in all of the samples from gastric biopsies and saliva (34). The results of the present study, along with the previous work, suggest that saliva may serve as a method of infection transmission and may also lead to gastric re-infection after treatment of the disease.
Some studies have suggested that the detection of H. pylori in saliva and dental plaque by PCR may reflect the secretion of dead bacteria from the digestive tract. However, the fact that six patients in the current study produced gastric biopsies negative for H. pylori by PCR and Southern blotting while testing positive for H. pylori in their oral cavity corroborates the hypothesis that the mouth may be a natural reservoir of H. pylori.
Berroteran et al. (35) also reported that H. pylori was detected in the oral cavity of five patients whose gastric biopsies were negative for H. pylori by PCR whereas H. pylori was also present in 15% of (three of 20) asymptomatic subjects. Li et al. (36) suggest that this phenomenon may occur because H. pylori may persist in low numbers in the oral cavity for many months or years without colonizing the stomach or because the oral cavity is the initial site of infection.
The vacA s1 alelle was found in 53% of the H. pylori isolates from saliva and 48% of those from dental plaque, while the cagA gene was detected in 13 (50%) saliva samples and only 7 (24.1%) dental plaque samples. Unlike the gastric biopsy samples, no association was found between vacA s1 alleles and the presence of cagA gene in saliva or dental plaque samples. We detected four saliva and seven dental plaque samples with vacA alelles s1 and s2 simultaneously, suggesting the presence of multiple H. pylori strains in the oral cavity of the same subject. Wang et al. (32) found that the cagA gene was present in 23% of the saliva samples whereas the vacA s1 allele was present in 77% of the saliva samples. We found the cagA gene was highly prevalent in the saliva samples from our subject pool, but the prevalence of the gene was only 23% in the dental plaques. In all, approximately 70% of the oral-cavity samples were positive; however, there was a distinct difference between such positivity in saliva and dental plaques.
We also found significant genotypic diversity among H. pylori cytotoxins from the stomach, saliva and dental plaque samples, which corroborates the work of Wang et al. (32). The heterogeneity of H. pylori may be due to genotypic variation among strains and or variations in H. pylori populations within an individual host, as proposed by Blaser (37).
In the present study we verified that the strains found in the stomach are apparently more virulent than those in the oral cavity, an observation not explored in the literature. Thus, we can suggest that this selection is attributable to two explanations: H. pylori cagA + and "s1" have efficient adaptive mechanisms, which allow a rapid and effective colonization of the stomach, thus inhibiting the growth of less adapted organisms and suggesting a possible biological interaction called amensalism among different strains of H. pylori. The second explanation may be that the oral cavity can harbor at least 400 different types of bacteria and, given such bacterial heterogeneity, the adaptive mechanisms might not be efficient, thus allowing the growth of other H. pylori strains.
The frequency of H. pylori in the oral cavity differed in relation to previous studies. The difference in the presence of the pathogen in the oral cavity may be the result of differences in study populations, oral health status, clinical presence of H. pylori infection, the type and number of clinical samples, complexity of the oral flora or detection methods (38). Kignel et al. (16) reported that the levels of the bacterium in the oral cavity may be too low to be detected by one round of PCR and they emphasized that the region of the mouth from which the samples are collected can influence the prevalence of the microorganism. Song et al. (27) determined that the presence of H. pylori was 82% in the molar region, 64% in pre-molar region and 59% in the incisor region. Loster et al. (39), suggested that this discrepancy may be attributable to the fact that the dental plaque in the posterior region of the oral cavity is less oxygenated, thus providing optimal conditions for the survival of H. pylori.
In summary, there is a high prevalence of H. pylori and the main virulence factor genes in Brazilian H. pylori isolates, namely the cagA gene, appears to be associated with vacA s1 alleles in gastric biopsies. Our results suggest a correlation between gastric infection and the presence of the bacterium in the oral cavity. However, H. pylori was present in the oral cavity with variable cytotoxin genotype cagA and vacA alleles; furthermore, there was a variable distribution between saliva and dental plaque, suggesting the existence of a reservoir of the species, which can lead to gastric re-infection. Additionally, more than one H. pylori strain may exist in the saliva, dental plaque and stomach of the same patient.
The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES), Sacred Heart University (USC), Marília Medical School (FAMEMA) and The State of São Paulo Research Foundation (FAPESP) for their support.
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Spencer Luiz Marques Payão
Laboratório de Genética, Hemocentro, FAMEMA
Rua Lourival Freire, 240, Bairro Fragata
CEP 17519-050, Marília, SP, Brasil
Phone: +55 14 34021856. Fax: +55 14 34330148
Received: October 4, 2011.
Accepted: November 30, 2011.
Abstract published online: January 18, 2012.
Full paper published online: May 31, 2012.
CONFLICTS OF INTEREST: The authors declare no conflicts of interest.
FINANCIAL SOURCE: The State of São Paulo Research Foundation (FAPESP) provided the financial grants (process n. 06/60836-1). ETHICS COMMITTEE APPROVAL: The present study was approved by the Ethics Research Committee of Sacred Heart University (process n. 056/2005). Moreover, all study subjects signed an informed consent in order to be included in the study.