Abstract: Clinical outcome of Helicobacter pylori infection might be associated with specific virulence-associated bacterial genotypes. The distribution of different bacterial genotypes varies geographically. The aim of this study, is based on determining any associations between the Helicobacter pylori vacA research in causing gastritis and peptic ulcer, in Shahrekord, Iran. Gastric biopsies were collected from patients that were referred to endoscopy unit of Hajar hospital Shahrekord, Iran. H. pylori was detected in these biopsies by culture test. Then polymorphism of vacA gene was genotyped by Polymerase Chain Reaction (PCR). The vacA genotypes were significantly different among gastritis, peptic ulcer and gastric cancer patients. Also, the vacA s1 allele was more frequently identified than the vacA s2 allele among gastrointestinal patients. Furthermore, the severest genotype of vacA gene in these patients was s1/m2. The results of our study illustrated that s1/m2 genotypes were associated with peptic ulceration in patients that infected by H. pylori.

INTRODUCTION

The gram-negative bacterium Helicobacter pylori is a highly successful pathogen persistently colonizes the human stomach (Scott-Algood and Cover, 2006; Allen et al., 2005) and remains one of the most common infections of humans worldwide (E-Khayat et al., 2007). H. pylori infection occurs in approximately 50% of the world's population (Yucel et al., 2008; Anderson et al., 2002) and it's prevalence is estimated at around 25% in developed countries and at >80% in developing countries (Jafarzadeh et al., 2007; Lin et al., 2004).

In the absence of antibiotic therapy, H. pylori can persist in the human stomach for decades or for an entire lifetime (Scott-Algood and Cover, 2006). H. pylori induced gastric inflammation does not cause symptoms in most infected persons but is associated with an increased risk for development of duodenal and gastric ulcer diseases (Scott-Algood and Cover, 2006) and is an important risk factor for the development of gastric cancer and gastric lymphoma (Miciuleviciene et al., 2008). There is no doubt that the eradication of H. pylori infection leads to healing of duodenal ulceration and the risk of recurrence is greatly reduced (Tovey et al., 2006). Variation in the clinical outcome of H. pylori induced pathology is multifactorial, involving a complex interplay between the host immune responses, pathogen virulence factors and niche characteristics (Kamali-Sarvestani et al., 2006) several potential markers of pathogenicity have been described in H. pylori and some of them seem to be associated with more severe clinical outcomes of the infection (Miciuleviciene et al., 2008). Several genes have been identified that may play a role in the pathogenesis of H. pylori, such as CagA, vacA, iceA and babA (Kamali-Sarvestani et al., 2006).

There are 2 proteins of H. pylori recognized as most virulent that are crucial in the formation of lesions of gastric mucosa: vacA (vacuolating cytotoxin gene A) and CagA (cytotoxin-associated geneA). Both of them, take part in the colonization and modulation of inflammatory response and in the development of inflammatory changes, peptic ulcer and gastric carcinoma (Maciorkowska et al., 2007).

The cagA gene is part of the cag pathogenicity island, which is a region consisting of approximately 30 genes and this genes encode for an immunodominant protein that is injected via a type IV secretion system into epithelial cells and is capable of interaction with signal transduction pathways in epithelial cells leading to inhibition of apoptosis and a number of cellular changes (E-Khayat et al., 2007). The vacA is the important virulence factor of H. pylori (Roche et al., 2007). The vacA gene is highly polymorphic and consists of 2 possible signal regions s1 and s2 and 2 possible midregions m1 and m2. The production of the vacuolating cytotoxin is governed by the mosaic combination of s and m subtypes (E-Khayat et al., 2007). In this study we investigated H. pylori genotypes at molecular level in gastro-duodenal disease population within September 2007 to August 2008 in Shahrekord, Iran.

MATERIALS AND METHODS

Patient samples: H. pylori were isolated from gastric biopsy obtained after inform consents from patient of both sexes who had under gone gastrointestinal endoscopy. In total, 250 patients from Shahrekord, Iran who have been referred for upper gastrointestinal endoscopy to the endoscopy unit of Hajar hospital in Shahrekord were enrolled in this study. Gastric biopsies were obtained from antrum and corpus of the stomach of these patients and before long placed in urea medium in order to assay by Rapid Urease Testing (RUT). These biopsy specimens were used as the come on process.

Bacterial strains and culture: All H. pylori strains were grown on pH 6 trypticase brucella agar containing 5% sheep blood under microaerophilic conditions (5% O₂, 7% CO₂, 88% N₂) (Allen et al., 2000). After 5 days of culture on selective agar plates, the organisms were identified as H. pylori by Gram staining, colony morphology and positive oxidase, catalase and urease reactions.

DNA preparation: After culture, H. pylori colonies were pooled from the plates and washed by phosphate-buffered saline. H. pylori genomic DNA was prepared after bacterial cell lysis using SDS and proteinase K solution and phenol chloroform extraction (Sambrook and Russel, 2001).

Analysis of bacterial vacA genotypes: All primer sets used were selected from the published literature and were synthesized. The forward primer for signal sequence of vacA region was: VA2-F: 5'-CAATCTGTCCAATCAA GCGAG-3' and the reverse primer was: VA2-R: 5'-GCG TCTAAATAATTCCAAGG-3' The amplification of a 259 or 286 bp fragments were expected from genotype s1 or s2, respectively. The middle region of vacA gene was analyzed with forward primer as the sequence of VA1-F: 5'- ATGGAAATACAACAAACACAC-3' and Reverse primer as the sequence of VA1-R: 5'- CTGCTTGAATGC GCCAAAC-3', which amplified 570 bp fragments for m1 and 645 bp fragments for m2.

The PCR assay was performed in a 25 μL reaction mixture containing 100 ng of genomic DNA, 1 x PCR buffer (Promega, Madison WI), 2 mmol L^-1 MgCl2, 0.2 mmol L^-1 deoxynucleotide triphosphates, 1.5 unit of Taq polymerase (Promega) and 0.2 mmol L^-1 of each primers. Cycling profile for amplification of vacA was one starting cycle at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, 1 min in 55°C for s region and in 52°C for m region and 72°C for 1 min. The final extension at 72°C was performed for 5 min. PCR products were examined by 2% agarose gel electrophoresis and photographed using an ultraviolet reflection analyzer.

Statistical analysis: The association between genotypes and the clinical symptoms was analyzed by using the Fisher's exact. The difference between the patient group such as gastritis, ulceration and adenocasinoma with bacterial was calculated by independence sample test.

RESULTS

We have detected H. pylori positive biopsy samples according to culture test. Out of 250 samples were collected in this study 178 specimens (71.2%) were grown on selective agar. H. pylori colonies were identified through H. pylori identification. Then genomic DNA was extracted from colonies of the bacterium.

The vacA gene was identified in all of the H. pylori strains (Miciuleviciene et al., 2008) and the primers on the allele s1, s2 and m1, m2 used in this study enabled the differentiation and characteristics alleles of s region and m region in vacA genotype isolated from examined H. pylori strains (Fig. 1). The most frequently observed H. pylori vacA gene signal region allele was s1 allele. Allele s1 of the signal region from the examined gene was determined in 143 specimens out of 178 DNA isolates (80.64%), whereas allele s2 found in 35 specimens (20.27%). H. pylori vacA gene m1 or m2 alleles of mid region were found in whole analyzed DNA.

Fig. 1:	Lane 1: 259 bp fragment related to s1 amplification. Lane 2: 286 bp fragment related to s2 amplification. Lane 3: 570 bp fragment related to m1 amplification. Lane 4: 645 bp fragment related to m2 amplification. Lane 5: negative control

Table 1:	The association of clinical outcomes and molecular detection of vacA genotype

Among them m1 allele was shown in 129 specimens out of 178 samples (72.97%) and allele m2 in 49 specimens (27.02%) was detected. And the mid region type m1 was significantly more frequent in H. pylori strains with genotype s1 (p<0.01) and m2 alleles in most cases were detected in vacA s1 strains too. The vacA s1/m1 combination was found in 36 (20.27%) of typable strains; s1/m2 in 96 (54.05%) and s2/m2 was present in 39 (21.62%), although the combination of s2m1 is uncommon (E-Khayat et al., 2007) in this study it have been found in 7 specimens (4.05%). While estimating relationship between potentially virulent H. pylori strains and clinical outcomes, significant differences (p<0.01) were found between isolates from DU and CG patients. The vacA s1 allele without either the m1 or m2 allele was the most common genotype in patient with Peptic Ulcer Disease (PUD). Furthermore the vacA genotype in patient with gastric cancer was s1/m2. As it is shown in Table 1, 11.11% of the strains possess vacA s1/m1 genotype with NUD (Non Ulceration Disease) and 66.66% of this genotype was involved with PUD. Furthermore, 3.1 and 58.33% of s1/m2 strains was NUD and PUD, respectively. However s2 typing shows no significant correlation with PUD in this study. The relationship between the clinical symptoms and H. pylori vacA is shown in Table 1.

DISCUSSION

H. pylori infection is thought to play an etiologic role in several gastroduodenal diseases (Jafarzadeh et al., 2007). The present study, reports on the vacA genotypes of H. pylori that were identified in gastric biopsies.

The vacA gene was identified in all of the H. pylori strains (Miciuleviciene et al., 2008) and the clinical relevance of putative vacA genes of H. pylori is still a matter of controversy. This gene is considered to be an important virulence factor that contributes to epithelial cell injury and peptic ulceration in the host cells (Roche et al., 2007). Several studies, have demonstrated that the genotype varies among H. pylori strains isolated from different geographic regions and conclusions derived from one geographic region may not be true for others (Faundez et al., 2002).

Type s1 forms of vacA are active in many in vitro assays of toxin activity, whereas type s2 forms of vacA lack detectable activity in most in vitro assays (Allen et al., 2000; Tomasini et al., 2003). Infection with H. pylori strains containing type s1 forms of vacA is associated with an increased risk of gastric cancer or peptic ulcer disease compared to infection with strains containing type s2 forms of vacA (Tomasini et al., 2003; Cao and Cover, 2002) E-Khayat et al. (2007) reported, the s1 allele obtained from PUD patients expressed the vacA gene, while the transcription of the less virulent allele, s2 was detected in a few patients with PUD or gastritis who harbored this gene (E-Khayat et al., 2007). The vacA s2 allele was detected in <30% in the studied population from Europe, Central and South America and East Asia (Nimri et al., 2006). A study in Kuwait reported that vacA s1 and s2 types were detected in approximately equal numbers In biopsies obtained from patients (Al-Qabandi et al., 2005) and s1 alleles present almost identical percentage (65-100%) in North America and is observed in 77% of H. pylori strains in Eastern Asia and is the most frequently occurring subtype in H. pylori strains in Japan (Maciorkowska et al., 2007). Similarly, infection with H. pylori strains containing type m1 forms of vacA is associated with an increased risk of gastric cancer compared to infection with strains containing type m2 forms of vacA (Basso et al., 1998). The vacA m1 allele was more often observed (>80%) in South America, Portugal, Spain and Japan while in a study from China, the allele m2 of the midregion was the one most frequently observed (42.1% in children and 50% in adults) (Maciorkowska et al., 2007).

There are different allelic types of vacA, Based on the presence of a combination of the vacA s and m alleles. Some reports indicated that the vacA s1 allele was significantly associated with vacA m1 (Nimri et al., 2006). According to the previous studies, the type s1/m1 are toxic and strongly associated with duodenal ulceration, mostly in countries with a relatively low prevalence of H. pylori infection (Tovey et al., 2006; Faundez et al., 2002), for example the results of Shimoyama et al., show that all strains of Japanese population are s1/ml genotype (Shimoyama et al., 1998). But in this study, there was a significant correlation in combination of s1/m2 (54.05%), while in another study from Jordan, it was reported that vacA s1/m2 genotype was detected only in 7.7% of the vacA s/m combination (Nimri et al., 2006) and our study has revealed that s1/m2 strains are more characteristic for PUD. In general, vacA type s1m1 and s1m2 strains produce high and moderate levels of toxin, respectively, whereas s2/m2 strains produce little or no toxin (Van-Doorn et al., 1998). And in a study from Tennessee, all positive combination of vacA region were identified, with the exception of s2m1 (Atherton et al., 1995) and in this study the proportion of s2m1 was 4.05%.

CONCLUSION

In conclusion, results of our study suggest that s1/m2 genotypes are associated with peptic ulceration in patients from Shahrekord, Iran that infected by Helicobacter pylori.