Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients
© Ben Mansour et al; licensee BioMed Central Ltd. 2010
Received: 19 November 2009
Accepted: 19 March 2010
Published: 19 March 2010
Distinct virulence factors of H. pylori have been described: the vaculating cytotoxin (vacA), the cytotoxin associated gene (cagA), the induced by contact with epithelium factor Antigen (iceA gene) and the outer membrane protein oipA. In Tunisia, there are no data regarding the pattern of H. pylori genotypes; therefore, this prospective and multicentre study was the first to be done in Tunisia and aimed to investigate the prevalence of the vacA, cagA, iceA and oipA genotypes of H. pylori isolates from Tunisian patients with peptic ulceration, gastric cancer, MALT lymphoma and gastritis.
H. pylori was cultured from endoscopic biopsies obtained from 281 Tunisian patients. The vacA alleles, cagA, iceA and oipA genotypes were determined by PCR.
The vacA s1m1, s1m2 and s2m2 were respectively found in 10.7%, 12.5% and 45.6% of strains. The s2m1 genotype was not detected in our study. The cagA was found in 61.6% of isolates. The iceA1 and the iceA2 genotypes were respectively isolated in 60.2% and in 16% of strains. The oipA genotype was detected in 90.8% of strains. Considering the vacA and iceA genotypes, the presence of multiple H. pylori strains in a single biopsy specimen was found respectively in 31.4% and 23.8%. The comparison between strains isolated from antrum and fundus showed that Tunisian patients were infected with two or more strains of different cagA, vacA, iceA and oipA genotypes and the discordance was respectively in 9.6%, 4.6%, 8.9% and 8.5% of strains.
Our results showed that in 46% (131 strains among 281), the H. pylori strains were highly virulent in relation of the three or four virulent factors they could carry. These finding were described before in the literature. Tunisian patients were colonized by one or multiple strains of H. pylori in the same time in relation of presence of vacA m1/m2 and iceA1/iceA2 in the same biopsy. The discordance between strains isolated from antrum and fundus was high, and it is in favour of multicolonization.
Helicobacter pylori (H. pylori) is one of the most common bacterial pathogens of humans and has a worldwide distribution. Infection by H. pylori is associated with the development of chronic gastritis, gastric or duodenal ulcer, gastric cancer and MALT-lymphoma . Different virulence genes have been described in H. pylori infection such as cagA, vacA, iceA and oipA genes. The cytotoxin-associated gene (cagA) is frequently associated with cytotoxin production and the induction of interleukin 8 (IL8) by gastric epithelial cells . Several studies have suggested that cagA is a useful marker for the most virulent strains that are associated with peptic ulcer, atrophic gastritis and adenocarcinoma [3–5]. The cag pathogenicity island (PaI) encodes a type IV secretory system and delivers CagA into the host cytosol where becomes phosphorylated on tyrosine residue. Phosphorylated CagA interacts with the phosphatase SHP-2 causing dephosphorylation of cortactin and cytoskeletal rearrangements forming the "hummingbird" phenotype . The vacuolating cytotoxin gene (vacA) is an important virulence factor encoding the vacuolating toxin and it is present in all strains [7, 8]. There is a considerable variation in vacuolating activities among strains [9–11] due to the sequence heterogeneity at the middle (m) and the signal (s) regions within the vacA gene. The middle region has m1 and m2 allelic types and the signal region located at the 5' end of the gene has an s1 (s1a, s1b and s1c) or an s2 allele. Strains carrying the s1ml mosaic combination of the gene vacA exhibit higher levels of cytotoxic activity than s1m2 strains, while s2m2 strains do not secrete the vacuolating cytotoxin . Another virulence gene designated iceA (for induced by contact with epithelium) has been recently described. The iceA gene has two main allelic variants iceA 1 and iceA 2  but the function of these variants is not yet clear [14–18]. iceA 1 is upregulated upon contact of H. pylori with the gastric epithelium and has been regarded as a marker for peptic ulcer disease . Recently, a novel putative virulence factor has been identified, the oipA (outer inflammatory protein) gene, which encodes one of the outer membrane proteins and is an inflammation-related gene located approximately 100 kb from the cag PAI on the H. pylori chromosome . oipA induces IL-8 secretion by epithelial cells and active OipA protein production may be "on" or "off" depending on the number of CT dinucleotide repeats in the signal sequence of the oip A gene (HP0638) .
In Tunisia, there are no data regarding the pattern of H. pylori genotypes in patients; therefore, our prospective and multicentre study was the first to be done in Tunisia and to our knowledge the first one in North Africa. It aimed to investigate the prevalence of the vacA, cagA, iceA and oipA genotypes of H. pylori isolates from Tunisian patients.
Materials and methods
Biopsy samples were obtained over a 2 years-period (March 2005 to August 2007) from patients referred for endoscopy at 6 centers of gastroenterology in Tunisia. 281 patients, who had H. pylori positive cultures, were enrolled in this study. The mean age of the patients was 39.9 (range, 2 to 88) and 54.4% were women. Regarding macroscopic aspect of the mucosa and histological routine results, patients were distributed into gastritis in 195 cases (69.3%), peptic ulceration in 78 cases (27.7%) (duodenal ulcer in 67 cases, gastric ulcer in 11 cases), MALT lymphoma of the stomach in one case (0.3%) and gastric cancer in 7 cases (2.5%). One endoscopic biopsy specimen taken from the antrum and one taken from the fundus were immediately frozen and kept separately at -80°C. In addition, one biopsy specimen from the antrum and one from the fundus were used for the culture. In our study, we also obtained colonies isolated separately from two biopsy sites (antrum and fundus) to determine if one patient can be colonized with one or more strains of H. pylori. All subjects were answering to questionnaire related to age, sex, gastric or duodenal peptic ulcer diseases upon endoscopy.
Culturing of H. pylori and DNA extraction
The biopsy specimens were cut into small pieces, homogenized in a Petri dish with a sterile scalpel and were smeared on the surface of Columbia agar plates containing 10% horse blood and Skirrow (Oxoid, England) supplement. Incubation was performed in microaerophilic conditions at 37°C for a maximum of 6 days. The H. pylori colonies were smooth, translucent and small (2 mm of diameter). In our study, the antral and fundic biopsy specimens were studied separately. Colonies that exhibited the described characteristic morphologies were identified as H. pylori if they were Gram negative and shaped bacilli, and urease, catalase and oxidase positive. From the primary growth, seven or eight colonies were pooled together, and genomic DNA was extracted with the QIAamp DNA mini kit (Qiagen, Germany) according to the manufacturer's instructions. The isolated DNA was eluted in 200 μl of 1× TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and stored at -20°C until use.
H. pylori genotyping
PCR primers for amplification of cagA, vacA, iceA and oipA sequences
Primer sequence (5'-3')
Size of PCR product (bp)
Data were analyzed using X2 test. A p value of < 0.05 was considered to be statistically significant.
Prevalence of H. pylori genotypes detected in strains
iceA 1/iceA 2
The vacA s- and m- region genotype were determined in all strains studied. In the m-region, 88 strains contained both m1 and m2 alleles. In the strains containing one single vacA m allele, the m1 allele was found in 30 isolates (10.7%) and m2 in 163 one (58%).
For the s-region, in strains where a single vacA s allele was found, the majority 128 (45.6%) contained the s2 allele. In 65 isolates contained s1 allele (23.2%), 50 (77%) were subtype s1b while only 15 (23%) were subtype s1a.
Considering strains with only one single vacA genotype, and taking vacA s- and m-region together, three genotypes were found: s1/m1 (10.7%), s1/m2 (12.5%) and s2m2 (45.6%). The s2m1 genotype was not found in our study.
The discordance between strains taken from antrum and those from fundus was found in 13 cases (4.6%) (Table 2).
The oipA genotype was detected in 255 (90.8%) from 281 strains, and only 26 strains (9.2%) were oipA negative. The discordance between strains obtained from antrum and fundus was determined in 24 strains (8.5%) (Table 2). The 401-bp PCR product indicating the presence of the oipA gene was shown in figure 1.
Combined cagA, vacA, iceA and oipA genotypes
Combined vacA, cagA, iceA and oipA genotypes
cagA, vacA, iceA and oipA status of H. pylori strains with different gastric diseases: a comparison between patients with peptic ulceration, and with gastritis
Relation between the age of patients and virulence factors:
Relation between the sex of patients and virulence factors:
The predominant genotype in strains that were positive for H. pylori by PCR was the oipA (90.8%), followed by the cagA gene (61.6%), and the iceA 1 gene (60.2%), while the iceA 2 was amplified only in 45 strains (16%). Our results were in agreement with other studies conducted in Europe, Central and South America, and East Asia where a higher prevalence (67% or more) of the cagA genotype was reported . In Turkey , Korea and Japan, the prevalence of the iceA 1 genotype in patient with duodenal ulcer were respectively 68.8%, 69.8% and 62.5% which is similar to our study, whereas iceA 2 was prevalent in the USA and Columbia . The already mentioned similitude can be defined by the epidemiological resemblance in these countries.
For the vacA genotype, and when considering a single combined genotype, our results showed that the vacA s2 allele was predominant (45.6%) followed by the vacA s1 allele (23.2%). A study in Kuwait reported that vacA s1 and s2 types were detected in approximately equal numbers in biopsies obtained from patients of Middle-Eastern origin, while North Africans were predominantly infected with the s2 type .
The prevalence of cagA+ strains in Tunisian peptic ulcer and those with gastritis was similar to that shown in the study from South Africa .
In a previous report by van Doorn et al (19), an association was found between the iceA 1 allele and peptic ulcer disease. Other studies from Asia were suggesting that vacA, cagA and iceA genotypes were not associated with peptic ulcer disease . These findings may reflect important geographic differences between H. pylori strains and patients. As reported by van Doorn et al, H. pylori genotypes are not uniformly distributed over the world . When considering the vacA and oipA genes, our results showed that there is a significant difference between the two studied groups (p < 0.001), but, cagA and iceA genes were not associated with gastroduodenal diseases in our study (p > 0.05). The oipA (HpO638) outer membrane protein expression was linked to severe inflammation and the induction of IL-8 secretion. In one Turkish study, oipA (HpO638) gene was highly associated with peptic ulcer disease (92.9%) than with gastritis (80.9%) . In other studies, no difference in prevalence of cagA genotypes was found between peptic ulcers and other gastroduodenal diseases . Yamaoka et al reported that there was no association between the iceA, vacA or cagA status and clinical outcome in studied patients.
In our study, the oipA gene was found in 100% of gastric ulcer. Several studies failed to show a relationship between the gene status and clinical symptoms in several patient populations, this might be due to that patient selection is extremely important and the study group should be sufficiently large and diverse with respect to genotypes and clinical symptoms.
As for age, we have demonstrated that the difference between children and adults was statistically significant about the cagA and iceA genes, but not with the vacA and oipA genes. Regarding the distribution of gene virulent factors according to sex, no difference seen in term of cagA gene, but the difference was statistically significant among the vacA, iceA and oipA genes. In other reported studies, there were no significant differences in frequencies of H. pylori virulence-associated genes, between children and adults, and the strains from men exhibited genotypes similar to those in women . Laila FN et al have concluded in their study that the presence of certain genotypes was not significantly associated with the age or gender of the patient.
The detection of multiple genotypes implies the presence of multiple strains in a clinical sample. Considering vacA and iceA genes in our study, the presence of multiple H. pylori strains in a single biopsy specimen was found respectively in 31.4% and 23.8%. Then, when comparing the genetic differences in isolates of H. pylori from the antrum and the fundus, we determined that patients were infected with two or more strains of different cagA, vacA, iceA and oipA, and the percentage of discordance was respectively in 9.6%, 4.6%, 8.9% and 8.5%. Consequently, if multiple genotypes are found, this is a strong indication for the presence of multiple strains. It may be speculated that more than one strain may be acquired in childhood, especially in countries with a very high prevalence of H. pylori, but, it is not known whether multiple strains colonize simultaneously (coinfection) or at different time points (superinfection), and several studies demonstrated that coinfection or superinfection are common [32, 33]. The co-existence of more than one strain in the same individual may reflect the capacity of H. pylori to evolve genetic variations during the long-term colonization from childhood , and the dynamics of co-colonization by multiple strains has been studied in animal models . Since the prevalence of multiple strains colonization is clinically important in our study, there is a clear need to be considered when planning therapeutic strategies to avoid the risk of the emergence of such strains.
The present study showed that patients with peptic ulcer disease and those with gastritis were almost infected similarly by multiple strains of H. pylori. This underlines that the positive associations between H. pylori genotypes and peptic ulcer disease do not imply that patients without ulcer disease cannot be infected with high-risk H. pylori genotypes.
The combination of the distinct vacA, cagA, iceA and oipA genotypes illustrated the mosaic composition of the H. pylori genome. Strains typed as vacA s2/cagA+/iceA+/oipA+ were more prevalent than those typed as vacA s1/cagA+/iceA+/oipA+ and which are associated to severe pathologies.
In conclusion, we have examined the prevalence of vacA, cagA, iceA and oipA genotypes of H. pylori strains clinically isolated in Tunisia. No significant correlation was found between the expression of cagA and iceA genes and the two groups of studied patients, but the difference was statistically significant with the vacA and oipA genes. As for age, the difference between children and adults was statistically significant with the cagA and iceA genes, but not with oipA and vacA genes. Regarding the distribution of virulence genes to sex, the difference was statistically significant among oipA, iceA and vacA genes, but not with cagA gene.
In Tunisia, it is so possible that the high prevalence of infection with virulent factors contributes to the characteristics of H. pylori infection, but not used to discern the risk of developing serious gastroduodenal diseases in the host. Our results showed that Tunisian patients were colonized by one or more strains of H. pylori in relation of presence of vacA m1/m2 and iceA 1/iceA 2 in the same biopsy, than, the discordance between strains isolated from antrum and fundus was higher, and it is in favour of multicolonization.
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