Association of <Emphasis Type="Italic">iceA</Emphasis> and <Emphasis Type="Italic">babA</Emphasis> genotypes in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> strains with patient and strain characteristics

Data on the geographic prevalence of Helicobacter pylori iceA and babA alleles in Eastern Europe are still relatively scant. The aim of this study was to evaluate the prevalence of iceA and babA genotypes in Bulgarian symptomatic patients. The iceA and babA genotypes were evaluated by PCR with pure cultures in strains from 196 and 181 patients, respectively. Mixed infections were found in 10.2% of all 196 patients. Prevalence of H. pylori genotypes in patients with single-strain infections was 69.3% for iceA1, 30.7% for iceA2, 82.4% for cagA ⁺, 89.2% for vacA s1, 10.8% for vacA s2, 39.8% for vacA m1, 60.2% for vacA m2 and 48.8% for babA2. Within the iceA1 positive strains, 94.3% and 88.5% were also vacA s1a and cagA positive, respectively. Of the babA2 positive strains, 100.0%, 92.4% and 72.2% were also vacA s1a, cagA and iceA1 positive, respectively. Ulcer patients had more often strains with cagA positive status and vacA s1a allele. Although neither iceA1 nor babA2 were more common in ulcer patients, the combination of both alleles was more frequent (48.1%) in the ulcer patients than in the rest (28.7%). Clarithromycin susceptible strains had more often iceA1 allele (74.4%) than the resistant strains (55.3%). In conclusion, the results demonstrated a high prevalence of virulent H. pylori in Bulgaria. Both iceA1 and babA2 genotypes were associated with other virulence factors of H. pylori and, in addition, the iceA1 allele was associated with the strain susceptibility.     

Clinical relevance of cagL gene and virulence genotypes with disease outcomes in a Helicobacter pylori infected population from Iran

Helicobacter pylori infection is common in Iran as in other developing countries. Certain genotypes of H. pylori have been associated with increased occurrence of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. The aim of this study was to investigate the clinical relevance of cagL gene and other virulence genotypes of H. pylori isolates with clinical outcomes in Iranian patients. Totally, 126 symptomatic patients who underwent gastroduodenal endoscopy were enrolled in the study. Sixty-one H. pylori strains were isolated from the patients studied. The presence of the cagL, cagA, vacA, iceA, babA2 and sabA genes in the corresponding H. pylori isolates were determined by polymerase chain reaction and the results were compared with clinical outcomes and histopathology. The cagL, cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA ₂ , and sabA genotypes were detected in 96.7, 85.2, 75.4, 24.6, 29.5, 70.5, 42.6, 23, 96.7, and 83.6 % of the isolates, respectively. The three genotypic combinations, cagL/cagA/vacAs1m1/iceA1/babA2/sabA, cagL/cagA/vacAs1m2/iceA1/babA2/sabA, and cagL/cagA/vacAs1m2/iceA2/babA2/sabA were determined as the most prevalent combined genotypes. There was a significant correlation between the presence of cagL gene and cagA positivity (P = 0.02). No significant correlation was found between the various genotypes and clinical outcomes (P > 0.05). The present study showed a very high prevalence of cagL genotype among the H. pylori isolates from Iranian patients. Our results demonstrated that neither single genotype nor combination genotypes of virulence-associated genes was significantly helpful markers for predicting the severity of gastroduodenal disease associated with H. pylori infection in Iranian patients.     

Association of Malaysian Helicobacter pylori Virulence Polymorphisms with Severity of Gastritis and Patients' Ethnicity

Background and aim: Polymorphisms of Helicobacter pylori cagA and vacA genes do exist and may contribute to differences in H. pylori infection and gastroduodenal diseases among races in the Malaysian population. This study was conducted to characterize the polymorphisms in H. pylori cagA and vacA in Malaysian population. Methods: A total of 110 H. pylori isolates were genotyped by PCR and sequenced for cagA and PCR‐RFLP for vacA. Results: East Asian cagA was predominantly detected (64.5%), whereas vacA s1m1 and s1m2 alleles were detected in 60.9 and 37.3% of strains, respectively. A statistical association between cagA type with patients’ ethnicity (p < .0001) and age group >50 years old (p = .027) was identified. vacA alleles showed significant association with age group >50 years old (p = .017) and increased neutrophil activity in gastric mucosa (p = .028 and p = .016 for moderate and marked activity, respectively). Further identification of vacA polymorphism revealed that 84% of strains from Malays and Indians showed one RFLP pattern (RFLP‐1), whereas more than one RFLP patterns (RFLP‐2, 3, 4, 5, 6, and 8) were predominantly observed in strains from Chinese (82%) (p < .0001). Increasing severity of gastric inflammation was observed in gastric mucosa infected with strains carrying RFLP‐2, 3, 4, 5, and 6 (p = .037). About 86.6% of H. pylori strains with East Asian cagA were vacA RFLP‐2, 3, 4, 5, 6, and 8, and 88% of Western cagA strains were vacA RFLP‐1 (p < .0001). Chinese and Indians are susceptible to different virulence genotypes of H. pylori, whereas Malays showed a mixed virulence genotypes. Conclusion: Marked differences in the polymorphisms of cagA and vacA were observed among strains in Malaysian population. This provides a new insight into the pathogenicity of H. pylori in multiracial population.     

Helicobacter pylori bab Paralog Distribution and Association with cagA,vacA, and homA/B Genotypes in American and South Korean Clinical Isolates

Helicobacter pylori genetic variation is a crucial component of colonization and persistence within the inhospitable niche of the gastric mucosa. As such, numerous H. pylori genes have been shown to vary in terms of presence and genomic location within this pathogen. Among the variable factors, the Bab family of outer membrane proteins (OMPs) has been shown to differ within subsets of strains. To better understand genetic variation among the bab genes and to determine whether this variation differed among isolates obtained from different geographic locations, we characterized the distribution of the Bab family members in 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). Overall, we identified 23 different bab genotypes (19 in AH and 11 in KH), but only 5 occurred in greater than 5 isolates. Regardless of strain origin, a strain in which locus A and locus B were both occupied by a bab gene was the most common (85%); locus C was only occupied in those isolates that carried bab paralog at locus A and B. While the babA/babB/- genotype predominated in the KH (78.8%), no single genotype could account for greater than 40% in the AH collection. In addition to basic genotyping, we also identified associations between bab genotype and well known virulence factors cagA and vacA. Specifically, significant associations between babA at locus A and the cagA EPIYA-ABD motif (P<0.0001) and the vacA s1/i1/m1 allele (P<0.0001) were identified. Log-linear modeling further revealed a three-way association between bab carried at locus A, vacA, and number of OMPs from the HOM family (P<0.002). En masse this study provides a detailed characterization of the bab genotypes from two distinct populations. Our analysis suggests greater variability in the AH, perhaps due to adaptation to a more diverse host population. Furthermore, when considering the presence or absence of both the bab and homA/B paralogs at their given loci and the vacA genotype, an association was observed. Our results highlight the multifactorial nature of H. pylori mediated disease and the importance of considering how the specific combinations of H. pylori virulence genes and their multiple interactions with the host will collectively impact disease progression.     

Helicobacter pylori Induces Apoptosis of T- and B-Cell Lines and Translocates Mitochondrial Apoptosis-Inducing Factor to Nucleus

Immune cell apoptosis may play a role in human persistent Helicobacter pylori infection. We planned to study the apoptosis of T and B cells by H. pylori strains. T (Jurkat) and B (Raji) cell lines were co-cultured with cagA-positive H. pylori strains carrying different vacA genotypes (s1a/m1, s1a/m2, and s2/m2). Apoptosis was detected by microscopy, DNA fragmentation assay, and flow cytometry. Apoptosis-inducing factor (AIF) transfer from mitochondria to nucleus was studied by immunoblot analysis. Apoptosis of T and B cells was significantly higher in H. pylori-infected cells than in uninfected controls (s1a/m1 80%, s1a/m2 78%, s2m2 69% vs. control 16% for T cells, P < 0.001; s1 a/m1 78%, s1a/m2 73%, s2m2 62% vs. control 24% for B cells, P < 0.001 by flow cytometry) with no difference among the genotypes. AIF transfer from mitochondria to nucleus was demonstrated in both apoptotic cell lines. Thus, H. pylori induces apoptosis in T- and B-cell lines and translocates AIF. T and B cells deletion through apoptosis may explain the persistence of H. pylori infection; its role in pathogenesis needs further research.     

Presence of the cagA Gene in the Majority of Helicobacter pylori Strains Is Independent of Whether the Individual Has Duodenal Ulcer or Asymptomatic Gastritis

Background Helicobacter pylori infection presents as many different diseases, including asymptomatic gastritis, peptic ulcer disease, and gastric cancer. Although the virulence factor(s) responsible for different H. pylori-related diseases have not been identified, several candidate genes are being investigated for such an association. The polymerase chain reaction (PCR) frequently is used to assess the presence of genetic factors associated with pathogenesis of disease; the cagA gene and its product have been postulated to have a disease-specific relationship to peptic ulcer and gastric cancer because of differential expression in these diseases compared to histological gastritis alone. Materials and Methods. Genomic DNA was amplified by PCR, using synthetic oligonucleotide primers to the cagA gene to determine the prevalence of the cagA gene in 60 H. pylori isolates obtained from well-documented duodenal ulcer or asymptomatic gastritis patients (30 each). Results were confirmed by hybridization with a 1.4-Kb cagA probe. Results. The expected PCR product was obtained in 90% of isolates from duodenal ulcer patients, compared to 70% of isolates from individuals with asymptomatic gastritis. The PCR products were polymorphic in size, suggesting cagA gene sequence differences among isolates. Evaluation for the presence of the cagA gene by hybridization with a 1.4-Kb cagA probe showed a homologous product in 29 of 30 strains [96.7%; 95% confidence interval (CI) = 83–100%] from duodenal ulcer patients versus 25 of 30 strains (83.3%; 95% CI = 65–94%) obtained from individuals with asymptomatic gastritis (p= 0.19). Conclusions. The high prevalence of the cagA gene in asymptomatic gastritis suggests that it will not prove to be a useful marker to distinguish more virulent or disease-specific H. pylori strains. The genetic heterogeneity among H. pylori strains makes PCR an unwise choice as the single method to determine prevalence of a putative virulence factor. In evaluation of the prevalence of a gene or genetic factor in a population of H. pylori, hybridization with extended probes might be important to ensure that the results are representative of the organism's genotype.     

Comparison of genotyping of Helicobacter pylori cagA and vacA virulence genes from gastric biopsies and stool specimens

Background. We compared results of genotyping of Helicobacter pylori cagA and vacA virulence genes in DNA from gastric biopsies, both paraffin‐embedded and frozen, and from stool samples, in order to evaluate the comparative sensitivity of the stool assay. Methods. Genomic DNA from paraffin‐embedded biopsies, unfixed frozen biopsies, and stool samples of the same 20 patients was amplified for the cagA gene, an empty site (which provides a positive signal for cagA negative strains) and for the s and m alleles of the vacA gene. Composite genotypes were determined by combining data from analysis of all three materials. Results. Analysis of none of the materials taken singly showed all of the genotypes revealed by all three materials taken together, probably because of sampling error. Analysis of paraffin biopsies revealed 83.5%, that of frozen biopsies revealed 74.7% and that of stools revealed 75.9% of the genotypes. There was no significant difference in the percentage of the H. pylori genotypes identified from the three materials. Analysis of combinations of frozen biopsies and stools revealed 89.9% of the composite genotypes, and that of paraffin biopsies and stools revealed 96.2% of the composite genotypes. Evidence of multiple genotypes was found in 10 of 20 (50%) of the cases. Conclusions. Any one of the investigated biological materials can be used for detection of cagA and vacA genes, but no single assay provided a complete genotype. The use of a combination of two materials may generate a more accurate representation of H. pylori genotypes in each individual.     

Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children

The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric population, to further clarify its significance in causing inflammation of gastric mucosa in children and to verify whether cagA, vacA, iceA and babA genes work independently or synergistically in causing gastritis. A total of 163 H. pylori isolates obtained from the same number of children were tested for the presence of cagA, vacA and iceA genes using previously established methods, while the babA2 gene was determined using novel polymerase chain reaction assay targeting a 139-bp fragment of the central region of babA2. The babA2 gene was detected in 47.9 % of H. pylori samples. The presence of the babA2 gene was strongly associated with cagA, vacA s1 and vacA m1 genotype. The babA2 status correlated positively with bacterial density score, activity of inflammation and chronic inflammation of gastric mucosa. No significant correlation was found between the babA2 status and the presence of atrophy or intestinal metaplasia. In addition, the activity of gastric inflammation and density score were significantly associated with the coexpression of the cagA, vacA s1, vacA m1 and babA2 genes. The study, which included the largest number of pediatric H. pylori samples to date, confirmed that babA2 gene plays an important role in the pathogenesis of H. pylori gastritis in children. Furthermore, our results suggest that babA2, cagA and vacA s1 and m1 gene products may work synergistically in worsening the inflammation of gastric mucosa.     

Mixed infection with cagA positive and cagA negative strains of Helicobacter pylori lowers disease burden in The Gambia

Background

The prevalence of Helicobacter pylori including strains with putatively virulent genotypes is high, whereas the H. pylori-associated disease burden is low, in Africa compared to developed countries. In this study, we investigated the prevalence of virulence-related H. pylori genotypes and their association with gastroduodenal diseases in The Gambia.

Methods and Findings

DNA extracted from biopsies and H. pylori cultures from 169 subjects with abdominal pain, dyspepsia or other gastroduodenal diseases were tested by PCR for H. pylori. The H. pylori positive samples were further tested for the cagA oncogene and vacA toxin gene.One hundred and twenty one subjects (71.6%) were H. pylori positive. The cagA gene and more toxigenic s1 and m1 alleles of the vacA gene were found in 61.2%, 76.9% and 45.5% respectively of Gambian patients harbouring H. pylori. There was a high prevalence of cagA positive strains in patients with overt gastric diseases than those with non-ulcerative dyspepsia (NUD) (p = 0.05); however, mixed infection by cagA positive and cagA negative strains was more common in patients with NUD compared to patients with gastric disease (24.5% versus 0%; p = 0.002).

Conclusion

This study shows that the prevalence of H. pylori is high in dyspeptic patients in The Gambia and that many strains are of the putatively more virulent cagA⁺, vacAs1 and vacAm1 genotypes. This study has also shown significantly lower disease burden in Gambians infected with a mixture of cag-positive and cag-negative strains, relative to those containing only cag-positive or only cag-negative strains, which suggests that harbouring both cag-positive and cag-negative strains is protective.     

Environmental Isolates of Aeromonas spp. Harboring the cagA-Like Gene of Helicobacter pylori

We investigated the presence of cagA-like gene of Helicobacter pylori in environmental isolates of Aeromonas spp. from different water samples of Calcutta, India, by colony hybridization using a cagA-specific DNA probe and by PCR with cagA-specific primers. Nucleotide sequencing of five PCR products revealed 97 to 98% homology to canonical cagA of H. pylori 26695 as well as to four clinical H. pylori strains from Calcutta. The cagA-like gene of the environmental isolates was unstable in laboratory conditions and tended to be lost upon subculturing.     

Infection of less virulent Helicobacter pylori strains in asymptomatic healthy individuals in Thailand as a potential contributing factor to the Asian enigma

In Thailand, gastric cancer incidence is considerably low despite the high prevalence of Helicobacter pylori infection. We investigated the prevalence of H. pylori infection and the genotypes of cagA by using 179 stool specimens obtained from asymptomatic Thai individuals. In this study, the prevalence of H. pylori infection was 43.6%, and the detection rate of cagA-positive strains was 43.5%. In addition, the proportion of the highly virulent East-Asian type of cagA was 7.2%. These results indicate that the low prevalence of cagA-positive H. pylori strain as well as the low prevalence of East-Asian genotype cagA-positive strains may contribute to the low gastric cancer incidence.     

Non-invasive genotyping of Helicobacter pylori cagA, vacA, and hopQ from asymptomatic children

Background: Helicobacter pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non‐invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim is to genotype cagA, hopQ, and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, to avoid selection bias resulting from endoscopy. Methods: H. pylori status of 86 asymptomatic children was assessed by ¹³C‐urea breath test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ, and vacA genes were amplified from stool DNA samples and sequenced. Results: UBT was positive in 69 (80.2%) of 86 children; in stool DNA analysis, 78.3% were positive by 16S rRNA PCR. cagA, vacA, and hopQ were detected in 66.1%, 84.6%, and 72.3% of stool DNA samples from 16S rRNA‐positive children. Of the children’s DNA samples, which revealed vacA and hopQ alleles, 91.7% showed vacA s1 and 73.7% showed type I hopQ. Type I hopQ alleles were associated with cagA positivity and vacA s1 genotypes (p < 0.0001). Conclusions: Using stool DNA samples, virulence markers of H. pylori were successfully genotyped in a high percentage of the asymptomatic infected children, revealing a high prevalence of genotypes associated with virulence. Type I hopQ alleles were associated with the presence of cagA and the vacA s1 genotype.     

Helicobacter pylori and the birth cohort effect: evidence for stabilized colonization rates in childhood

Background: The prevalence of Helicobacter pylori has declined over recent decades in developed countries. The increasing prevalence with age is largely because of a birth cohort effect. We previously observed a decline in H. pylori prevalence in 6‐ to 8‐year‐old Dutch children from 19% in 1978 to 9% in 1993. Knowledge about birth‐cohort‐related H. pylori prevalence is relevant as a predictor for the future incidence of H. pylori‐associated conditions. Aim: The aim of this study was to investigate whether the birth cohort effect of H. pylori observed between 1978 and 1993 continued in subsequent years. Methods: Anti‐H. pylori IgG antibodies and anti‐CagA IgG antibodies were determined in serum samples obtained in 2005/2006 from 545 Dutch children aged 7–9 years who participated in the Prevention and Incidence of Asthma and Mite Allergy birth cohort. The H. pylori and CagA antibodies were determined by enzyme‐linked immunosorbent assays that have been extensively validated in children, with a 94% sensitivity for H. pylori colonization and a 92.5% sensitivity for colonization with a cagA‐positive strain. Results: Of the 545 children (M/F 300/245), most (91.5%) were of Dutch descent. The H. pylori positivity rate was 9% (95% CI 6.6–11.4%). The prevalence of CagA antibodies was 0.9% (95% CI 0.1–1.6%). No significant differences were demonstrated in H. pylori and cagA prevalence in relation to gender or ethnicity. Conclusion: The prevalence of H. pylori in childhood has remained stable in the Netherlands from 1993 to 2005, suggesting a stabilization of the previously decreasing trend in subsequent birth cohorts. This finding may reflect stabilization in determinants such as family size, housing, and hygienic conditions (or offset by day care). If confirmed in other populations in developed countries, it implies that colonization with H. pylori will remain common in the coming decades. Remarkably however, the rate of colonization with cagA⁺H. pylori strains has become very low, consistent with prior observations that cagA⁺ strains are disappearing in Western countries.     

High Diversity of vacA and cagA Helicobacter pylori Genotypes in Patients with and without Gastric Cancer

Background

Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. The aim of this study was to assess the topographical distribution of H. pylori in the stomach as well as the vacA and cagA genotypes in patients with and without gastric cancer.

Methodology/Principal Findings

Three gastric biopsies, from predetermined regions, were evaluated in 16 patients with gastric cancer and 14 patients with dyspeptic symptoms. From cancer patients, additional biopsy specimens were obtained from tumor centers and margins; among these samples, the presence of H. pylori vacA and cagA genotypes was evaluated. Positive H. pylori was 38% and 26% in biopsies obtained from the gastric cancer and non-cancer groups, respectively (p = 0.008), and 36% in tumor sites. In cancer patients, we found a preferential distribution of H. pylori in the fundus and corpus, whereas, in the non-cancer group, the distribution was uniform (p = 0.003). A majority of the biopsies were simultaneously cagA gene-positive and -negative. The fundus and corpus demonstrated a higher positivity rate for the cagA gene in the non-cancer group (p = 0.036). A mixture of cagA gene sizes was also significantly more frequent in this group (p = 0.003). Ninety-two percent of all the subjects showed more than one vacA gene genotype; s1b and m1 vacA genotypes were predominantly found in the gastric cancer group. The highest vacA-genotype signal-sequence diversity was found in the corpus and 5 cm from tumor margins.

Conclusion/Significance

High H. pylori colonization diversity, along with the cagA gene, was found predominantly in the fundus and corpus of patients with gastric cancer. The genotype diversity observed across systematic whole-organ and tumor sampling was remarkable. We find that there is insufficient evidence to support the association of one isolate with a specific disease, due to the multistrain nature of H. pylori infection shown in this work.     

Helicobacter pylori in a poultry slaughterhouse: Prevalence,genotyping and antibiotic resistance pattern

Although Helicobacter pylori (H. pylori) is a highly significant pathogen, its source remains unclear. Many people consume chicken daily as a source of animal protein worldwide; thus, hygienic methods of supplying chickens for consumption are critical for public health. Therefore, our study examined the distribution of the glmM (ureC), babA2, vacA and cagA virulence genes in H. pylori strains in chicken meat and giblets (gizzards and livers) and the resistance of the strains to various antibiotics. Ninety chicken meat, gizzard and liver samples were obtained from a semi-automatic abattoir in Sadat City, Egypt, and were cultured and preliminarily analyzed using biochemical tests. The presence of the ureC, babA2, vacA and cagA genotypes was tested for in samples positive for H. pylori by multiplex polymerase chain reaction (Multiplex-PCR). The resistance of H. pylori to various antimicrobial drugs was tested using the disc diffusion method. In total, 7 of the 90 chicken samples were positive for H. pylori (7.78%); in 3/7 (42.85%) samples, the bacteria were found in the chicken liver, while the bacteria were found in the meat in 2/7 (28.57%) and in the gizzard in 2/7 (28.57%) samples. The total prevalence of both the ureC and babA2 genes in the isolated H. pylori strains was 100%, while the prevalence of the vacA and cagA genes was 57.1% and 42.9%, respectively. The resistance of H. pylori to the antibiotics utilized in our study was 100% for streptomycin; 85.7% for amoxicillin and penicillin; 71.4% for oxytetracycline, nalidixic acid and ampicillin; 57.1% for sulfamethoxazole and erythromycin; and 42.9% for neomycin, chloramphenicol and norfloxacin. In conclusion, the chicken meat and giblets were tainted by H. pylori, with a higher occurrence of the ureC, babA2, vacA and cagA genotypes. Future investigations should investigate the resistance of H. pylori to various antimicrobial agents in Egypt.     

Susceptibility to Helicobacter pylori infection: results of an epidemiological investigation among gastric cancer patients

The aim of this study was to identify the clinical, demographic, lifestyle factors and selected genetic polymorphisms that affect the susceptibility towards Helicobacter pylori (H. pylori) infection in gastric cancer patients. Histological confirmed gastric adenocarcinoma cases that underwent curative gastrectomy between 2002 and 2012 were included. Gastric biopsy samples were obtained to determine the H. pylori status, and further cagA status and vacA m and s genotypes by polymerase chain reaction. Patients were interviewed with structured questionnaires, and blood samples were collected for EPHX1, GSTM1, GSTT1, IL1B, IL1-RN, MTHFR and p53 genotyping. Proportions were compared in univariate analysis, while the relation between putative risk factors and H. pylori status and genotype were measured using logistic regression analysis. One hundred forty-nine gastric cancer patients were included, of which 78.5 % were H. pylori positive. Among positive patients 50 % were cagA+, 72.5 % vacA m1 and 80.7 % vacA s1. The presence of cagA was less frequent among vacA m1 (p = 0.031) and vacA s1 (p = 0.052) subtypes. The presence of father history for any cancer was a significant risk factor for H. pylori infection [adjusted odds ratio (OR) = 8.18, 95 % confidence interval (CI) 1.04–64.55]. EPHX1 exon 3 T > C (OR = 0.35, CI 95 % 0.13–0.94), IL1B-511 T > C (OR = 0.38, CI 95 % 0.15–0.97) and IL1-RN VNTR (OR = 0.19, CI 95 % 0.06–0.58) polymorphisms were protective towards H. pylori infection in the univariate analysis. Wine consumption was associated with higher risk of carrying the H. pylori vacA m1 virulent subtype (p = 0.034). Lastly, cardiovascular diseases were less common among cagA positive subjects (p = 0.023). Father history of any cancer is a risk factor for H. pylori infection. Polymorphisms in IL1B-511, IL1-RN and EPHX1 exon 3 genes might be protective towards H. pylori infection.     

Production of Calcite Nanocrystal by a Urease-Positive Strain of Enterobacter ludwigii and Study of Its Structure by SEM

Helicobacter pylori (H. Pylori) is an actively dividing spiral bacterium that changes to coccoid morphology under stressful environments. The infectivity of the coccoids is still controversial. The aim of this study was to determine the viability and expression of two important virulence genes (babA and cagE), in antibiotic-induced coccoid forms. Three strains of H. pylori, the standard 26695 and two clinical isolates (p1, p2) were converted to coccoid form by amoxicillin. Coccoids were identified according to Gram-staining and microscopic morphology. The viability of the cells was analyzed by flow cytometry. The expression of cagE and babA in coccoid forms were evaluated and compared to the spirals by quantitative PCR assay. The coccoid forms were developed after 72 h exposure of H. pylori to ½ MIC of amoxicillin, and the conversion form was completed (100 %) at 144 h in all of three isolates. Flow cytometry analyses showed that the majority of the induced coccoids (90–99.9 %) were viable. Expression of cagE and babA was seen in coccoids; however, in lower rate (cagE, ~3-fold and babA, ~10-fold) than these in spiral forms. Coccoid forms of two clinical isolates significantly expressed higher rate of cagE and babA than standard 26695 strain (P = 0.01). These results suggest that the induced coccoid form of H. pylori is not a passive entity but can actively infect the human by expression of the virulence genes for long time in stomach and probably play a role in chronic and severe disease.     

Benefits of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">cagE</Emphasis> genotyping in addition to <Emphasis Type="Italic">cagA</Emphasis> genotyping: a Bulgarian study

Associations of Helicobacter pylori cagE status with complex patient characteristics remain to be elucidated in Eastern Europe. The aim of this study was to assess the frequencies of cagE gene and cagA/cagE combinations in H. pylori strains from symptomatic Bulgarian patients and to improve cagA detection. cagA and cagE genotypes were evaluated in 219 patients with single-strain infections. In total, 84.9% of strains were cagA ⁺, while 68.5% were cagE ⁺. cagA ⁺, cagE ⁺, and cagA ⁺/cagE ⁺ strains were more prevalent in peptic ulcer (93.8%, 84.4%, and 84.4%) compared with nonulcer patients (81.3%, 61.9%, and 61.3%, respectively). In elderly patients, cagE ⁺ and cagA ⁺/cagE ⁺ strains were 1.9-fold more common than in the 12 children evaluated. Only 10% of the elderly subjects harbored low-virulence cagA ⁺/cagE ⁻ strains compared with 16.8% of adults and 41.7% of children. Intriguingly, prevalence of the cagA ⁺/cagE ⁻ genotype was 2.1-fold lower in men than in women, suggesting a higher frequency of more virulent strains in men. The presence of both cag genes and combinations was not linked to strain susceptibility to clarithromycin or metronidazole, place of residence, or prior therapy. Use of an extra primer pair increased cagA detection in 14.7% of 31 cagA ⁻ strains. In conclusion, use of a second primer pair for the cagA gene can be recommended in countries with common cagA ⁺ strains. Although both cag genes were linked to severe diseases in Bulgarian patients, the best discrimination of virulent strains was obtained by the cagA/cagE combination or by the cagE gene alone. cagE prevalence increased gradually with patient age, while the cagA ⁺/cagE ⁻ genotype, implying a disrupted cag pathogenicity island, was associated with both younger age and female gender.