Emergence of recombinant strains of Helicobacter pylori during human infection

Genetic recombination can be important evolutionarily in speeding the adaptation of organisms to new environments and in purging deleterious mutations. Here, we describe polymerase chain reaction (PCR), hybridization and DNA sequence-based evidence of six such exchanges between two strains of Helicobacter pylori during natural mixed infection of a patient in Lithuania. One parent strain contained the 37 kb long, virulence-associated cag pathogenicity island (PAI), and the other strain lacked this PAI. Most H. pylori from the patient had descended from the cag+ parent, but had become cag- during infection. This had resulted from transfer of DNA containing the 'empty site' allele from the cag- strain and homologous recombination, not from excision of the cag PAI without DNA transfer. Other cases of recombination involved genes for an outer membrane protein (omp5 and omp29; also called HP0227 and HP1342) and a putative phosphoenolpyruvate synthase (ppsA; HP0121). Replacement of a short patch of DNA sequence (36-124 bp) was also seen. As the chance of forming any given recombinant is small, the abundance of recombinants in this patient suggests selection for particular recombinant genotypes during years of chronic infection. We suggest that genetic exchange among unrelated H. pylori strains, as documented here, is important because of the diversity of this gastric pathogen and its human hosts. Certain H. pylori recombinants may grow better in a given host than either parent. The vigour of growth, in turn, could impact on the severity of disease that infection can elicit.     

Association of virulent Helicobacter pylori strains with ischemic heart disease

BACKGROUND: Previous studies have reported an association between chronic Helicobacter pylori infection and ischemic heart disease. However, it is not clear whether this association is really due to the virulence of the bacterium or is merely the result of confounding factors (in particular, age and social class). METHODS AND RESULTS: We assessed the prevalence of infection by Helicobacter pylori and by strains bearing the cytotoxin-associated gene-A (CagA), a strong virulence factor, in 88 patients with ischemic heart disease (age, 57+/-8 years; 74 men) and in 88 age- and sex-matched controls (age, 57+/-8 years; 74 men) with similar social background. Prevalence of Helicobacter infection was significantly higher in patients than in controls (62% versus 40%; P=.004), with an odds ratio of 2.8 (95% CI, 1.3 to 7.4; P<.001) adjusted for age, sex, main cardiovascular risk factors, and social class. Patients with ischemic heart disease also had a higher prevalence of CagA-positive strains (43% versus 17%; P=.0002), with an adjusted odds ratio of 3.8 (95% CI, 1.6 to 9.1; P<.001). Conversely, prevalence of CagA-negative strains was similar in patients and controls (19% versus 23%), with an adjusted odds ratio of 0.8 (95% CI, 0.4 to 1.4). CONCLUSIONS: The association between Helicobacter pylori and ischemic heart disease seems to be due to a higher prevalence of more virulent Helicobacter strains in patients. These results support the hypothesis that Helicobacter pylori may influence atherogenesis through low-grade, persistent inflammatory stimulation.     

The Helicobacter pylori fatty acid cis-9,10-methyleneoctadecanoic acid stimulates protein kinase C and increases DNA synthesis of gastric HM02 cells

Protein kinase C (PKC) has been implicated in the control of epithelial proliferative activity and in the process of malignant transformation. Helicobacter pylori (H.p.) infection is associated with increased gastric epithelial cell proliferation and has been linked with gastric carcinoma. In the present study, we report that the H.p. fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) directly activates PKC (Ka 3.3 microM). The effect of MOA upon PKC activation was Ca2+ dependent but did not require phosphatidylserine as phospholipid cofactor. MOA increased the stimulatory effect of phosphatidylserine at low Ca2+ (1 microM) concentrations. These findings indicate that MOA interacts at the phospholipid- and the diacylglycerol-binding domain to elicit PKC activation. Treatment of gastric mucous cells HM02 caused translocation of PKC from the cytosol to the nuclear, mitochondrial and membrane fraction. Furthermore, MOA stimulated [3H]thymidine incorporation into the DNA of HM02 cells. Our results show that the H.p. fatty acid MOA activates PKC and increases DNA synthesis in gastric epithelial cells.     

Helicobacter pylori: 6-day triple therapy in duodenal ulcer

OBJECTIVES: To investigate the risk of lymphatic and haematopoietic malignancies in deck crew on tankers exposed to cargo vapours. METHODS: The study design was as a nested case-referent study in two cohorts of male Swedish seamen 20-64 years of age at the national census 1960 (n 13,449) and 1970 (n 11,290), respectively. Cases were detected by record linkage with the Swedish Cancer Register 1961-79 and 1971-87, respectively. For each case, three to five age matched referents from the population were selected. Exposure was assessed from data in the Swedish Registry of Seamen and from a register of Swedish ships. RESULTS: Seamen in the 1970 cohort, who had been exposed to cargo vapours for at least one month on chemical or product tankers, had an increased risk of lymphatic and haematopoietic malignancies (Mantel-Haenszel odds ratio (OR) 2.6, 95% confidence interval (95% CI) 1.1 to 5.9)) with a significant exposure-response relation (conditional logistic regression analysis, p = 0.04). The ORs were increased for both lymphoma (3.2), multiple myeloma (4.0), and leukaemia (1.6), but the increase was only significant for non-Hodgkin's lymphoma (OR 3.3, 95% CI 1.1 to 10.6). There were no significantly increased risks for the 1960 cohort or for seamen exposed only on crude oil tankers, but these groups had few exposed cases and low cumulative exposure to benzene and other light petroleum products. CONCLUSIONS: Seamen exposed to cargo vapours from gasoline and other light petroleum products on chemical or product tankers had an increased incidence of lymphatic and haematopoietic malignancies. One possible cause is exposure to benzene during loading, unloading, and tank cleaning operations.     

REP-PCR fragments as biomarkers for differentiating gastroduodenal disease-specific Helicobacter pylori strains

We previously identified four potential putative gastroduodenal disease fragments by using the interspersed repetitive extragenic palindromic DNA sequence based PCR (REP-PCR) technique. We investigated these fragments with regard to their disease specificity. The putative disease-specific REP-PCR fragments were cloned, mapped by restriction enzymes, cross-hybridized, and confirmed by Southern hybridization. The four fragments were also used as probes against REP-PCR amplicons from H. pylori isolates obtained from gastritis (N = 20), duodenal ulcer (N = 30), and gastric cancer patients (N = 30). Three of these fragments (1.4- and 0.76-kb for gastritis; 1.35 kb for duodenal ulcer) were amplified without any discrimination between any disease-specific H. pylori isolates. However, amplification following hybridization with the fourth 0.81-kb fragment was observed only from gastritis (60%) and duodenal ulcer (52%) but with none (0%) of gastric cancer patients. Nucleotide sequence analysis of the 0.81-kb fragment revealed that it was an open reading frame of the hypothetical protein HP0373 matched to the position of 380,966 to 383,068 nucleotides of the H. pylori complete genome sequence. Hence, the REP-PCR sequence was not a extragenic palindromic DNA sequence. The hypothetical protein was also present in all the tested isolates. The REP-PCR fingerprinting technique is useful to differentiate disease-specific H. pylori strains based on the interspersed repetitive extragenic palindromic DNA sequences; however, it may not be useful to identify disease-specific virulence determinant(s) without being confirmed by DNA sequence analysis and functional studies.     

Emodin-induced inhibition of growth and DNA damage in the Helicobacter pylori

Studies were conducted to examine the dose effects of emodin on inhibition of growth versus DNA damage events in H. pylori from patients who had peptic ulcer disease. Inhibition of growth study from H. pylori demonstrated that emodin elicited dose-dependent growth inhibition in H. pylori cultures; that is, the greater the concentration of emodin, the greater the growth inhibition to H. pylori. However, S1 nuclease sensitivity analysis studies revealed that emodin induced dose-dependent DNA damage in H. pylori. Collectively, these results suggest that there was a possible relationship between the dose response to emodin and the inhibition of growth and DNA damage in H. pylori.     

Evidence for a conjugation-like mechanism of DNA transfer in Helicobacter pylori

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.     

Helicobacter pylori in 1997

In this review Helicobacter pylori (H. pylori) infection and its relation to different diseases is presented. H. pylori doesn't cause inconvenience to most infected people, though all infected persons have chronic active gastritis. The 10 year risk of peptic ulcer for people infected with H. pylori is about 10%. Randomized double-blinded trials have shown that eradication of H. pylori can cure most patients with peptic ulcer disease. Some people infected with H. pylori develop atrophic gastritis which is a risk factor for development of gastric cancer. It is not known if H. pylori screening and eradication would have a prophylactic effect against gastric cancer. It is also unknown if persons with non-organic dyspepsia and persons in long-term treatment with proton-pump-inhibitors would benefit from H. pylori eradication.     

Helicobacter pylori in children

Helicobacter pylori is the major cause of antral gastritis in children, however, it is not always associated with symptoms. The exception to this occurs in duodenal ulcer disease with which H. pylori is linked in children albeit less strongly than in adults. Duodenal ulcers do not recur in older children following eradication of H. pylori. The importance of asymptomatic carriage of H. pylori in children, particularly in relation to the duration of this infection and the subsequent development of gastric cancer, remain to be established. Helicobacter pylori is associated with both hypochlorhydria and persistent diarrhoea in children in developing countries, but the significance of this association is still unknown. Although there is no consensus on the optimal regimen for treating H. pylori infection in children, dual therapy with amoxycillin and bismuth subcitrate for 2 weeks followed by monotherapy with bismuth subcitrate for a further 6 weeks will eradicate H. pylori infection in the majority of children. Those who relapse may be treated with a repeat course plus metronidazole for 4 weeks. Compliance with such regimens is a problem and shorter treatment courses that are equally effective in children need to be defined. Similarly, studies are required on the influence of the intrafamilial reservoir of H. pylori infection on relapse after treatment and the need for whole family eradication therapy.     

Detection and typing of the virulence determinants cagA and vacA of Helicobacter pylori directly from biopsy DNA: are in vitro strains representative of in vivo strains?

BACKGROUND: The relationship of Helicobacter pylori genotypes to gastrointestinal disease has relied on cultured isolates. This assumes that cultured strains are representative of in vivo strains. OBJECTIVE: To detect and type the cagA status and the vacA genotypes directly from biopsy DNA without the need for culture, and to further define the relationship between H. pylori genotypes and gastroduodenal pathology. METHODS: Fifty-two Caucasian patients undergoing routine endoscopy for dyspepsia had additional biopsies taken from four gastric sites and one duodenal site for biopsy DNA preparation. An antral sample was taken for biopsy culture. All recruited patients were H. pylori-positive on rapid urease test for Campylobacter-like organisms (CLO test) and/or histology. By polymerase chain reaction (PCR), the cagA status and the vacA s and m types were detected directly from biopsy DNA. RESULTS: H. pylori isolates were cultured from 28/52 patients in whom infection was detected by PCR. Two isolate types differed from biopsy types. Fifty of the 52 patients, strains were typable from all four gastric sites and in 51/52 the same strain predominated throughout. The cancer strains were all cagA-positive/vacA s1 type. There was a correlation between cagA positivity and vacA s1 (41/43). There was no difference between the cagA-positive/vacA s1 strains and the presence or absence of ulcers. There were only 5/52 vacA s2 m2 and four were in the non-ulcer dyspeptic group. CONCLUSION: cagA status and the vacA genotyping was successful with tissue samples taken directly from gastric and duodenal biopsies. Discrepancies between isolate and biopsy strain types stress the need for caution when interpreting in vitro strain types and suggest that direct PCR of biopsies is the preferred typing technique. The cagA status and the s1 vacA allele are unreliable as single markers in determining the risk of developing peptic ulcer disease.     

Isolation and characterization of Helicobacter pylori strains from peptic ulcer patients in Dhaka, Bangladesh

To study the association of Helicobacter pylori with peptic ulcer and the associated histopathological changes, to characterize the isolated strains in terms of their protein profile, 83 peptic ulcer cases were studied. A high association of H pylori with peptic ulcer (duodenal ulcer 77%, gastric ulcer 75%) and gastritis (74%) was observed. Age and smoking did not have any relationship with H pylori infection. The infection was predominantly associated with the 'quiescent' form of chronic gastritis. Comparative sodium dodecyl sulfate polyacrylamide gel electrophoresis of whole cell extracts of the local isolates and a reference strain from Australia showed a general homogeneity between the strains with obvious interstrain differences. However, the difference between the local isolates and the reference strain was more marked. Significant association of H. pylori with peptic ulcer along with strain variations were observed.     

Binding of the Helicobacter pylori vacuolating cytotoxin to target cells

The vacuolating cytotoxin of Helicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.     

Helicobacter pylori in South America

Interviews of a representative sample of 201 physicians (general practitioners, gynecologists, pediatricians) with a mean age of 47 years show: there is a unmanageable amount of information about drugs and their risks. The physicians prefer practical experience rather than specialized literature. As a result they plead for a central authority of information with periodical reports on the current knowledge of drug-therapy and with the possibility of therapeutical consultation.     

Helicobacter pylori infection in children

BACKGROUND: Although immigrants to the United States are usually ethnic minorities and socioeconomically disadvantaged, foreign-born women generally have lower rates of low birth weight infants than do US-born women. OBJECTIVE: To measure the relationship between maternal birthplace, ethnicity, and low birth weight infants. DESIGN: Retrospective cohort study of birth certificate data. SETTING: California, 1992. SUBJECTS: Singleton infants (n = 497 868) born to Asian, black, Latina, and white women. MAIN OUTCOME MEASURES: Very low birth weight (500-1499 g), moderately low birth weight (1500-2499 g), and normal birth weight (2500-4000 g, reference category). RESULTS: Foreign-born Latina women generally had less favorable maternal characteristics than US-born Latinas, yet foreign-born Latina women were less likely to have moderately low birth weight infants (odds ratio, 0.91; 95% confidence interval, 0.86-0.96) than US-born Latinas after adjusting for maternal age, education, marital status, parity, tobacco use, use of prenatal care, and gestational age. While foreign-born Asian women generally had a less favorable profile of maternal characteristics than US-born Asians, there was no statistically significant difference in the odds of very low birth weight or moderately low birth weight infants between foreign- and US-born Asian women. Foreign-born black women had more favorable maternal characteristics than US-born women, but there was no significant nativity difference in very low birth weight or moderately low birth weight between foreign- and US-born black women after adjusting for maternal and infant factors. CONCLUSIONS: The relationship between maternal birthplace and low birth weight varies by ethnicity. Further study is needed to understand the favorable pregnancy outcomes of foreign-born Latina women.     

Analysis and typing of the vacA gene from cagA-positive strains of Helicobacter pylori isolated in Japan

Approximately 50% of Helicobacter pylori strains produce a cytotoxin that is encoded by vacA and that induces vacuolation of eukaryotic cells. Mosaicism in vacA alleles was reported, and there are three different families of vacA signal sequences (s1a, s1b, and s2) and two different families of middle-region alleles (m1 and m2). In addition, the vacA genotype of a strain is associated with its cytotoxin phenotype and its capacity to induce peptic ulceration. To clarify the strain diversity of H. pylori in Japan, 87 Japanese clinical isolates of H. pylori (40 from patients with chronic atrophic gastritis, 25 from patients with duodenal ulcer, 16 from patients with gastric ulcer, 3 from patients with both duodenal and gastric ulcers, and 3 from patients with intestinal type gastric cancer) were characterized by vacA typing by PCR and DNA sequencing. Eighty-four of the 87 isolates were s1a/m1, one was s1b/m1, and two could not be typed. Moreover, all isolates in this study were cagA positive. There were no distinct differences between the cytotoxin-producing strains and cytotoxin-nonproducing strains within the 0.73-kb middle region. Japanese strains were highly homologous, with more than 96% identity in this region, in which maximum divergence has been reported. In addition, there were no associations between the specific vacA types and the level of in vitro cytotoxin activity or the clinical consequences. These results indicate that the cagA-positive, s1a/m1-type strains are common in Japan, regardless of the vacA phenotype or clinical outcome.     

Comparison of five PCR methods for detection of Helicobacter pylori DNA in gastric tissues

Five different PCR methods for the detection of Helicobacter pylori were evaluated. The results of this study indicate that of the five PCR methods examined, the ureC (glmM) gene PCR is the most sensitive and specific for the detection of H. pylori in gastric biopsy specimens.