Lack of a relationship between Lewis antigen expression and cagA, CagA, vacA and VacA status of Irish Helicobacter pylori isolates

The cagA gene, vacA gene, CagA (cytotoxin-associated gene A product) and VacA (vacuolating cytotoxin) status of a collection of Helicobacter pylori isolates from the geographically distinct Irish population was determined, the potential association of these traits with Lewis (Le) antigen expression was assessed, and the relationship between these bacterial properties and the pathology associated with H. pylori infection was evaluated. Of the 57 isolates, a higher proportion from ulcer than from non-ulcer patients expressed VacA (71% vs. 53%). H. pylori isolates which were cagA-positive were no more significantly associated with peptic ulcers than non-ulcer disease (71% vs. 67%, P = 0.775), nor were CagA-positive isolates (57% vs. 50%, P = 0.783), but 80% of the isolates from duodenal ulcer patients were cagA-positive. Thirty-seven of the 57 isolates were tested for Le antigen expression. No statistically significant relationship (P > 0.05) was found between the occurrence and level of expression of Le(x) or Le(y) and cagA, vacA, or VacA status. This lack of an association in the Irish H. pylori isolates contrasts with that previously reported for predominantly North American isolates, and may be attributable to the adaptation of H. pylori strains with differing attributes to different human populations.     

Role of futC slipped strand mispairing in Helicobacter pylori Lewisy phase variation

The O antigen of the Helicobacter pylori lipopolysaccharide is composed of repeating units of fucosylated Lewis (Le) antigens. The alpha(1,2)-fucosyltransferase (futC) of H. pylori, which catalyzes the conversion of Le(x) to Le(y) by addition of fucose, is subject to slipped-strand mispairing involving a homonucleotide (poly-C) tract. To explore the distribution of Le phenotypes within H. pylori cells grown in vitro, 379 single colonies of strain J166 were examined for Le expression. Two major populations with reciprocal Le(x)/Le(y) phenotypes were identified. Phenotypes correlated with futC frame status, suggesting that strain J166 represents a mixed population with respect to futC poly-C tract length, which was confirmed by a translational reporter. After hundreds of generations in vitro, phenotypes did not change significantly, indicating that the observed J166 Le diversity reflects the founding population. Since slipped-strand mispairing in the futC poly-C tract was postulated to explain the Le(y) phenotypic change observed in J166 derivative strain 98-169 isolated 10 months after rhesus monkey challenge, in trans complementation with in-frame futC was performed. Le(y) synthesis was restored and Le(x) expression was reciprocally lowered. From these studies, we confirmed the principal role of futC slipped-strand mispairing in Le antigenic variation in vitro and in vivo.     

兰州地区幽门螺杆菌分离株主要毒力基因的研究

本文首次报道了兰州地区胃病患者幽门螺杆菌分离株主要毒力基因ｕｒｅＡ ｖａｃＡ 和ｃａｇＡ的 ＰＣＲ 检测情况。共获 ４１ 株Ｈｐ 分离株,分别来自于慢性胃炎病人（３２ 株）、胃－十二指肠溃疡病人（７株）和胃癌病人（２ 株）。检测结果表明,４１ 株Ｈｐ 分离株的ｕｒｅＡ,ｖａｃＡ 及ｃａｇＡ 的阳性率分别为１００％ ,１００％ 和９７．６％ ;含有ｕｒｅＡ,ｖａｃＡ 和ｃａｇＡ 基因的Ｈｐ 与人类胃部疾患密切相关,而ｃａｇＡ 基因的存在可能与更加严重的胃部疾病有关。Ｈｐ 毒力基因的检测结果与其它地区Ｈｐ 分离株的检测结果相似。作者建议,对ｕｒｅＡ 基因的ＰＣＲ 检测可以作为鉴定Ｈｐ 的一个指标。     

Genotypic,phenotypic, and clinical characteristics of isolates of<Emphasis Type="Italic">Helicobacter pylori</Emphasis> from San Luis,Argentina

The vacA and cagA genotypes of Helicobacter pylori exhibited distinct geographic distribution and correlation with severity of disease. In the above genotypes (obtained from 150 H. pylori-positive patients--139 with gastritis, 10 with ulcer and 1 patient with gastric cancer) combinations vacA s1/m1 and s2/m2 were detected using PCR in 75 and 25% of isolates, respectively, in patients with chronic gastritis. The of s1/m1 and s2/m2 combinations were also detected from ulcers (60 and 40%, respectively). The cagA was detected in 30% of isolates. Concentrated culture supernatants of 7 (64%) out of 11 H. pylori strains induced vacuolization in Vero cells in titers ranging from 1:5 to 1:40. The vacA s1 genotype was significantly associated with, but not predictive of the presence of vacuolating cytotoxin activity and the cagA gene.     

Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil

Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified. This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H. pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil. Gastric biopsy of 61 H. pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction. Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT). The prevalence of H. pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p=0.02). The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2. However, the prevalence of the vacA s1 genotyping was the same in either DU or GT group. The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b. In resume there was a strong association between the H. pylori cagA+ gene and DU. However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominant.     

Carbohydrate-dependent inhibition of Helicobacter pylori colonization using porcine milk

Breast milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. In a previous study, we showed that pigs express the Helicobacter pylori receptors, sialyl Lewis x (Le x) and Le b, on various milk proteins. Here, we investigate the pig breed- and individual-specific expression of these epitopes, as well as the inhibitory capacity of porcine milk on H. pylori binding and colonization. Milk proteins from three different pig breeds were analysed by western blotting using antibodies with known carbohydrate specificity. An adhesion assay was used to investigate the capacity of pig milk to inhibit H. pylori binding to neoglycoproteins carrying Le b and sialyl-di-Le x. alpha1,3/4-fucosyltransferase transgenic FVB/N mice, known to express Le b and sialyl Le x in their gastric epithelium, were colonized by H. pylori and were subsequently treated with Le b- and sialyl Le x-expressing or nonexpressing porcine milk, or water (control) only. The degree of H. pylori colonization in the different treatment groups was quantified. The expression of the Le b and sialyl Le x carbohydrate epitopes on pig milk proteins was breed- and individual specific and correlated to the ability of porcine milk to inhibit H. pylori adhesion in vitro and H. pylori colonization in vivo. Milk from certain pig breeds may have a therapeutic and/or prophylactic effect on H. pylori infection.     

Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA, babA2 genotypes and correlation with clinical outcome in Turkish patients with dyspepsia

BACKGROUND: Distinct virulence factors of Helicobacter pylori have been associated with clinical outcome of the infection; however, considerable variations have been reported from different geographic regions and data on genotypes of Turkish H. pylori isolates are sparse. AIM: To determine the prevalence of specific genotypes of H. pylori in Turkish patients with dyspepsia. MATERIALS AND METHODS: Ninety-three H. pylori-positive patients [30 with non-ulcer dyspepsia (NUD), 30 with duodenal ulcer (DU), and 33 with gastric cancer (GC)] who were admitted to our endoscopy unit due to dyspepsia were enrolled in the study. H. pylori infection was confirmed in all patients by histology and rapid urease test (RUT). The presence of vacA alleles, cagA, cagE, iceA, and babA2 genotypes were determined by polymerase chain reaction (PCR). Chi-squared test and Fisher's exact test were used for statistical comparisons and multivariate regression analysis was performed to find out independent predictors of different clinical outcomes. RESULTS: Turkish strains examined predominantly possessed the vacA s1,m2 (48.4%) and s1,m1 (40.7%) genotypes. The vacA s1a genotype was detected in 66.7, 96.4, and 87.9% of isolates from patients with NUD, DU, and GC, respectively, and its presence was significantly associated with that of DU (p = .004), GC (p = .043), and cagA gene (p = .021). None of the cases was found to harbor the s1c genotype. The frequencies of the cagA and cagE genes among studied isolates were 73.6 and 59.3%, respectively. The cagA gene was significantly associated with the presence of DU (p = .004) and GC (p = .003), and the cagE gene, too, was significantly associated with the presence of DU (p = .002) and GC (p = .000). All H. pylori isolates possessed the iceA gene. In all, 68 isolates (74.7%) were positive for iceA1 and 23 (25.3%) for iceA2. The frequency of icea1 gene was significantly higher in cases with GC (85%) than in cases with NUD (60%) (p = .026). The frequency of babA2 gene was 23.3, 46.4, and 87.9% in isolates of patients with NUD, DU, and GC, respectively. When compared to cases with NUD (p = .000) and DU (p = .000), the presence of babA2 gene was significantly higher in cases with GC. Multivariate regression analysis disclosed cagE (p = .006) and vacA s1a (p = .027) genotypes to be independent predictors of DU and babA2 (p = .000) and cagE (p = .013) genotypes to be independent predictors of GC. CONCLUSIONS: H. pylori vacA s1a, cagA, cagE genotypes have significant relations with the presence of DU and GC, and iceA1, babA2 with GC in Turkish patients with dyspepsia, whereas cagE and vacA s1a genotypes are independent predictors of DU, and babA2 and cagE genotypes are independent predictors of GC.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

High-level genetic diversity in the vapD chromosomal region of Helicobacter pylori.

Helicobacter pylori isolates from different patients are characterized by diversity in the nucleotide sequences of individual genes, variation in genome size, and variation in gene order. Genetic diversity is particularly striking in vacuolating cytotoxin (vacA) alleles. In this study, five open reading frames (ORFs) were identified within a 4.2-kb region downstream from vacA in H. pylori 60190. One of these ORFs was closely related to the virulence-associated protein D (vapD) gene of Dichelobacter nodosus (64.9% nucleotide identity). A probe derived from vapD of H. pylori 60190 hybridized with only 19 (61.3%) of 31 H. pylori strains tested. Sequence analysis of the vapD region in vapD-negative H. pylori strains revealed that there were two different families of approximately 0.5-kb DNA segments, which were both unrelated to vapD. The presence of vapD was not associated with any specific family of vacA alleles. These findings are consistent with a recombinational population structure for H. pylori.     

A structural comparison of lipopolysaccharides from two strains of Helicobacter pylori, of which one strain (442) does and the other strain (471) does not stimulate pepsinogen secretion.

Lipopolysaccharides (LPSs) from strains of Helicobacter pylori (442 and 471), which differed in stimulation of pepsinogen secretion, were isolated as water-soluble material of high-M(r), and as water-insoluble gels of low-M(r). Chemical and spectroscopic analyses of soluble LPS and oligosaccharides liberated from the gels led to proposed structures with Lewis (Le) antigen termini connected to N -acetyllacto-saminoglycans of alternating 3-linked beta-D-Gal and 4-linked beta-D-GlcNAc residues with various laterally attached glycosyl substituents. The LPS of H.pylori 442 was similar to previously examined strains (NCTC 11637 and P466) in having partially glycosylated chains with alpha-L-Fuc units attached to O-3 of the majority of GlcNAc residues in Le(x)units, and in chain termination with Le(x)or Le(y)determinants. In contrast, terminal Le(y)units occurred in LPS of H.pylori 471 and glycosaminoglycan chains carried a smaller proportion of alpha-L-Fuc units, but at O-6 of a majority of nonfucosylated GlcNAc residues, there was a novel type of branching with alpha-D-Gal substituents. Evidence for the branched regions was obtained from(1)H-NMR spectra and from characterization of oligosaccharides formed by the action of endo-beta-galactosidase. Examination of oligosaccharides liberated from water-insoluble LPS gels of H.pylori 442 and 471 provided evidence for similar core OS structures to those from other H.pylori strains but interesting differences were observed.     

Isolation and genotyping of Helicobacter pylori from untreated municipal wastewater

For this study, we isolated Helicobacter pylori from wastewater by a series of steps beginning with immunomagnetic separation and cell culture. After Gram staining and three standard microbial tests, the 16S rRNA sequences of a total of 23 out of 37 putative H. pylori isolates were verified by PCR. Eleven H. pylori isolates were genotyped and fell into four vacA classes: those with the vacA allelic variants s1a and m1, s1b and m1, s2 and m2, or s2 and m1. Most H. pylori isolates were of the vacA s1a/m1 type, which has been shown to be associated with advanced diseases based on genotyping of H. pylori from gastric cancer patients. These results demonstrated that H. pylori survives in water and may be a potential source of H. pylori transmission, especially where water is not adequately treated.     

Binding of lactoferrin and free secretory component to enterotoxigenic Escherichia coli

To compare the genomic variation of Helicobacter pylori in samples obtained from patients with perforated peptic ulcer, living in the same area of Estonia but belonging to different nationalities, 50 non-consecutive patients (32 Estonians and 18 Russians) admitted in the Tartu University Hospital in 1997-1999 were studied. Gastric samples of antral mucosa were obtained during operation and analysed histologically and with PCR for detection of different genotypes of H. pylori (cagA and vacA s and m subtypes). Among the 50 perforated peptic ulcer patients with histologically proven H. pylori colonisation no sample of gastric mucosa showed the s1b subtype of the vacA gene. The perforated peptic ulcer patients were mainly infected with cagA (82%) and s1 (98%) genotypes of H. pylori. The distribution of s1a/m1, s1a/m2 and s2/m2 subtypes of vacA genes was statistically different in Estonian and Russian patients (P<0.05). In conclusion differences in the distribution of vacA s and m subtypes of H. pylori were revealed between Estonian and Russian patients with perforated peptic ulcer from Southern Estonia.     

Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants

Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le(b)) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le(b) positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le(a), Le(b), Le(x), and Le(y)) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le(a) and Le(b) positive. 1C5 expressed Le(b) on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le(b) on N-, O-glycans, and GLs. Furthermore, both clones expressed Le(a) on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le(y) on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le(x) on GLs. A Le(b)-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le(b)-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.     

Diversity of Helicobacter pylori cagA and vacA genes in Costa Rica: its relationship with atrophic gastritis and gastric cancer

BACKGROUND: Associations between Helicobacter pylori gene diversity and gastric cancer have not been reported on in Costa Rica, despite its being one of the countries with the highest gastric cancer incidence and mortality rates in the world. The aim of this study was to determine the prevalence of H. pylori cagA and vacA genes and investigate whether it could be correlated with atrophic gastritis (AG) and gastric cancer (GC) in Costa Rica. MATERIALS AND METHODS: Genomic DNAs from isolates of 104 patients classified into two groups: non-atrophic gastritis group (n = 68) and atrophic gastritis group (n = 36), were subjected to PCR-based genotyping of cagA and vacA genes and their correlation with clinical outcome was investigated. Total DNA extractions from gastric tissues of 25 H. pylori-infected gastric cancer patients were utilized for comparative purposes. RESULTS: The presence of cagA (75.3%), vacA s1b (75.3%), and vacA m1 (74.2%) was detected, and colonization by strains with different vacA genotypes in the same stomach was found in 9.7% of the patients. Age- and sex-adjusted vacA s1b and vacA m1 were associated with GC while only vacA m1 was significantly associated with AG. A tendency for association between cagA and vacA s1b, and AG was reported. CONCLUSIONS: The prevalence status of the cagA and vacA (s1/m1) genes in Costa Rica seems to fall between that found in European/North American and East Asian countries, and both cagA and vacA seem to have clinical relevance in this country.     

Phenotypic variation in molecular mimicry between Helicobacter pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms. Acid-induced phase variation in Lewis(x) and Lewis(y) expression by H. Pylori lipopolysaccharides.

Helicobacter pylori is an important gastroduodenal pathogen of humans whose survival in the gastric environment below pH 4 is dependent on bacterial production of urease, whereas above pH 4 urease-independent mechanisms are involved in survival, but that remain to be elucidated fully. Previous structural investigations on the lipopolysaccharides (LPSs) of H. pylori have shown that the majority of these surface glycolipids express partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine (LacNAc) O-polysaccharide chains containing Lewis(x) (Le(x)) and/or Lewis(y) (Le(y)), although some strains also express type 1 determinants, Lewis(a), Lewis(b), and H-1 antigen. In this study, we investigated acid-induced changes in the structure and composition of LPS and cellular lipids of the genome-sequenced strain, H. pylori 26695. When grown in liquid medium at pH 7, the O-chain consisted of a type 2 LacNAc polysaccharide, which was glycosylated with alpha-1-fucose at O-3 of the majority of N-acetylglucosamine residues forming Le(x) units, including chain termination by a Le(x) unit. However, growth in liquid medium at pH 5 resulted in production of a more complex O-chain whose backbone of type 2 LacNAc units was partially glycosylated with alpha L-fucose, thus forming Le(x), whereas the majority of the nonfucosylated N-acetylglucosamine residues were substituted at O-6 by alpha-D-galactose residues, and the chain was terminated by a Le(y) unit. In contrast, detailed chemical analysis of the core and lipid A components of LPS and analysis of cellular lipids did not show significant differences between H. pylori 26695 grown at pH 5 and 7. Although putative molecular mechanisms affecting Le(x) and Le(y) expression have been investigated previously, this is the first report identifying an environmental trigger inducing phase variation of Le(x) and Le(y) in H. pylori that can aid adaptation of the bacterium to its ecological niche.     

Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules,the selectins--a preliminary report

The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer. We are exploring the possibility that carbohydrate components on H. pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. Lipopolysaccharides of most H. pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens. Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes. Here we investigate H. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins. Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.     

IgG antibodies to Lewis type 2 antigens in serum of H. pylori-infected and noninfected blood donors of different Lewis(a,b) blood-group phenotype

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.     

Clinical and pathological importance of vacA allele heterogeneity and cagA status in peptic ulcer disease in patients from North Brazil

We have examined the prevalence of gene cagA and vacA alleles in 129 patients, 69 with gastritis and 60 with peptic ulcer diseases from North Brazil and their relation with histopathological data. vacA and cagA genotype were determined by polymerase chain reaction. Hematoxylin-eosin staining was used for histological diagnosis. 96.6% of the patients were colonized by Helicobacter pylori strains harboring single vacA genotype (nont-mixed infection). Among them, 11.8% had subtype s1a, 67.8% had subtype s1b, and 17% subtype s2. In regard to the middle region analysis, m1 alleles were found in 75.4% and m2 in 21.2% of patients. The cagA gene was detected in 78% patients infected with H. pylori and was associated with the s1-m1 vacA genotype. The H. pylori strains, vacA s1b m1/cagA-positive, were associated with increased risk of peptic ulcer disease and higher amounts of lymphocytic and neutrophilic infiltrates and the presence of intestinal metaplasia. These findings show that cagA and vacA genotyping may have clinical relevance in Brazil.     

Conservation and microdiversity of the phospholipase A (pldA) gene of Helicobacter pylori infecting dyspeptics from different countries

Phospholipase activity is important in bacterial pathogenicity and could contribute to the pathogenic role of Helicobacter pylori by degradation of the gastric mucus, and in maintaining long-term colonisation. Our aim was to determine the degree of variation in the phospholipase A gene (pldA) of H. pylori from different geographic locations, and to investigate links between pldA genotype and clinical disease severity, as well as with variation in cagA status and vacA genotypes. PCR-restriction fragment length polymorphism (RFLP) analysis with MboI and HaeIII was used to study 124 isolates from 10 countries that included the two genome-sequenced strains (26695 and J99), as well as Tx30a and NCTC 11637 (type strain). The 925-bp pldA fragment was amplified with a frequency of 90%. The presence of pldA was confirmed in the other strains using an alternative forward primer. Isolates were distinguished by PCR-RFLP analysis with 10 MboI and four HaeIII restriction patterns that combined to give 25 distinct pldA RFLP types. The pldA M2H2 strain genotype was most common (20%) in the UK but similar strains came from several other countries. Microdiversity was evident in pldA sequences of strains representing different RFLP types, and five M2H2 strains each had a distinct pldA sequence type. Intragenic variation was independent of gastric disease severity as well as strain cagA status and vacA genotype, with the exception of eight geographically diverse strains all with the pldA M4H3/cagA+/vacA s1m1 genotype predominantly from peptic ulcer patients. The study indicated a spectrum of genotypic variants and was supportive of a pldA function in H. pylori colonisation and persistence rather than in chronicity of infection.