Helicobacter pylori cag pathogenicity island (PAI) is a major determinant of gastric injury via induction of several matrix metalloproteinases (MMPs). In the present study, we examined the influence of the cag PAI on gastric infection and MMP-9 production in mice and in cultured cells. A new mouse colonizing Indian H. pylori strain (AM1) that lacks the cag PAI was used to study the cag PAI importance in inflammation. Groups of C57BL/6 mice were inoculated separately with H. pylori strains AM1 and SS1 (cag ؉ ), gastric tissues were histologically examined, and bacterial colonization was scored by quantitative culture. Mice infected with either cag ؉ or cag ؊ H. pylori strains showed gastric inflammation and elevated MMP-3 production. Significant up-regulation of pro-MMP-9 secretion and gene expression in H. pylori infected gastric tissues indicate dispensability of cag PAI for increased pro-MMP-9 secretion and synthesis in mice. In agreement, cell culture studies revealed that both AM1 and SS1 were equipotent in pro-MMP-9 induction in human gastric epithelial cells. Both strains showed moderate increase in MMP-2 activity in vivo and in vitro. In addition, increased secretion of tumor necrosis factor (TNF)-␣, interleukin (IL)-1, and IL-6 induced pro-MMP-9 secretion and synthesis in AM1 or SS1 strain-infected mice suggesting elicitation of pro-inflammatory cytokines by both cag ؊ and cag ؉ genotype. Moreover, tissue inhibitors of metalloproteinase-1 expression were decreased with increase in pro-MMP-9 induction. These data show that H. pylori may act through different pathways other than cag PAI-mediated for gastric inflammation and contribute to upregulation of MMP-9 via pro-inflammatory cytokines.Helicobacter pylori is a microaerophilic bacterium with an extraordinary ability to chronically infect human stomachs for years together despite gastric mucosal turnover and other host defenses (1). It colonizes more than half of all people worldwide and is still unknown why most remain asymptomatic (2). Persistent H. pylori infection is associated with chronic gastritis and gastric cancer, one of the most lethal of malignancies worldwide (2-4). It may also cause childhood malnutrition and increase the risk or severity of infection by other gastrointestinal pathogens such as Vibrio cholerae, especially in developing countries (5, 6). H. pylori appears to be one of the most genetically diverse of bacterial species and shows significant geographic differences among strains (7-11). Studies of strains from Europe and North America indicated that H. pylori genotypes can be important in colonization and disease outcome (12). Recent studies revealed that Indian H. pylori strains are genetically distinct from European and East Asian strains (9, 10). Variation in the clinical outcome of H. pylori infection seems to be multifaceted and involves a complex interplay between virulence factors, host immune responses, and other features of the H. pylori gastric mucosal niche. Prominent among the H. pylori virulence-associated determin...
We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing countries.
SUMMARYBackground: Helicobacter pylori infection is very common in India, as in other developing countries, but few data exist on the susceptibility of H. pylori to antimicrobial agents commonly used for eradication here. Aim: To determine the antimicrobial susceptibility of H. pylori strains from Kolkata, in eastern India. Methods: A total of 67 H. pylori strains isolated from gastritis and peptic ulcer patients of Kolkata were examined in the study. Minimum inhibitory concentration to the antibiotics was determined by the agar dilution method.Results: Most of the strains (85%) were resistant to at least 8 lg/mL of metronidazole and 7.5% strains were resistant to tetracycline, which was high when compared with other reports in India. All Kolkata strains were highly sensitive to clarithromycin, furazolidone and amoxicillin. Conclusions: Our results differed significantly from the few available reports on drug sensitivity profile of H. pylori from other parts of India, namely, Hyderabad, Mumbai and Lucknow. This finding supports the need for rigorous susceptibility testing as a guide to empirical treatment and more generally, to define the resistance patterns of H. pylori in particular geographical areas.
Helicobacter pylori is one of the most diverse bacterial species that chronically infects more than 70% of Indian population. Interestingly, data showing microdiversity of the H. pylori strains within a particular gastric niche remained scarce. To understand the extent of genetic diversity among H. pylori strains within a given host, 30 patients with gastro-duodenal problems were subjected to endoscopy and from each patient 10 single colonies were isolated. Characterization of each of these 10 single colonies by DNA fingerprinting as well as genotyping of several important genetic markers viz. cagA, vacA, iceA, vapD, cag PAI empty site, IS605, RFLP and two other genetic segments within cag PAI revealed that all of the 30 patients were infected with more than one strain and sometimes strains with 5 to 6 types of genetic variants. Analyses of certain genetic loci showed the microdiversity among the colonies from single patient, which may be due to the recombination events during long-term carriage of the pathogen. These results suggest that most of the patients have acquired H. pylori due to repeated exposure to this pathogen with different genetic make-up, which may increase the possibility of super infections. Genetic exchanges between these unrelated H. pylori strains may support certain H. pylori variant to grow better in a given host than the parental strain and thereby increasing the possibility for the severity of the infection.
BackgroundInfection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India.ResultsSeventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants.ConclusionOur analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.
Several genes including the cagA in the cag pathogenicity island (cag PAI) of Helicobacter pylori are thought to be associated with the gastroduodenal diseases and hence variation in the genetic structure of the cag PAI might be responsible for different clinical outcomes. Our study was undertaken to characterize the cag PAI of H. pylori strains from duodenal ulcer (DU) patients and asymptomatic or non-ulcer dyspepsia (NUD/AV) subjects from Kolkata, India. Strains isolated from 52 individuals (30 DU and 22 NUD/AV) were analyzed by PCR using 83 different primers for the entire cag PAI and also by dot-blot hybridization. Unlike H. pylori strains isolated from other parts of India, 82.6% of the strains used in this study had intact cag PAI, 9.6% had partially deleted cag PAI, and 7.7% of the strains lacked the entire cag PAI. Dot-blot hybridization yielded positive signals in 100% and 93.8% of PCR-negative strains for HP0522-523 and HP0532-HP0534 genes, respectively. An intact cagA promoter region was also detected in all cagApositive strains. Furthermore, the expression of cagA mRNA was confirmed by RT-PCR for the representative strains from both DU and NUD/AV subjects indicating the active cagA promoter regions of these strains. A total of 66.7% of Kolkata strains produced a ~390-bp shorter amplicon than the standard strain 26695 for the HP0527 gene, homologue of virB10. However, sequence analyses confirmed that the deletion did not alter the reading frame of the gene, and mRNA transcripts were detected by RT-PCR analysis. The strains isolated from DU and NUD/AV express CagA protein and possess a functional type IV secretion system, as revealed by Western blot analyses. Interestingly, no significant differences in cag PAI genetic structure were found between DU and NUD/AV individuals suggesting that other bacterial virulence factors, host susceptibility, and environmental determinants also influence the disease outcome at least in certain geographical locations.
The prevalence of different H. pylori genotypes in various geographical regions indicates region-specific adaptations during the course of evolution. Complete genomes of H. pylori from countries with high infection burdens, such as India, have not yet been described. Herein we present genome sequences of two H. pylori strains, NAB47 and NAD1, from India. In this report, we briefly mention the sequencing and finishing approaches, genome assembly with downstream statistics, and important features of the two draft genomes, including their phylogenetic status. We believe that these genome sequences and the comparative genomics emanating thereupon will help us to clearly understand the ancestry and biology of the Indian H. pylori genotypes, and this will be helpful in solving the so-called Indian enigma, by which high infection rates do not corroborate the minuscule number of serious outcomes observed, including gastric cancer.
RESULTS: Intraglandular neutrophil infiltration, a hallmark of activity of gastritis, was present in 34 (94%) of tribals (TRs) and 42 (84%) of DU individuals infected with H pylori . Lymphoid follicles and aggregates, which are important landmarks in H pylori infection, were positive amongst 15 (41%) of TRs and 20 (40%) of DU subjects. Atrophic changes were observed in 60% and 27.7%, respectively, among DU cases and tribals (P > 0.003). Metaplastic changes were detected in low numbers in both groups. Moderate to severe density distribution of H pylori in the gastric mucosa was 63% among TRs, whereas it was 62% in DU subjects. There were no significant differences in the distribution of virulence-associated genes like cagA , vacA and iceA of H pylori strains carried by these two populations. CONCLUSION:Our study showed almost similar distribution of inflammatory cells among asymptomatic tribals and DU Bengali patients. Interestingly, the tribal population are free from any clinical symptoms despite evidence of active histologic gastritis and infection with H pylori strains carrying similar virulence markers as of strains isolated from patients with DU. There was an increased cellular response, especially in terms of neutrophil infiltration, but much lower risk of developing atrophy and metaplastic changes among the tribal population.
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