Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique

Kansau, Imad; Raymond, Josette; Bingen, Édouard; Courcoux, P.; Kalach, Nicolas; Bergeret, M.; Braimi, N.; Dupont, Christophe; Labigne, Agnès

doi:10.1016/0923-2508(96)84023-x

Cited by 126 publications

(86 citation statements)

References 18 publications

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“…The data also indicated that small deletions, such as the ones that we observed, do not usually affect DNA fragment migration; the result is similar RAPD patterns (1). Identity among strains was confirmed by sequencing of the internal 294-bp fragment of the glmM gene, which has been shown to be a highly polymorphic region in all H. pylori strains (19). The association between the presence of single genes of the cag island and both serological expression and clinical expression of infection with cagA-positive H. pylori strains has been studied (34,30).…”

Section: Discussionmentioning

confidence: 99%

Heterogeneity of cag Genotypes in Helicobacter pylori Isolates from Human Biopsy Specimens

Tomasini¹,

Zanussi²,

Sozzi

et al. 2003

J Clin Microbiol

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The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene (cag) island may have different compositions in infecting strains. In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA-positive H. pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA-negative strains. Three loci in the cag island (cagA, cagE, and virB11) and the conserved gene glmM (ureC) were investigated by PCR. The levels of anti-H. pylori and anti-CagA antibodies in patient sera were also measured. For subjects infected with cagA-negative strains, all sweep isolates were also negative for cagE and virB11, suggesting the complete absence of the cag island. For subjects infected with cagA-positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island. cagA, cagE, and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies. The deletion of virB11 was always associated with the deletion of cagA and/or cagE. H. pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment. Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical. All of the patients had detectable antibodies against H. pylori, and 9 of 10 had anti-CagA antibodies. In conclusion, we show that a single infecting H. pylori strain may include variable proportions of colony subtypes with different cag genotypes. The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H. pylori infections.Helicobacter pylori is a gram-negative spiral bacterium that colonizes the human stomach. Infection with H. pylori is associated with chronic gastritis, peptic ulcer, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma (25).Although the pathogenesis of H. pylori infection is not completely understood, several putative virulence factors may contribute to mucosal damage (5, 26). The cytotoxin-associated gene (cag) island, an approximately 40-kb cluster of genes in the H. pylori chromosome, probably was inherited by horizontal transfer from an unknown microorganism. Many of the cag genes have homology to those of other bacteria encoding virulence factors and a type IV secretion system. Therefore, they may play an important role in microbial virulence and pathogenicity (6, 10). Among H. pylori isolates, the presence of insertion elements causes differences in the compositions of the cag island; the consequent cag instability may produce d...

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Section: Discussionmentioning

confidence: 99%

Heterogeneity of cag Genotypes in Helicobacter pylori Isolates from Human Biopsy Specimens

Tomasini¹,

Zanussi²,

Sozzi

et al. 2003

J Clin Microbiol

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“…Antibiotics for the selection of recombinant E. coli strains were used at the following final concentrations (in g ml ¹1 ): ampicillin, 100; kanamycin, 50; and tetracycline, 20. H. pylori strains, N6 , 85P (Labigne et ᮊ 1997 Blackwell Science Ltd, Molecular Microbiology, 25, 989-998 al., 1991) and the 45 clinical isolates (Kansau et al, 1996) were grown on a horse blood (10%) agar medium containing antibiotics at the following final concentrations: vancomycine, 10 g ml…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Skouloubris

Labigne

Reuse

1997

Molecular Microbiology

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SummaryWe report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37 746 Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6-836. In a N6-urease negative mutant, amidase activity was two-to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.

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“…In most cases, Helicobacter-like organisms were identified to be H. felis, and H. pylori was not detected in any of these animals before experimental infection. After H. pylori infection, rapid urease test, PCR, and culture were used to check gastric infestation according to procedures described elsewhere by ourselves and others (21,23,30). Distinction between H. felis and H. pylori was based, if necessary, on two PCR tests, with primer sequences chosen for amplification on 16S RNA and U3 urease genes as previously described (22,30,45).…”

Section: Bacteriamentioning

confidence: 99%

Putative effect ofHelicobacter pyloriand gastritis on gastric acid secretion in cat

Sobhani

Cañedo

Alchepo

et al. 2002

American Journal of Physiology-Gastrointestinal and Liver Physi

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Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique

Cited by 126 publications

References 18 publications

Heterogeneity of cag Genotypes in Helicobacter pylori Isolates from Human Biopsy Specimens

Heterogeneity of cag Genotypes in Helicobacter pylori Isolates from Human Biopsy Specimens

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Putative effect ofHelicobacter pyloriand gastritis on gastric acid secretion in cat

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