Draft Genome Sequences of Helicobacter pylori Strains HPARG63 and HPARG8G, Cultured from Patients with Chronic Gastritis and Gastric Ulcer Disease.

Helicobacter pylori colonizes the human gastric mucosa, leading to a spectrum of gastric diseases in susceptible populations. Here we announce the draft genome sequences of strains HPARG8G and HPARG63. The data for both genome sequences provide insights regarding the diversity in gene content and rearrangement of the genomic islands commonly harbored by H. pylori.

Helicobacter pylori heterogeneity in patients with gastritis and peptic ulcer disease.

Genetic diversification allows Helicobacter pylori to persist during chronic colonization/infection. We investigated the intra-host variation of several markers that suggested microevolution in patients with chonic gastritis (CG) and peptic ulcer disease (PUD). One-hundred twenty-six isolates recovered from 14 patients with CG and 13 patients with PUD were analysed. cag pathogenicity island (cagPAI), oipA, vacA, bab gene status and the presence of jhp0926, jhp0945, jhp0947, jhp0949 and jhp0940 genes from the genomic Plasticity Zone (PZ) were taken into accout to investigate intra-host variation. lspA-glmM-RFLP was performed to identify mixed infections. Only one patient was colonised/infected by two ancestrally unrelated strains. Among the 126 isolates, a significant association among cagPAI genotypes, oipA status and vacA alleles was indicated. Complete cagPAI, oipA "on", and vacA s1-m1 variants were significantly found in patients with PUD, without intra-host variations. Isolates from 7/14 patients with CG lacked babA in all chromosomal loci. In contrast, isolates from all or several biopsies of PUD patients carried babA, but in one patient only, the isolates showed positive Lewis b (Leb) binding assay. Considering cagPAI, vacA, oipA, bab genotypes, intra-host variation was also significantly higher in patients with CG. Conversely, a similarly high intra-host variation in almost PZ genes was observed in isolates from patients with CG and PUD. In conclusion, the lowest intra-host variation in cagPAI, oipA, vacA, and bab genes found in patients with PUD suggests the selection of a particular variant along the bacteria-host environment interplay during ulceration development. However, the predominance of this variant may be a refletion of the multifactorial etiology of the disease rather than the cause, as it was also found in patients with CG. The intra-host variation in PZ genes may predict that this genomic region and the other markers of microevolution studied evolve under diverse pressure(s).

Helicobacter pylori oipA, vacA and dupA genetic diversity in individual hosts.

Helicobacter pylori putative virulence factors can undergo a continuously evolving mechanism as an approach to bacterial adaptation to the host changing environment during chronic infection. oipA, vacA and dupA genetic diversity among isolates from multiple biopsies (niches) from the antrum and corpus of 40 patients was investigated. A set of 229 isolates was examined. Direct DNA sequence analysis of amplified fragments was used to study oipA 'on/off' expression status as well as the presence of C or T insertion in jhp0917 that originates a continuous (jhp0917-jhp0918) dupA gene. vacA alleles were identified by multiplex PCR. Different inter-niches oipA CT repeat patterns were observed in nine patients; in six of these, 'on' and 'off' mixed patterns were found. In three of these nine patients, different vacA alleles were also observed in a single host. Inter-niche dupA differences involved the absence and presence of jhp0917 and/or jhp0918 or mutations in dupA, including those that may originate a non-functional gene, and they were also present in two patients with mixed oipA CT patterns and in another seven patients. Evidence of mixed infection was observed in two patients only. In conclusion, oipA and dupA genes showed similar inter-niche variability, occurring in approximately 1/4 patients. Conversely, vacA allele microevolution seemed to be a less common event, occurring in approximately 1/10 patients, probably due to the mechanism that this gene evolves 'in vivo'.

Helicobacter pylori amoxicillin heteroresistance due to point mutations in PBP-1A in isogenic isolates.

OBJECTIVES: To investigate the Helicobacter pylori amoxicillin resistance rate, the occurrence of heteroresistance, and their related molecular mechanisms. METHODS: Eighty-seven H. pylori-positive patients were included: 45/87 with single biopsy and 42/87 with multiple biopsies. MICs were determined, and sequencing analysis of pbp1A gene and the variable regions of seven hop porins was performed in resistant and susceptible isolates. Clonal relationships were determined by lspA-glmM-RFLP and by random amplification of polymorphic DNA-PCR. An isogenic amoxicillin-susceptible isolate was transformed with pbp1A PCR products from the resistant isolates. RESULTS: Amoxicillin-resistant (MIC 2 mg/L) and amoxicillin-susceptible (MIC 0.06 mg/L) isolates, belonging to the same strain, were observed in different biopsies in one patient (inter-niche heteroresistance). Isolates from the remaining patients were amoxicillin-susceptible. Sequencing analysis of the pbp1A of two amoxicillin-resistant isolates and their susceptible partners revealed the same two point mutations: (i) in the third PBP motif of the resistant isolates (C1667G); (ii) a nonsense mutation at the 3' end of the gene. Replacement of pbp1A of a susceptible isolate by pbp1A from a resistant isolate increased the transformants MICs (2 mg/L). A similar MIC was observed when a pbp1A DNA fragment including both point mutations was transformed. Transfer of the smallest fragment (C1667G region only) yielded slightly lower MICs (0.5-1 mg/L). Identical hop gene sequences were observed in paired susceptible and resistant isolates. CONCLUSIONS: A low resistance rate was observed. However, inter-niche heteroresistance could hinder amoxicillin resistance detection when only one biopsy is obtained. Alteration in PBP-1A seems to be enough to reach an MIC of 2 mg/L in our resistant isolates.

Helicobacter pylori cag pathogenicity island genotype diversity within the gastric niche of a single host.

cag pathogenicity island (PAI) integrity was investigated in isolates from multiple biopsies recovered from 40 patients in an attempt to determine the co-existence of a varying cagPAI-positive to cagPAI-negative ratio in a single host. Six biopsies were obtained from each patient during the same endoscopic session. cagPAI analysis included amplification of seven loci (cagA, cagE, cagG, cagM, cagT, HP0527 and HP0524) and the left end of cagII (LEC). Absence of the island was confirmed by empty-site PCR. lspA-glmM RFLP and random amplified polymorphic DNA PCR were used for strain delineation. The number of biopsies with Helicobacter pylori-positive culture ranged from three to six per patient and a total of 218 isolates were recovered. Mixed infection was only found in two patients. Nearly one-third of the 40 patients harboured isolates with an intact cagPAI in all niches, another third of the isolates were empty-site-positive in all niches, whilst the remaining third of the isolates had a disrupted cagPAI in all or at least one of the niches. Co-existence of variants of the same strain with different cagPAI genotypes was observed in one-quarter of patients. The variations in cagPAI genotype included co-existence of: diverse cagPAI deletions in different niches, variants with intact and with partially deleted islands, variants with empty-site-positive and with partially deleted cagPAIs, and variants with an intact cagPAI and with empty-site-positive. Half of the patients with different cagPAI genotypes harboured an intact cagPAI in at least one niche. Co-existence of diverse genotypes of putative virulence factors in a single host must be considered when drawing a correlation with clinical presentation.

DNA sequence analysis of rdxA and frxA from paired metronidazole-sensitive and -resistant Helicobacter pylori isolates obtained from patients with heteroresistance.

Genomic variations of rdxA and frxA genes in eight pairs of metronidazole (MTZ)-sensitive and -resistant isolates of Helicobacter pylori were investigated. The paired strains from each single biopsy had identical lspA-glmM restriction fragment length polymorphism profiles, and the differences in MTZ susceptibility were mostly accounted for by mutations in the rdxA gene. Truncation of RdxA is associated with a minimum inhibitory concentration of 64-128 mg/L. Early truncation of FrxA was observed both in susceptible and resistant isolates of seven paired strains. Inactivation of frxA might play a significant role only in one low-level MTZ-resistant isolate where a missense mutation was found in the rdxA gene.