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1 Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
2 Servicio de Endoscopia, Hospital General de Agudos Juan A Fernández, Buenos Aires, Argentina
3 Servicio de Gastroenterología, Hospital Escuela ‘Don José de San Martín’ Facultad de Medicina, Buenos Aires, Argentina
Correspondence
Mariana Catalano
catalanoma{at}gmail.com.ar
Received 10 August 2006
Accepted 9 January 2007
Abbreviations: LEC, left end of the cagPAI; PAI, pathogenicity island; RAPD, random amplified polymorphic DNA.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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It is known that initial colonization of the gastric mucosa by a founder H. pylori strain leads to persistent infection (Marshall et al., 1998). Over a period of time, bacterial subpopulations with highly similar genomes colonizing different regions of the stomach can emerge (Marshall et al., 1998; Blaser & Berg, 2001). Rearrangement of the cagPAI appears to be a prevalent phenomenon, and its constituent genes could be under more selection pressure than other regions in the chromosome (Kauser et al., 2004). Thus a varying ratio of cagPAI-positive to cagPAI-negative isolates could co-exist within a single host as a way of avoiding host constraints and ensuring H. pylori persistence. In an attempt to demonstrate this co-existence, PCR-based genotyping approaches were used to investigate cagPAI integrity in paired isolates recovered from multiple biopsies of single hosts.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Six biopsy specimens were obtained from each patient using FB-24KR Olympus biopsy forceps: A1, from the mid greater curvature of the antrum; A2, from the greater curvature facing the incisura angularis; A3, from the antral lesser curvature within 2 cm of the pylorus; C1, from the middle portion of the greater curvature of the corpus; C2, from the greater curvature within 3 cm proximal to the antral–corpus border; and C3, from the lesser curvature within 3 cm of the Z line.
Bacterial culture and DNA extraction. All biopsies were cultured on blood agar base no. 2 (Oxoid) containing 7 % (v/v) defibrinated horse blood with triphenyltetrazolium sodium salt (40 µg ml–1) and sodium pyruvate (0.025 %), and also supplemented with vancomycin (10 µg ml–1) and amphotericin B (5 µg ml–1). Plates were incubated at 37 °C under microaerophilic conditions for up to 5 days. DNA was extracted from confluent cultures with fewer than three in vitro passages by standard protocols, using SDS, lysozyme and proteinase K, followed by phenol/chloroform extraction and ethanol precipitation.
Strain genotyping and cagPAI PCR analysis. lspA-glmM RFLP using AluI and HhaI (Leanza et al., 2004) and random amplified polymorphic DNA (RAPD)-PCR using primer A04 (5'-ATCAGCGCACCA-3'; Konno et al., 2005) were performed for strain delineation. Restricted fragments were electrophoresed in a 3.5 % agarose gel and RAPD-PCR products in a 1.5 % agarose gel. Banding patterns were analysed visually and all loci were scored for the presence or absence of a band. Fingerprint similarities were assessed using the Dice coefficient, as described previously (Leanza et al., 2004). A cut-off level of 80 % similarity was considered to delineate different strains (Leanza et al., 2004). Roman numerals were used to name strains determined by each genotyping method.
cagPAI PCR analysis was carried out with eight oligonucleotide pairs specific for the cagA, cagE, cagG, cagM, cagT, orf10 and orf13 genes and for LEC (Ikenoue et al., 2001; Hsu et al., 2002). Table 1
shows the nucleotide sequences of primers used for each gene or DNA region amplification. The absence of the cagPAI was confirmed by amplification of a 550 bp fragment using primers specific to a region flanking this island (empty-site PCR; Mukhopadhyay et al., 2000). The PCR program comprised a 3 min pre-incubation at 95 °C, followed by 38 cycles of 1 min at 95 °C, 1 min at the annealing temperature indicated in Table 1 and 1 min at 72 °C. Final extension was performed for 7 min at 72 °C. For each gene amplification, PCRs of isolates from different biopsies of single patients were carried out simultaneously. H. pylori strain 26695 DNA was used as a positive control and three DNAs from single colonies of clinical isolates that were positive for empty-site PCR were used as negative controls. PCR was performed at least twice for each sample with basically identical results.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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shows the 42 different fingerprints determined by lspA-glmM RFLP with HhaI and the 37 profiles shown by RAPD-PCR. Inter-niche mixed infection was detected in only two patients. In one patient, the strain denoted lspA-glmM RFLP-XI was isolated from the three antral biopsies and from one biopsy from the corpus, and strain lspA-glmM RFLP-XII was isolated from the remaining two corpus biopsies. All isolates from the antrum and corpus showed the same profile by RAPD-PCR (Fig. 1a, b, lanes 10 and 11). In the other patient with mixed infection, strain lspA-glmM RFLP-XXIII was identified in the three antral biopsies and in two of the corpus biopsies, and strain lspA-glmM RFLP-XXIV was recovered from the remaining corpus biopsy. All isolates were also determined by RAPD-PCR to be a single strain (Fig. 1a, b, lanes 21 and 22; Table 2, patient 9C).
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In one patient, isolates with an intact cagPAI were recovered from the antrum whilst isolates showing absence of the island were recovered from the corpus. Conversely, in another patient, isolates with an intact island were identified from the corpus and isolates that were empty-site-positive from the antrum. In both patients, the cagPAI-positive and cagPAI-negative isolates were variants of the same strain (Table 2
, patients 60F and 63F).
For the remaining 12 patients, isolates showing partial deletion within cagPAI were recovered from all or at least from one of the different niches. From four of these, isolates with the same cagPAI rearrangement were recovered homogeneously from the antrum and corpus (Table 2, patients 19F, 32F, 38C and 37F), whereas in the remaining eight patients, different cagPAI genotypes were found in one or more niches of a single host (Table 2, patients 29F, 35C, 52F, 58F, 42C, 48C, 8C and 9C). In four of these eight patients, variants of the same strain were identified with diverse cagPAI deletions in different niches (Table 2, patients 29F, 35C, 52F and 58F). In addition, co-existence of variants with intact and partially deleted islands in different niches was identified in two patients (Table 2, patients 42C and 8C), whilst variants of the same strain that were empty-site-PCR positive and had a partially deleted cagPAI were also found in another patient (Table 2, patient 48C). From the patient harbouring lspA-glmM-RFLP strains XXIII and XXIV described above, two cagPAI variants of strain XXIII were identified. From the antrum and proximal to the antral–corpus border, strain XXIII isolates were found to be empty-site-positive, whereas from the greater curvature of the corpus, co-existence of isolates with an empty site and isolates with cagPAI lacking the cagG gene were identified (Table 2, patient 9C). Both variants, recognized by expansion of single colonies from the stored sweep of the original culture plates after PCR results, showed the presence of different cagPAI genes simultaneously with empty-site-positive PCR results. Strain XXIV recovered from the lesser curvature of the corpus carried an intact cagPAI (Table 2, patient 9C). This case could support the hypothesis that strains differing in virulence potential can colonize in a mixed infection, thus allowing recombinant variants to emerge (Kersulyte et al., 1999).
With respect to cagPAI rearrangement, 18 different genotypes were found. Among the eight analysed loci, the most frequently deleted gene was cagM (10/18), whereas the least frequently deleted ones were orf10, orf13 and LEC (3/18, 5/18 and 6/18, respectively). cagA, cagT and cagE showed the same deletion rate (7/18). The finding of a conserved LEC agrees with the report of Kauser et al. (2004) showing that this region was rearranged more frequently in isolates linked to severe pathology worldwide. The indication that the cagE gene is a good marker of an intact cagPAI in Japanese isolates (Ikenoue et al., 2001) contrasts with our finding of simultaneous deletion of cagA, cagT and cagE. However, deletion of cagA, cagE and cagT has been reported to be more frequent in isolates linked to benign infections than in isolates recovered from patients with severe ulcers and gastric cancers (Kauser et al., 2004).
The results shown in this study indicated that nearly one-third of the patients harboured isolates with an intact cagPAI homogeneously in all positive niches, another one-third of the isolates were empty-site-PCR positive in all positive niches and the remaining one-third had a disrupted cagPAI in all or at least one niche. These results may be associated with the fact that most of the patients in the study population had superficial gastritis. Nilsson et al. (2003) suggested that the presence of a complete cagPAI gives a fivefold increased risk of severe disease outcome compared with an intermediate cagPAI. These authors found no significant difference in the risk of developing severe disease among patients infected with cagPAI-negative strains and those with strains carrying the intermediate genotype. However, investigation of H. pylori cagPAI genotypes from different human populations has demonstrated that the cagPAI appears to be disrupted in the majority of patients worldwide, with a range of conservation from 57.1 % in Japanese strains to 4–15 % in European isolates (Kauser et al., 2004). In this study, the finding of an intact cagPAI recovered homogeneously from multiple paired antrum/corpus biopsies in almost 30 % of patients is high considering the European ethnic origin of the study population.
Co-existence of variants of the same strain with different cagPAI genotypes was observed in one-quarter of patients. This occurrence may reflect the physiological differences among gastric regions of a given host that would select for derivatives that adapted better in other available locations (Akada et al., 2003). A recent study in which two paired biopsies were analysed, one from the antrum and another from the corpus, also demonstrated the occurrence of variants of the same strain differing with respect to polymorphism of the cagA locus (Carroll et al., 2004). The identification of isolates from a single host sharing an ancestral relationship undergoing independent genomic alterations reinforces the phenomenon termed microevolution displayed by H. pylori during persistent colonization (Marshall et al., 1998; Blaser & Berg, 2001). Although these ten patients had superficial gastritis, no conclusions can be drawn about disease association, as only nine of the 40 patients had erosion, and just five had ulceration.
No association was found between the presence or absence of inter-niche variation and previous eradication therapy administration. Among these ten patients showing co-existence of different cagPAI genotypes, five harboured an intact cagPAI in one or several niches. Considering the multiple processes initiated or supported by cagPAI, such as the induction of the innate immune response, cell-cycle control, cytoskeletal reorganization, disruption of cell–cell adhesion and cell motility, it is tempting to speculate that severe disease could be the result of the cumulative effect of multiple interactions between the bacteria and its host that select a variant with high virulence potential. However, the absence of the cagPAI among strains from varied clinical outcomes could also denote that cagPAI may not be the specific virulence factor associated with disease outcome.
In conclusion, the present study demonstrated the co-existence of a varying ratio of cagPAI-positive to cagPAI-negative isolates in a single host. The presence of variants located at separate niches emphasizes the potential complexity of the gastric ecosystem (physiological differences between regions of a given host’s gastric mucosa such as pH and temperature, in addition to local chemical milieu including host-defence molecules after microbial colonization) and how local differences may promote H. pylori genetic divergence during chronic infection. However, most of the patients in the study population harboured isolates with an intact cagPAI, or that were empty-site-positive, or isolates with a disrupted cagPAI, homogeneously in all of the niches with positive culture. Therefore, the presence or absence of variants with different cagPAI genotypes in a single host may be related to the genetic characteristics of both the H. pylori colonization founder cell and the host, and could be the result of multiple bacteria–host interactions. Nevertheless, the possibility of the co-existence of diverse genotypes of putative virulence factors in different stomach niches of a single host must be considered when drawing a correlation with clinical presentation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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B and ERK-signaling pathways contribute to the gene expression induced by cag PAI-positive-Helicobacter pylori infection. World J Gastroenterol 21, 6134–6143.This article has been cited by other articles:
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  M. J. Matteo, G. Granados, M. Olmos, A. Wonaga, and M. Catalano Helicobacter pylori amoxicillin heteroresistance due to point mutations in PBP-1A in isogenic isolates J. Antimicrob. Chemother., March 1, 2008; 61(3): 474 - 477. [Abstract] [Full Text] [PDF]
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