Population genetics of Helicobacter pylori in the southern part of Switzerland analysed by sequencing of four housekeeping genes (atpD, glnA, scoB and recA), and by vacA, cagA, iceA and IS605 genotyping

Nadia Maggi Solcà1, Marco V. Bernasconi1, Claudio Valsangiacomo1, Leen-Jan Van Doorn2 and Jean-Claude Piffaretti1

Istituto Cantonale Batteriosierologico, Via Ospedale 6, 6904 Lugano, Switzerland1
Delft Diagnostic Laboratory, R. de Graafweg 7, 2625 AD Delft, The Netherlands2

Author for correspondence: Jean-Claude Piffaretti. Tel: +41 91 923 25 22. Fax: +41 91 922 09 93. e-mail: jean-claude.piffaretti{at}ti.ch


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The population biology of 78 Helicobacter pylori strains (71 from Swiss Italian, 4 from East Asian and 3 from South African patients) was investigated by sequence analysis of four housekeeping genes: atpD, scoB, glnA and recA. The vacA genotype, the presence of cagA and IS605, the iceA allelic type, and the resistance to metronidazole, clarithromycin and amoxycillin were determined. A high percentage of DNA polymorphic sites (19·8% for atpD, 21·3% for scoB, 23·7% for glnA and 20·3% for recA) was found. The phylogenetic trees based on the nucleotide sequences of the four gene fragments showed different topologies and were incongruent. The virulence-associated markers were distributed over the dendrograms and no association was found with phylogenetic clusters or clinical manifestations (chronic gastritis, gastric or duodenal ulcer, MALT lymphoma). Moreover, the H ratios (calculated with the homoplasy test) ranged from 0·742 to 0·799, depending on the gene fragment examined. All these observations suggest that H. pylori exists as a recombinant population. The clustering of the strains according to their geographical origin (USA/Europe, East Asia, South Africa) that has recently been demonstrated elsewhere could only be confirmed for the East Asian vacA s1c strains. In contrast, the South African strains clustered together only in the atpD tree. Presumably, recombination at the different loci has masked the evolutionary relationship among the strains.

Keywords: H. pylori, housekeeping genes, antibiotic resistance

Abbreviations: MALT lymphoma, gastric mucosa-associated lymphoid tissue lymphoma

The GenBank accession numbers for the sequences reported in this paper are AY004351–AY004662


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Helicobacter pylori is a microaerophilic, Gram-negative, slow-growing, spiral-shaped and flagellate micro-organism. Its ecological niche is the human gastric mucosa, where it can survive in an acid environment thanks to a powerful urease activity (Blaser, 1997 ). The bacterium, discovered in 1983 by Robin Warren and Barry Marshall, has been associated with various gastropathologies such as chronic atrophic gastritis, peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue lymphoma (MALT lymphoma) (Blaser et al., 1995 ; Roggero et al., 1995 ). Once acquired, H. pylori usually persists for life, unless eradicated by antimicrobial therapy. Even though the prevalence of infection may be very high (70–90% in developing countries, 25–50% in developed countries), most humans infected with H. pylori are asymptomatic and only a few patients develop peptic ulcer or gastric cancer (Pounder, 1995 ). Host genetic predisposition and local environmental factors, together with bacterial genotypes, may play an important role in the development of disease.

Since H. pylori is involved in gastric pathology, researchers have tried to identify specific virulence factors or markers associated with different clinical manifestations of the infection. This led to the discovery of the VacA cytotoxin (which induces vacuolation in eukaryotic cells), the high-molecular-mass antigen CagA and the iceA gene, which is induced by contact with the epithelium (Censini et al., 1996 ; Cover et al., 1994 ; Peek et al., 1998 ). The vacA gene exhibits various signal sequence types (e.g. s1a, s1b, s1c and s2) and middle region types (e.g. m1a, m1b, m1c, m2a and m2b) (Atherton et al., 1995 ; Pan et al., 1998 ; Mukohpadhyay et al., 2000 ; Van Doorn et al., 1998a , c ). The cagA gene is a marker for the presence of a pathogenicity island that may be present or absent (Censini et al., 1996 ; Van der Ende et al., 1998 ; Van Doorn et al., 1998a , c ). The iceA gene has two allelic forms, either iceA1 or iceA2 (Van Doorn et al., 1998c ; Figueiredo et al., 2000 ). Furthermore, H. pylori strains may also be phenotypically distinguished according to their resistance to metronidazole, clarithromycin and amoxycillin (the three antibiotics used in different combinations in anti-H. pylori therapy). Finally, another factor differentiating H. pylori strains is the presence of the transposon-like IS605 element. Because of their role in promoting DNA rearrangements, transposable elements in H. pylori may explain part of the diversity in the genome organization encountered in this species (Hook-Nikanne et al., 1998 ).

In many populations of bacterial pathogens, particular clones are responsible for severe syndromes or for epidemic outbreaks (Musser, 1996 ; Piffaretti et al., 1989 ; Selander et al., 1986 ). Among the different techniques used, DNA sequencing of appropriate targets in the genome is the most powerful tool to discriminate between different strains or species. Automated DNA sequencing of housekeeping genes has extended the use of these techniques to genotyping and phylogenetic studies (Busse et al., 1996 ; Maiden et al., 1998 ). For instance, genes such as atpD (Christensen & Olsen, 1998 ), recA (Eisen, 1995 ), hbb (Valsangiacomo et al., 1997 ) and glnA (Kumada et al., 1993 ), have been widely used for population genetic studies. The population structure of H. pylori has been investigated with MLEE (Go et al., 1996 ) and with other methods based on DNA sequence analysis (Achtman et al., 1999 ; Akopyanz et al., 1992a , b ; Forbes et al., 1995 ; Gibson et al., 1998 ; Salaun et al., 1998 ; Suerbaum et al., 1998 ; Tee et al., 1992 ; Van Doorn et al., 1999a , b ; Yamaoka et al., 1998 ), and the results suggested a panmictic nature for H. pylori. However, apart from MLEE, most of these methods considered the genomic diversity in pathogenicity-associated genes, e.g. vacA and cagA (Salaun et al., 1998 ; Suerbaum et al., 1998 ; Van Doorn et al., 1999a , b ; Yamaoka et al., 1998 ), or in colonization factors such as the urease genes (Akopyanz et al., 1992a ; Salaun et al., 1998 ) and the flaA and flaB genes (Forbes et al., 1995 ; Salaun et al., 1998 ; Suerbaum et al., 1998 ), while other studies analysed a limited number of strains (Achtman et al., 1999 ; Gibson et al., 1998 ; Salaun et al., 1998 ). In the present study, the population structure of H. pylori has been investigated by comparative sequence analysis of four housekeeping genes (atpD, scoB, glnA and recA) and on a relatively large number of isolates.

With the aim of better characterizing our population of H. pylori, we also determined the vacA and the iceA status, and the presence of cagA and IS605. In addition, we tested all strains for their susceptibility to metronidazole, clarithromycin and amoxycillin.

Most of the strains used in this study (71/78) were isolated from patients living in the southern part of Switzerland with various gastropathologies. The prevalence of infection in this area, together with the northern part of Italy, is the highest found in Europe (Doglioni et al., 1992 ; EUROGAST Study Group, 1993 ). These regions are also of particular interest because of a significant incidence of gastric MALT lymphoma and gastric cancer. Finally, we looked at the geographical distribution of H. pylori genotypes that has been recently discovered (Achtman et al., 1999 ; Campbell et al., 1997 ; Miehlke et al., 1996 ; Van der Ende et al., 1998 ; Van Doorn et al., 1999a , b). For this reason, three strains from South Africa and four from East Asia were also included in this study.


   METHODS
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INTRODUCTION
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Patients.
Gastric biopsies from 356 patients living in the southern part of Switzerland were tested for H. pylori by culture. For each patient, gastroenterologists were asked to complete a questionnaire on the gastroduodenal disease, age, sex, family gastric pathology history and previous treatment of H. pylori infections. The endoscopic diagnosis of the disease was confirmed by histopathological examination of the gastric biopsy performed at the Istituto Cantonale di Patologia (Locarno, Switzerland). H. pylori strains could be cultured from 178 (50%) patients. Of these 178 isolates, 71 were randomly selected for the phylogenetic analysis, including strains from 41 (57·7%) patients with chronic gastritis, 4 (5·6%) with dyspepsia, 20 (28·8%) with duodenal ulcer, 4 (5·6%) with gastric ulcer and 2 (1·4%) with gastric MALT lymphoma. The Ethical Committee of the Cantone Ticino approved this work and informed consent was obtained from all patients.

H. pylori strains.
Biopsy specimens were collected into Portagerm medium (BioMérieux) and processed in our laboratory within 4 h of gastroduodenoscopy. H. pylori strains were isolated by streaking gastric biopsies (either from the antrum or from the corpus) onto Brain Heart Infusion Agar (Oxoid) supplemented with 5% sheep blood and Vitox (Oxoid) and Columbia Agar supplemented with 5% sheep blood and Skirrow’s supplement (Oxoid). Plates were incubated at 37 °C in 5% O2/10% CO2/ 85% N2 for up to 7 d. Isolates with typical colony morphology, Gram stain and biochemical tests positive for urease, catalase and oxidase were harvested in 25% peptone-glycerol and stored at -70 °C. A total of 142 strains were collected and were tested for antibiotic susceptibility, the results of which have been published elsewhere (Maggi-Solcà et al., 2000 ). Of these strains, 71 were randomly chosen for the phylogenetic analysis. S. Suerbaum (Würzburg, Germany) and M. J. Blaser (Nashville, TN, USA) kindly provided DNA of 3 South African strains and DNA of 4 East Asian strains, respectively. The characteristics of the strains are reported in Table 1.


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Table 1. H. pylori strains and their characteristics

 
Choice of the four housekeeping genes.
We considered the following housekeeping genes: atpD, scoB, glnA and recA. The beta subunit of the F1-F0 ATPase, encoded in H. pylori by the atpD gene, is one of the five subunits of the F1 catalytic portion of the enzyme, and it has the most conserved primary structure of all the F1-F0 subunits (McGowan et al., 1997 ). This gene has already been used to analyse bacterial phylogeny, generating trees congruent with those obtained with the 16S rDNA (Amann et al., 1988 ; Christensen & Olsen, 1998 ). The succinyl-CoA:acetoacetate CoA-transferase (SCOT)-encoding gene (sco) of H. pylori was discovered by Corthésy-Theulaz et al. (1997) . In contrast to the monomeric eukaryotic homologues, the H. pylori enzyme consists of two subunits, A and B. Both subunits were present in all strains tested. For the phylogenetic analysis we chose scoB, encoding the subunit B. The glutamine-synthetase-encoding glnA gene is well-conserved (Garner et al., 1998 ) and has also been employed for phylogenetic studies (Kumada et al., 1993 ). The RecA protein is involved in many essential biological processes, like homologous recombination, recombinational DNA repair, and activation of the SOS system. The primary sequence of recA is very well conserved among eubacterial species and it is also generally considered as an appropriate target for phylogenetic studies (Eisen, 1995 ).

DNA preparation, PCR and sequencing.
H. pylori strains were subcultured for 3 d on fresh Brain Hearth Infusion Agar supplemented with 5% sheep blood and Vitox. The cells were collected from the plates and DNA extraction and purification were performed in a single step using a commercial ion-exchange resin (InstaGene matrix; Bio-Rad), according to the manufacturer’s instructions. Specific primers (Table 2) were used to amplify the four target genes (i.e. atpD, scoB, glnA and recA). A 2 µl portion of DNA extract was used for the PCR in a total reaction volume of 50 µl. The reaction mixture contained 5 µl PCR buffer (Roche Molecular Biochemicals), each deoxynucleoside triphosphate at a concentration of 200 µM, the appropriate primers, each at a concentration of 0·5 µM, and 1 U Taq DNA polymerase (Roche Molecular Biochemicals). The thermal profile used for the amplification of atpD was 2 min at 94 °C, followed by 35 cycles consisting of 94 °C for 1 min, 53 °C for 1 min and 72 °C for 1·5 min; for the amplification of the scoB gene fragment the annealing temperature was 50 °C, and for both glnA and recA the annealing temperature was 52 °C. The PCR amplicons were used for cycle sequencing after purification with the Qiaquick PCR purification Kit (Qiagen). Cycle sequencing reactions were performed with the dRhodamine Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Biosystems) and with an automated DNA sequencer (ABI 310, Perkin Elmer). For the fragments with nucleotide substitutions resulting in an amino acid change, the DNA sequence of both strands was determined. The sequencing primers are listed in Table 2.


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Table 2. Primers for PCR and sequencing experiments

 
Analysis of the nucleotide sequences.
The sequences of the four housekeeping genes, atpD, scoB, glnA and recA, were handled and stored with the help of the Lasergene program EditSeq (1994 release; DNAstar) and aligned using Megalign (1994 release; DNAstar). Detailed information on the sequenced genes is reported in Table 3. The sequences were analysed using the neighbour-joining method (Saitou & Nei, 1987 ), with Kimura two-parameter distances as implemented in MEGA (Molecular Evolutionary Genetics Analysis 1.01, Kumar et al., 1993 ). The reliability of internal branches was assessed by bootstrapping (Felsenstein, 1988 ), with 500 pseudo-replicates. Percentages of the mean differences between pairs of strains at synonymous nucleotide positions (KS) and nonsynonymous positions (KA) were calculated with DNASP 3.0 (Rozas & Rozas, 1999 ) using Jukes & Cantor parameters. The homoplasy test (Maynard Smith & Smith, 1998 ) was performed using the HOMOPLASY program (Suerbaum et al., 1998 ) to test the importance of recombination (Achtman et al., 1999 ).


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Table 3. Characteristics of the fragments sequenced

 
vacA, cagA and iceA genotyping.
Typing of the vacA signal sequence and middle region of the iceA allele and detection of the cagA gene of the Swiss Italian strains were performed with a single-step procedure based on PCR and reverse hybridization (LiPA) as described by Van Doorn et al. (1998b ).

IS605 detection.
All 78 strains were screened for the presence of IS605 by PCR amplification of the two open reading frames (A and B) using published primers (Hook-Nikanne et al., 1998 ). The PCR program was 2 min at 94 °C, followed by 35 cycles consisting of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1·5 min. Reaction conditions were similar to those described above.

Statistical analysis.
Data were analysed by the chi-squared test.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Nucleotide sequence analysis
The nucleotide sequences of the fragments originating from the atpD, scoB, glnA and recA genes were determined for 78 H. pylori isolates of our collection (see Methods and Table 1). Among these sequences, 73 allelic variants were found for atpD, 78 for scoB, 78 for glnA, and 77 for recA. No deletions or insertions were found in any of the analysed gene fragments. The number of polymorphic sites (Table 4) was similar for all the four gene fragments (range 19·8–23·7%) and the substitutions were randomly distributed over the DNA sequences. The percentages of the mean differences between pairs of strains at synonymous nucleotide positions (KS) varied from 15·1% to 20·3%, and from 0·2% to 1% at nonsynonymous positions (KA). Most of the nucleotide replacements (77·7%–84·1%) were at the third codon position. According to the amino acid substitution frequencies, atpD and recA were the most conserved genes. In atpD, the region encoding the nucleotide-binding site (nucleotide positions 453–465) was highly conserved (data not shown). The amino acid substitutions in recA were mostly concentrated in a specific region arbitrarily designated as ‘group II defining region’ (see below). For scoB and glnA, there were no particular regions with a higher level of mutations. The sets of sequences were tested by the homoplasy test, which measures the importance of recombination (Maynard Smith & Smith, 1998 ). The resulting H ratios (Table 4) ranged from 0·742 to 0·799, indicating frequent recombination for all four gene fragments examined (H is a number whose expected value is 0 if the population is clonal and 1·0 if it is in linkage equilibrium).


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Table 4. DNA polymorphism in the four housekeeping genes analysed

 
Phylogenetic analyses
Phylogenetic trees constructed from sequence analyses of the four housekeeping genes, atpD, scoB, glnA and recA, of the 78 H. pylori strains are presented in Figs 1, 2, 3 and 4. Comparison of these phylogenetic trees provided further evidence for the high genomic diversity found at the nucleotide level. The topologies of the trees are very different and most of the internal nodes are supported by low bootstrap values. In general, no significant clustering of strains was found for the atpD, scoB and glnA genes (Figs 1, 2 and 3). In contrast, the tree generated with the recA gene was clearly divided into two groups (designated I and II) (Fig. 4).



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Fig. 1. Neighbour-joining tree with Kimura two-parameter distances based on atpD sequences. Bootstrap values from 500 pseudo-replicates are shown when they exceeded 30%. The strains are labelled with a or c, according to their origin, antrum or corpus, respectively. The clinical manifestation, the vacA type, the presence of cagA and IS605 are shown in parentheses. CG, chronic gastritis; GU, gastric ulcer; DU duodenal ulcer; D, dyspepsia; MALT, gastric MALT lymphoma. Strains for which the cagA and IS605 status is not mentioned lack these sequences. East Asian strains with the s1c-m1 vacA genotype are marked by black circles and that with the s2-m2a vacA genotype by a black triangle; South African strains are indicated by black squares.

 


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Fig. 2. Neighbour-joining tree with Kimura two-parameter distances based on scoB sequences. See Fig. 1 for more details.

 


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Fig. 3. Neighbour-joining tree with Kimura two-parameter distances based on glnA sequences. See Fig. 1 for more details.

 


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Fig. 4. Neighbour-joining tree with Kimura two-parameter distances based on recA sequences. See Fig. 1 for more details.

 
Cluster analysis of strains from other countries
The distribution of the East Asian and of the South African strains was variable and depended on the molecular marker used (atpD, scoB, glnA or recA gene). The three East Asian strains (HK9728, HPK28, HPK76) with the s1c vacA genotype, a newly discovered vacA type which is mainly found in the Asian population (Van Doorn et al., 1999a , b) were always grouped together in the four trees (Figs 1, 2, 3 and 4). However, only in the scoB and glnA trees (Figs 2 and 3) did they form a well-supported monophyletic group (bootstrap values 68% and 97%, respectively). In contrast, in the atpD (Fig. 1) and recA (Fig. 4) trees, they were paraphyletic with the inclusion of other strains, namely strain 111A for atpD and 125A2 for recA. Both strains had been isolated from Swiss Italian patients and were vacA type s1b and s2, respectively. The East Asian strain HPK154, which is vacA s2-m2, never clustered with the s1c Asian strains in any tree. The South African strains (CC28, CC33 and CC35) formed a well-supported cluster (75% bootstrap value) only in the atpD tree (Fig. 1). For the scoB gene (Fig. 2), the three South African strains were distributed throughout the tree, whereas in the glnA tree (Fig. 3), only strains CC33 and CC35 clustered together (bootstrap value 100%). In the recA tree (Fig. 4), even though all the South African strains belonged to ‘group II’, only strains CC35 and CC28 clustered together (bootstrap value 88%).

vacA, cagA and iceA genotyping
This analysis was only performed for the 71 Swiss Italian strains (Table 1). The vacA genotype and the presence of cagA for each strain are also indicated in parentheses beside the taxon name in the four trees (Figs 1, 2, 3 and 4). The vacA s1 type was slightly more common than s2 (54·9% vs 45%), with the s1a and s1b subtypes nearly equally distributed (28·2% vs 26·8%). Among the vacA middle region types, m2a was more prevalent than m1 (69% vs 31%). The s1c and the m2b alleles were not found in our collection of Swiss Italian strains. The following vacA type combinations were detected: s1a-m1 (17%), s1a-m2a (11%), s1b-m1 (14 %), s1b-m2a (13%) and s2-m2a (45%). The combination s2-m1 was not found. The cagA gene was present in 50% of the strains and the iceA1 allele was found more frequently than iceA2 (59% vs 41%).

There were no significant associations between the vacA, cagA, iceA status and particular clinical outcomes of the infection (Table 5). Apart from vacA s1, which was strongly associated with the presence of cagA (P<0·001), there was no correlation between any of the other virulence-associated markers.


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Table 5. Relationship of the different markers with the type of disease

 
IS605
IS605 ORFA and ORFB sequences were identified in the following 12 strains: 86C, 348C, 48A, 47A, 65A, 128A, 99A2, 84A, 192A, 163C, 18A, 35C (Table 1). However, in strain 322A, only the ORFA sequence and in strain 103A, only the ORFB sequence could be detected. According to Hook-Nikanne et al. (1998) , we considered these two strains as positive for IS605. The presence of IS605 was not correlated with specific clinical manifestations, but was associated with vacA s1 (P=0·009) and with the presence of cagA (P=0·02), as reported earlier (Hook-Nikanne et al., 1998 ).

Antibiotic resistance
Antibiotic susceptibility was tested for the 71 Swiss Italian strains (Table 1). No association was found between antibiotic resistance and other genetic markers investigated in this study.


   DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
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DNA polymorphism and recombination
The amount of polymorphic sites we found in the genes analysed was higher in H. pylori (range 19·8–23·7%) than in other pathogenic bacteria (e.g. 0·01% in Mycobacterium tuberculosis complex, 6·18% in Neisseria meningitidis, 11·77% in Escherichia coli) (Sreevatsan et al., 1997 ). However, the percentage of DNA polymorphism was similar to those found by Suerbaum et al. (1998) for vacA, flaA and flaB sequences of H. pylori. The number of allelic variants was very high: nearly every isolate contained a unique gene sequence (Table 4). The mean KS values (measuring the rate of synonymous changes) were higher than the KA values (non-synonymous changes), supporting previous studies (Alm et al., 1999 ; Suerbaum et al., 1998 ). Moreover, the KS/KA values were in the same range as those found for most of the housekeeping genes analysed by Achtman et al. (1999) . The KS/KA ratio was lower in virulence-associated genes (i.e. vacA and cagA) than in sequences encoding housekeeping enzymes (Achtman et al., 1999 ; Suerbaum et al., 1998 ), i.e. virulence genes presented a higher percentage of nonsynonymous changes. The importance of recombination was estimated by calculating the H ratios (Table 4), using the homoplasy test. The values obtained were similar to those previously reported for other genes (Achtman et al., 1999 ; Suerbaum et al., 1998 ) and clearly indicated frequent recombination. The homoplasy test is particularly appropriate when the sequences are rather similar, differing by 1–5% of nucleotides (Maynard Smith & Smith, 1998 ). However, this test has been successfully used also for sequences with much higher polymorphisms (Achtman et al., 1999 ; Suerbaum et al., 1998 ), as is the case for H. pylori.

The question why the genome of H. pylori has so much polymorphism still has to be clarified. Apparently H. pylori does not have a full DNA repair system (Tomb et al., 1997 ); this, together with a high recombination frequency, could offer a possible explanation. Further studies on the H. pylori repair system are required to understand this observation better.

Topology of the trees
In general, phylogenetic analyses of the four housekeeping genes revealed no significant clustering of strains, and most of the internal nodes in the four trees were characterized by extremely low bootstrap values. The observation that the grouping of the 78 strains, and their genetic distances, were clearly different in the four dendrograms (Figs 1, 2, 3 and 4) again suggests the existence of frequent recombination events, thus supporting the hypothesis of a panmictic structure of the H. pylori population.

Only the recA-based tree showed a subdivision into two groups, indicating the existence of two distinct allelic variants of this gene (Fig. 4). The two groups resulted from specific mutations at positions 309 (C->T; His->Tyr), 363–365 (GAT->AGC; Asp->Ser), 368 (A->G, silent) and 374–375 (GC->AG; Gln->Glu). These mutations were linked and defined these two allelic forms of recA. The existence of those groups is supported by the finding that sequences belonging to groups I and II had already been previously independently reported by Schmitt et al. (1995) and Thompson & Blaser (1995) , respectively.

Clustering according to geographical origin
The analyses of the four housekeeping genes were unable to resolve the geographical relationships among the H. pylori strains examined, with the exception of the East Asian strains in the scoB and glnA trees (Figs 2 and 3). Recently, Van Doorn et al. (1999a ) investigated the worldwide distribution of the vacA alleles and found a gradient: in Northern Europe the s1a genotype prevailed, whereas in France and Italy the s1a and s1b genotypes were nearly equally present and in Spain and in Portugal the s1b type was highly prevalent. The Swiss Italian strains apparently show the same distribution of vacA s-types as in France and Italy. The East Asian and the South African populations show particular features. In East Asia nearly all the H. pylori isolates are cagA+ and vacA s1c (Maeda et al., 1998 ; Van Doorn et al., 1999a ). These genotypes have been associated with ulcer disease and, in fact, the incidence of ulcer and gastric cancer in East Asia is the highest in the world (Maeda et al., 1998 ). In South Africa mostly the s1b vacA type is found (Letley et al., 1999 ) and, in spite of a high prevalence of H. pylori infection, the incidence of gastric cancer is low (this is called the ‘African enigma’; Holcombe, 1992 ). Furthermore, Suerbaum et al. (1998) reported a more conserved H. pylori population among South Africans. Concerning the South African strains analysed in the present study, only the vacA type of strain CC28 was known and it was unusual because it contained a hybrid of vacA s1a and s1b. This strain was previously included in a study by Achtman et al. (1999) dealing with the population genetics and geographical diversity of 20 H. pylori strains from different parts of the world. In this study, strain CC28 belonged to the weakly clonal group called ‘clone 2’, which included also one strain from Gambia, one strain from the USA and one strain from Guatemala.

vacA, cagA, iceA
Previous studies have indicated that the presence of the vacA s1, cagA+ and iceA1 genotypes is associated with a severe manifestation of the infection (Blaser et al., 1995 ; Peek et al., 1998 ; Van Doorn et al., 1998a , c ). We could not confirm any of these associations (Table 5). In addition, other authors recently failed to find a linkage between specific vacA and cagA genotypes and the severity of the disease (Go & Graham, 1996 ; Go et al., 1998 ; Maeda et al., 1998 ; Yamaoka et al., 1999 ). Two factors could have influenced the results. One is the problem of the reliability of the clinical data: for instance, it cannot be excluded that a patient with gastritis at the time of endoscopy has experienced ulcer disease in the past. The second factor is related to the fact that we analysed only one strain from each patient, although colonization with multiple strains is possible (Taylor et al., 1995 ). Additional analyses of a collection of isolates originating from other diseased people should clarify the existence of this association between particular traits and virulence.

IS605 and antibiotic resistance
The presence of IS elements is usually associated with genome rearrangements. Specific genome rearrangements may provide a selective advantage to some strains. Nevertheless, the presence of IS605 was not correlated with specific strain clusters on the dendrograms (Figs 1, 2, 3 and 4). The antibiotic-resistant phenotype, which also results from a selective process, was also not associated with particular groups of strains.

Conclusions
Various authors (Achtman et al., 1999 ; Go et al., 1996 ; Salaun et al., 1998 ; Suerbaum et al., 1998 ) have suggested the non-clonal nature of H. pylori. Our study, based on the sequences of four housekeeping genes and involving a considerable number of strains originating from a limited geographical region (South Switzerland), confirms and reinforces this finding. The hypothesis of a recombining structure in our H. pylori population is based on the following points: (i) the level of DNA polymorphism found was high; (ii) the topology of the trees generated from four housekeeping genes was different for each genetic marker; (iii) the distribution of the strains according to their geographical origin was different in the four trees; (iv) no association between the distribution of the strains on the dendrograms and any of the characteristics of the strains (clinical manifestation, virulence markers, antibiotic resistance and IS605) was found; (v) finally, the H ratios measuring the importance of recombination (homoplasy test) ranged from 0·742 to 0·799. The extensive recombination structure can be partially explained by the natural competence of H. pylori (Alm et al., 1999 ; Hofreuter et al., 1998 ). In a recombinant population, clonal groups are difficult to identify. It is therefore surprising that distribution of H. pylori strains according to the ethnic origin of the host (even though not homogeneous) could still be recognized (Achtman et al., 1999 ; Campbell et al., 1997 ; Suerbaum et al., 1998 ; Van Doorn et al., 1999a , b ). It is likely that, besides frequent recombination events, host characteristics as well as environmental conditions might influence the selection process, leading for instance to parallel or convergent evolution. Geographical clusters might also be in part preserved by geographical barriers to some extent hindering global, worldwide recombination among strains. The H. pylori population structure deserves further investigations, for instance by analysing more strains from different geographical origins and by collecting more information on the host’s predisposition.


   ACKNOWLEDGEMENTS
 
We are grateful to all the gastroenterologists who participated in this work: V. Saglini, B. Miazza, P. Wunderlich, I. Fumagalli and A. Bonetti. We thank also E. Pedrinis of the Istituto Cantonale di Patologia in Locarno for the histological examinations. For providing the DNA of the East Asian strains and of the South African strains, we are grateful to M. J. Blaser (Nashville, TN, USA) and to S. Suerbaum (Würzburg, Germany), respectively. For technical assistance we thank R. Sanna and A. Verschuuren (Delft, The Netherlands). Many thanks also to E. Roggero and E. Zucca for helpful discussions and to P. Kuhnert (Bern, Switzerland) for reading the manuscript. We also thank M. Achtman (Berlin, Germany) for providing the HOMOPLASY program. This research was supported by grant 31-45914.95 from the Swiss National Science Foundation, by the Helmut Horten Foundation, and by Astra Pharmaceutica (Dietikon, Switzerland).


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Achtman, M., Azuma, T., Berg, D. E. & 7 other authors (1999). Recombination and clonal groupings within Helicobacter pylori from different geographical regions. Mol Microbiol 32, 459–470.[Medline]

Akopyanz, N., Bukanov, N. O., Westblom, T. U. & Berg, D. E. (1992a). PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori. Nucleic Acids Res 20, 6221-6225.[Abstract]

Akopyanz, N., Bukanov, N. O., Westblom, T. U., Kresovich, S. & Berg, D. E. (1992b). DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 20, 5137-5142.[Abstract]

Alm, R. A., Ling, L. S., Moir, D. T. & 20 other authors (1999). Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176–180.[Medline]

Amann, R., Ludwig, W. & Schleifer, K. H. (1988). Beta-subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria. J Gen Microbiol 134, 2815-2821.[Medline]

Atherton, J. C., Cao, P., Peek, R. M.Jr, Tummuru, M. K., Blaser, M. J. & Cover, T. L. (1995). Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 270, 17771-17777.[Abstract/Free Full Text]

Blaser, M. J. (1997). Ecology of Helicobacter pylori in the human stomach. J Clin Invest 100, 759-762.[Free Full Text]

Blaser, M. J., Perez-Perez, G. I., Kleanthous, H., Cover, T. L., Peek, R. M., Chyou, P. H., Stemmermann, G. N. & Nomura, A. (1995). Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 55, 2111-2115.[Abstract]

Busse, H. J., Denner, E. B. & Lubitz, W. (1996). Classification and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics. J Biotechnol 47, 3-38.[Medline]

Campbell, S., Fraser, A., Holliss, B., Schmid, J. & O’Toole, P. W. (1997). Evidence for ethnic tropism of Helicobacter pylori. Infect Immun 65, 3708-3712.[Abstract]

Censini, S., Lange, C., Xiang, Z., Crabtree, J. E., Ghiara, P., Borodovsky, M., Rappuoli, R. & Covacci, A. (1996). Cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 93, 14648-14653.[Abstract/Free Full Text]

Christensen, H. & Olsen, J. E. (1998). Phylogenetic relationships of Salmonella based on DNA sequence comparison of atpD encoding the beta subunit of ATP synthase. FEMS Microbiol Lett 161, 89-96.[Medline]

Corthésy-Theulaz, I. E., Bergonzelli, G. E., Henry, H., Bachmann, D., Schorderet, D. F., Blum, A. L. & Ornston, L. N. (1997). Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family. J Biol Chem 272, 25659-25667.[Abstract/Free Full Text]

Cover, T. L., Tummuru, M. K., Cao, P., Thompson, S. A. & Blaser, M. J. (1994). Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains. J Biol Chem 269, 10566-10573.[Abstract/Free Full Text]

Doglioni, C., Wotherspoon, A. C., Moschini, A., de Boni, M. & Isaacson, P. G. (1992). High incidence of primary gastric lymphoma in northeastern Italy. Lancet 339, 834-835.[Medline]

Eisen, J. A. (1995). The RecA protein as a model molecule for molecular systematic studies of bacteria: comparison of trees of RecAs and 16S rRNAs from the same species. J Mol Evol 41, 1105-1123.[Medline]

EUROGAST Study Group (1993). An international association between Helicobacter pylori infection and gastric cancer. Lancet 341, 1359–1362.[Medline]

Felsenstein, J. (1988). Phylogenies from molecular sequences: inference and reliability. Annu Rev Genet 22, 521-565.[Medline]

Figueiredo, C., Quint, W. G., Sanna, R. & 7 other authors (2000). Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori. Gene 246, 59–68.[Medline]

Forbes, K. J., Fang, Z. & Pennington, T. H. (1995). Allelic variation in the Helicobacter pylori flagellin genes flaA and flaB: its consequences for strain typing schemes and population structure. Epidemiol Infect 114, 257-266.[Medline]

Garner, R. M., Fulkerson, J.Jr & Mobley, H. L. (1998). Helicobacter pylori glutamine synthetase lacks features associated with transcriptional and posttranslational regulation. Infect Immun 66, 1839-1847.[Abstract/Free Full Text]

Gibson, J. R., Slater, E., Xerry, J., Tompkins, D. S. & Owen, R. J. (1998). Use of an amplified-fragment length polymorphism technique to fingerprint and differentiate isolates of Helicobacter pylori. J Clin Microbiol 36, 2580-2585.[Abstract/Free Full Text]

Go, M. F. & Graham, D. Y. (1996). Presence of the cagA gene in the majority of Helicobacter pylori strains is independent of whether the individual has duodenal ulcer or asymptomatic gastritis. Helicobacter 1, 107-111.[Medline]

Go, M. F., Kapur, V., Graham, D. Y. & Musser, J. M. (1996). Population genetic analysis of Helicobacter pylori by multilocus enzyme electrophoresis: extensive allelic diversity and recombinational population structure. J Bacteriol 178, 3934-3938.[Abstract]

Go, M. F., Cissell, L. & Graham, D. Y. (1998). Failure to confirm association of vacA gene mosaicism with duodenal ulcer disease. Scand J Gastroenterol 33, 132-136.[Medline]

Hofreuter, D., Odenbreit, S., Henke, G. & Haas, R. (1998). Natural competence for DNA transformation in Helicobacter pylori: identification and genetic characterization of the comB locus. Mol Microbiol 28, 1027-1038.[Medline]

Holcombe, C. (1992). Helicobacter pylori: the African enigma. Gut 33, 429-431.[Medline]

Hook-Nikanne, J., Berg, D. E, Peek, R. M.Jr, Kersulyte, D., Tummuru, M. K. & Blaser, M. J. (1998). DNA sequence conservation and diversity in transposable element IS605 of Helicobacter pylori. Helicobacter 3, 79-85.[Medline]

Kumada, Y., Benson, D. R., Hillemann, D., Hosted, T. J., Rochefort, D. A., Thompson, C. J., Wohlleben, W. & Tateno, Y. (1993). Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90, 3009-3013.[Abstract]

Kumar, S., Tamura, K. & Nei, M. (1993). MEGA: Molecular Evolutionary Genetics Analysis, version 1.01. University Park, PA: Pennsylvania State University.

Letley, D. P., Lastovica, A., Louw, J. A., Hawkey, C. J. & Atherton, J. C. (1999). Allelic diversity of the Helicobacter pylori vacuolating cytotoxin gene in South Africa: rarity of the vacA s1a genotype and natural occurrence of an s2/m1 allele. J Clin Microbiol 37, 1203-1205.[Abstract/Free Full Text]

McGowan, C. C., Cover, T. L. & Blaser, M. J. (1997). Analysis of F1F0-ATPase from Helicobacter pylori. Infect Immun 65, 2640-2657.[Abstract]

Maeda, S., Ogura, K., Yoshida, H., Kanai, F., Ikenoue, T., Kato, N., Shiratori, Y. & Omata, M. (1998). Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan. Gut 42, 338-343.[Abstract/Free Full Text]

Maggi-Solcà, N., Valsangiacomo, C. & Piffaretti, J.-C. (2000). Prevalence of Helicobacter pylori resistant strains in the Southern part of Switzerland. Clin Microbiol Infect 6, 38-40.[Medline]

Maiden, M. C., Bygraves, J. A., Feil, E. & 10 other authors (1998). Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 95, 3140–3145.[Abstract/Free Full Text]

Maynard Smith, J. & Smith, N. H. (1998). Detecting recombination from gene trees. Mol Biol Evol 15, 590-599.[Abstract]

Miehlke, S., Kibler, K., Kim, J. G., Figura, N., Small, S. M., Graham, D. Y. & Go, M. F. (1996). Allelic variation in the cagA gene of Helicobacter pylori obtained from Korea compared to the United States. Am J Gastroenterol 91, 1322-1325.[Medline]

Mukhopadhyay, A. K., Kersulyte, D., Jeong, J. Y. & 9 other authors (2000). Distinctiveness of genotypes of Helicobacter pylori in Calcutta, India. J Bacteriol 182, 3219–3227.[Abstract/Free Full Text]

Musser, J. M. (1996). Molecular population genetic analyses of emerged bacterial pathogens: selected insights. Emerg Infect Dis 2, 1-17.[Medline]

Pan, Z. J., Berg, D. E., Van der Hulst, R. W., Su, W. W., Raudonikiene, A., Xiao, S. D., Dankert, J., Tytgat, G. N. & Van der Ende, A. (1998). Prevalence of vacuolating cytotoxin production and distribution of distinct vacA alleles in Helicobacter pylori from China. J Infect Dis 178, 220-226.[Medline]

Peek, R. M.Jr, Thompson, S. A., Donahue, J. P., Tham, K. T., Atherton, J. C., Blaser, M. J. & Miller, G. G. (1998). Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene, iceA, that is associated with clinical outcome. Proc Assoc Am Physicians 110, 531-544.[Medline]

Piffaretti, J.-C., Kressebuch, H., Aeschbacher, M., Bille, J., Bannerman, E., Musser, J. M., Selander, R. K. & Rocourt, J. (1989). Genetic characterization of the bacterium Listeria monocytogenes causing epidemic disease. Proc Natl Acad Sci USA 86, 3818-3822.[Abstract]

Pounder, R. E. (1995). The prevalence of Helicobacter pylori in different countries. Aliment. Pharmacol Ther 9 (suppl 2), 33–44.

Roggero, E., Zucca, E., Pinotti, G. & 7 other authors (1995). Eradication of Helicobacter pylori infection in primary low-grade gastric lymphoma of mucosa-associated lymphoid tissue. Ann Intern Med 122, 767–769.[Abstract/Free Full Text]

Rozas, J. & Rozas, R. (1999). DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15, 174-174.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.[Abstract]

Salaun, L., Audibert, C., Le Lay, G., Burucoa, C., Fauchere, J. L. & Picard, B. (1998). Panmictic structure of Helicobacter pylori demonstrated by the comparative study of six genetic markers. FEMS Microbiol Lett 161, 231-239.[Medline]

Schmitt, W., Odenbreit, S., Heuermann, D. & Haas, R. (1995). Cloning of the Helicobacter pylori recA gene and functional characterization of its product. Mol Gen Genet 248, 563-572.[Medline]

Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M., Gilmour, M. N. & Whittam, T. S. (1986). Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 51, 873-884.[Medline]

Sreevatsan, S., Pan, X., Stockbauer, K. E., Connell, N. D., Kreiswirth, B. N., Whittam, T. S. & Musser, J. M. (1997). Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci USA 94, 9869-9874.[Abstract/Free Full Text]

Suerbaum, S., Smith, J. M., Bapumia, K., Morelli, G., Smith, N. H., Kunstmann, E., Dyrek, I. & Achtman, M. (1998). Free recombination within Helicobacter pylori. Proc Natl Acad Sci USA 95, 12619-12624.[Abstract/Free Full Text]

Taylor, N. S., Fox, J. G., Akopyants, N. S. & 7 other authors (1995). Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting. J Clin Microbiol 33, 918–923.[Abstract]

Tee, W., Lambert, J., Smallwood, R., Schembri, M., Ross, B. C. & Dwyer, B. (1992). Ribotyping of Helicobacter pylori from clinical specimens. J Clin Microbiol 30, 1562-1567.[Abstract]

Thompson, S. A. & Blaser, M. J. (1995). Isolation of the Helicobacter pylori recA gene and involvement of the recA region in resistance to low pH. Infect Immun 63, 2185-2193.[Abstract]

Tomb, J. F., White, O., Kerlavage, A. R. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547.[Medline]

Valsangiacomo, C., Balmelli, T. & Piffaretti, J. C. (1997). A phylogenetic analysis of Borrelia burgdorferi sensu lato based on sequence information from the hbb gene, coding for a histone-like protein. Int J Syst Bacteriol 47, 1-10.[Abstract/Free Full Text]

Van der Ende, A., Pan, Z. J., Bart, A., van der Hulst, R. W., Feller, M., Xiao, S. D., Tytgat, G. N. & Dankert, J. (1998). cagA-positive Helicobacter pylori populations in China and The Netherlands are distinct. Infect Immun 66, 1822-1826.[Abstract/Free Full Text]

Van Doorn, L. J., Figueiredo, C., Sanna, R., Plaisier, A., Schneeberger, P., de Boer, W. & Quint, W. G. (1998a). Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori. Gastroenterology 115, 58-66.[Medline]

Van Doorn, L. J., Figueiredo, C., Sanna, R., Pena, S., Midolo, P., Ng, E. K., Atherton, J. C., Blaser, M. J. & Quint, W. G. (1998b). Expanding allelic diversity of Helicobacter pylori vacA. J Clin Microbiol 36, 2597-2603.[Abstract/Free Full Text]

Van Doorn, L. J., Figueiredo, C., Rossau, R., Jannes, G., van Asbroek, M., Sousa, J. C., Carneiro, F. & Quint, W. G. (1998c). Typing of Helicobacter pylori vacA gene and detection of cagA gene by PCR and reverse hybridization. J Clin Microbiol 36, 1271-1276.[Abstract/Free Full Text]

Van Doorn, L. J., Figueiredo, C., Mégraud, F. & 14 other authors (1999a). Geographic distribution of vacA allelic types of Helicobacter pylori. Gastroenterology 116, 823–830.[Medline]

Van Doorn, L. J., Figueiredo, C., Sanna, R., Blaser, M. J. & Quint, W. G. (1999b). Distinct variants of Helicobacter pylori cagA are associated with vacA subtypes. J Clin Microbiol 37, 2306-2311.[Abstract/Free Full Text]

Yamaoka, Y., Kodama, T., Kita, M., Imanishi, J., Kashima, K. & Graham, D. Y. (1998). Relationship of vacA genotypes of Helicobacter pylori to cagA status, cytotoxin production, and clinical outcome. Helicobacter 3, 241-253.[Medline]

Yamaoka, Y., Kodama, T., Gutierrez, O., Kim, J. G., Kashima, K. & Graham, D. Y. (1999). Relationship between Helicobacter pylori iceA, cagA, and vacA status and clinical outcome: studies in four different countries. J Clin Microbiol 37, 2274-2279.[Abstract/Free Full Text]

Received 14 July 2000; revised 25 January 2001; accepted 13 February 2001.