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
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ABSTRACT |
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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 AY004351AY004662
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INTRODUCTION |
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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.
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METHODS |
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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 Skirrows 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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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 manufacturers 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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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.
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RESULTS |
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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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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.
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DISCUSSION |
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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), 363365 (GAT
AGC; Asp
Ser), 368 (A
G, silent) and 374375 (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 hosts predisposition.
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ACKNOWLEDGEMENTS |
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Received 14 July 2000;
revised 25 January 2001;
accepted 13 February 2001.