| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Helicobacter Reference Unit, Laboratory of Enteric Pathogens, Health Protection Agency, 61 Colindale Avenue, London, NW9 5HT, UK#dReceived 11 June 2002 Accepted 6 January 2003
Correspondence: Robert J. Owen (rowen{at}phls.nhs.uk)
 |
Abstract |
|---|
 TOP Abstract INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
An understanding of the natural history of initial infection is provided by investigation of intrafamilial transmission. Several immunological studies have demonstrated clustering of infection amongst family members (Drumm et al., 1990; Malaty et al., 1991; Oderda et al., 1991; Dominici et al., 1999), including antibody profiling (Ng et al., 2001). The importance of adult–child transmission has been demonstrated, with parents, in particular infected mothers, playing a key role as a source of H. pylori (Rothenbacher et al., 1999; Malaty et al., 2000; Taneike et al., 2001). Most of these epidemiological investigations have relied on serology and urea breath-tests to define intrafamilial clustering, but genotyping provides more precise information about transmission of specific strains between individuals. The genomic heterogeneity of H. pylori is well-documented (Owen et al., 2001b), and isolates in families have been investigated using a variety of DNA-based methods that have included ribotyping (Nwokolo et al., 1992; Bamford et al., 1993; Taneike et al., 2001), arbitrarily primed (RAPD) typing (van der Ende et al., 1996), PCR-RFLP and PFGE analysis (Han et al., 2000) and sequencing of pathogenicity-associated genes such as vacA, flaA and flaB (Suerbaum et al., 1998). Population genetic analysis by multilocus sequence typing (MLST), which is based on the concept of defining isolates according to allelic variation using seven internal fragment sequences (about 500 bp) of housekeeping loci (Enright & Spratt, 1999), has demonstrated that H. pylori has a non-clonal (panmictic) population structure, due to horizontal gene transfer and frequent recombination resulting in extensive genetic rearrangements (Achtman et al., 1999; Maggi Solcà et al., 2001). A similar conclusion was reached from sequence analysis of several virulence-associated loci (Achtman et al., 1999). Even so, H. pylori appears to be clonal over short periods of time, as there is evidence that clonal descent is readily detected within some family groups in Germany (Suerbaum et al., 1998; Han et al., 2000) and in Japan (Malaty et al., 2000). Subsequent development of diversity and loss of clonality may occur during mixed colonization by unrelated strains (Falush et al., 2001; Israel et al., 2001).
Our aim in this study was to explore the existence of clonality in H. pylori infecting family groups by investigation of sequence diversity within core regions of three housekeeping genes (ureI, atpA and ahpC). To assess the discriminatory power of sequence typing for H. pylori and to obtain an indication of the overall background species diversity at these loci, we examined isolates from two gastric sites, as well as strains from single sites, within unrelated individuals. We also analysed inferred amino acid sequences to gain an insight into the frequency of non-synonymous variation, and we determined heterogeneity in two key pathogenicity-associated markers (cagA status and vacA allelic type) to assess their contribution to strain diversity within the family clusters.
|
METHODS |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Culture of isolates and template DNA preparation.
All strains of H. pylori and the type strains of H. nemestrinae and H. mustelae were cultured for 2–3 days on Columbia agar base (Oxoid) containing 10 % (v/v) defibrinated horse blood, and incubated at 37 °C under microaerophilic conditions (4 % O2, 5 % H2, 5 % CO2 and 86 % N2, by vol.) in a variable-atmosphere incubator (Don Whitley Scientific). Stock cultures were preserved (after minimum passages) on glass beads in Nutrient Broth (Oxoid) containing 10 % (v/v) glycerol over liquid N2 or at -80 °C. Genomic DNA was extracted from sweep cultures of each isolate using the cetyltrimethylammonium bromide (CTAB) method (Wilson, 1987).
Selection of loci for sequence typing.
The ureI and atpA loci, encoding enzymes for intermediary metabolism, were chosen according to the criteria defined by Achtman et al. (1999). The ureI locus (HP0071) is part of the urease gene cluster (Mobley, 2001), and its product is a urea accessory (transporter) protein that is thought to be an integral cytoplasmic membrane protein, forming a proton-gated urea channel regulating cytoplasmic urease (Weeks et al., 2000). The atpA locus (HP1134) encodes a homologue of the ATP synthase F1 
subunit, which has a key function in the synthesis of ATP. The ahpC gene was included here as it is a key housekeeping gene, although it was not used in previous MLST studies (Achtman et al., 1999; Maggi Solcà et al., 2001). The AhpC subunit, partially involved in alkyl hydroperoxide reductase (Ahp) activity, is encoded by ahpC (HP1563/JHP1471), a cysteine-based peroxidase homologue, which may be involved in oxidative stress by reduction of hydrogen peroxide and a wide range of organic peroxides to the corresponding alcohols (Kelly et al., 2001). AhpC was identified as a major 26 kDa antigen in H. pylori and was present in other species of Helicobacter (Lundstrom et al., 2001).
PCR and sequencing.
Diluted DNA (100 ng) was used as the target to amplify an internal fragment for each locus. For ureI, the following primers for amplification and sequencing were designed from the sequence (HP0071) of NCTC 12455 (Tomb et al., 1997): forward primer ureIS4, 5'-GGAAGGAAAAGGCAATGC-3' and reverse primer ureIAS2, 5'-CTAAACGCTCTATGATCA-3'. These were used as alternatives to the previously described primers (Achtman et al., 1999) which in our hands did not amplify ureI from every strain. For atpA (HP1134) and ahpC (HP1563), previously described primers (Achtman et al., 1999; Lundstrom et al., 2001) were used for amplification and sequencing of fragments of 627 and 541 bp, respectively. PCR amplifications were performed in a total volume of 50 µl containing 100 ng template DNA, 1.5 mM MgCl2, 0.05 mM each dNTP, 0.4 mM each oligonucleotide primer, 0.2 µl (1 U) Taq polymerase (Life Technologies) and 5 µl 10x buffer provided by the manufacturer. Automated sequencing from both strands of PCR products was then performed commercially (MWG Biotech) and on-site by standard protocols using a CEQ 2000XL instrument (Beckman Coulter). All new sequences were deposited in the EMBL database (see Table 1).
|
Analysis of sequence and allele and sequence type (ST) assignment.
The sequences used in the phylogenetic analyses were as follows. For ureI and atpA, these each comprised the five H. pylori reference strain sequences obtained from the GenBank database (Table 1) and 68 new H. pylori sequences. For ahpC, these comprised two H. pylori reference strain sequences from GenBank (Table 1) and 71 new sequences. The atpA and ureI sequences for H. nemestrinae NCTC 12491T and the atpA sequence for H. mustelae NCTC 12198T were also obtained from GenBank. The ahpC sequences for H. mustelae and H. nemestrinae were determined. All sequences were stored and aligned using BioEdit (Hall, 1999). Sequences were converted in SeqVerter (GeneStudio) for use in TREECON version 1.3 (Van de Peer & De Wachter, 1994), implementing CLUSTAL W multiple alignment to construct and draw phylogenetic trees. Nucleotide sequences were analysed by pairwise comparison and clustering using the neighbour-joining method of Saitou & Nei (1987) with the Jukes–Cantor correction. Bootstrap analysis was performed with 100 resampled datasets from evolutionary distance, based on alignments of nucleotide sequences (Van de Peer & De Wachter, 1994). Each tree was rooted by using the H. mustelae sequence as the outgroup.
cagA and vacA genotyping.
Details of the primers and PCR conditions for the two genotyping assays based on the H. pylori pathogenicity-associated factors cagA and vacA have been described elsewhere. Briefly, PCR conditions for the two assays for cagA (a marker for the right-hand end of the cag pathogenicity island and for the cagI region) were as described for the F1/B1 primers and the D008/R008 primers (Slater et al., 1999; Owen et al., 2001a). Genotyping based on vacA signal (s1 and s2) and mid-region (m1 and m2) alleles was also performed as described previously (Atherton et al., 1999).
|
RESULTS |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
|
Assignment of nucleotide STs
From analysis of the sequences for the 73 isolates of H. pylori and the reference strains of H. nemestrinae and H. mustelae, the ureI, atpA and ahpC regions were found to be highly polymorphic (giving a total of 62, 63 and 65 alleles, respectively) for H. pylori including H. nemestrinae, with the sequences differing at one or more nucleotide positions. Each gene-sequence variant within the three sets was designated an allele number (see Figs 1, 2 and 3), and these were combined for the 73 strains of H. pylori and for the strains of H. nemestrinae and H. mustelae to give a total of 66 nucleotide STs (numbered ST1–ST66). All isolates had different STs apart from those in paired (antrum/corpus) sets from five patients (Table 2), and in two family sets where there were common STs within families (Table 3).
|
|
Assignment of derived amino acid STs
The ureI, atpA and ahpC nucleotide sequences for H. pylori, H. nemestrinae and H. mustelae were translated into derived amino acid (gene product) sequences, and phylogenetic analyses were performed (data not shown). Clustering pattern differences predominantly reflected nucleotide substitutions at non-synonymous sites, and 26, 15 and 23 amino acid STs were identified for UreI, AtpA and AhpC, respectively. Each amino acid ST was assigned a number and the type frequencies are shown in Fig. 4. The designated amino acid profiles, representing the combined types for each of the six reference strains and the five identical paired isolate sets, are listed in Table 2. Although the combined profiles of strains from different individuals were unique, certain amino acid types were highly conserved within the dataset, notably UreI type 4 (n = 12), AtpA types 1 (n = 42) and 3 (n = 14), and AhpC type 9 (n = 12). AtpA profile 1 was the most highly conserved, despite the geographic diversity of those isolates, indicating selective constraints on amino acid replacements in that protein due to possible functional or structural requirements.
|
Analysis of family isolates
Isolates of H. pylori from five family clusters were examined (Table 3). Isolates with the same STs were present in two families from Belfast (K and M) and in both of these, the strain from the child had a different ST from those of the parents. Nevertheless, members of family K all shared the ureI allele 36 and at the amino acid level also shared AhpC amino acid type 9. Likewise, in family M, all members shared AtpA amino acid type 1. Strains H3022 and H3023 from family H had identical amino acid profiles, although the mother in that family was not infected with H. pylori. Strains H357 (child) and H3024 (mother) from family B had different STs, with no shared alleles or shared amino acid types. For the Coventry family group, all members were infected by strains with unique STs. However, some alleles were conserved, as ureI allele 34 was present in isolates from four members of three generations. Furthermore, AtpA amino acid type 1 was present in five members and AhpC amino acid type 1 was present in two members (grandfather and uncle).
Associations with cagA status and vacA genotype
The cagA status and vacA genotype were determined for all 73 strains of H. pylori in the study. The genotypes of the reference strains and multiple isolates are listed in Table 2 and those of the family isolates are listed in Table 3. In addition, H. nemestrinae NCTC 12491T had the typical cagA+/vacA s1m1 genotype. In the paired isolates of H. pylori, members of each set had identical cagA/vacA types, as did isolates in the families with matching STs, except for family Belfast-K where the isolates from the mother (H3021) and father (H3020) differed in vacA m-type.
|
DISCUSSION |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Members of the five families examined were colonized by more than one unrelated strain of H. pylori. The fact that strains with identical STs were present in two members of each of two of the five families gives support to the accepted model that intrafamilial transmission provides the main focus for H. pylori spread. However, at present there is no general agreement on the precise route: one possibility is spread between spouses, for which there is evidence from several serology-based studies (Brenner et al., 1999; Singh et al., 1999; Stone et al., 2000). However, genotyping provides conflicting results, with detection of the same strain in both partners apparently being uncommon in spouses examined in Singapore (Kuo et al., 1999) and Japan (Suzuki et al., 1999), whereas others reported the presence of identical strains in 8 of 18 couples in Greece (Georgopoulos et al., 1996), and in one of four couples in Germany (Suerbaum et al., 1998). The fact that we found strains with identical STs in husband and wife in two Belfast families (K and M) confirms that interspouse transmission is also a possibility in UK households, although the family K isolates had different vacA m-types. Pathogenicity-associated genes such as vacA, where change in m-type could be attributed to the loss or gain of a 75 bp insert in the mid-region, may therefore provide less stable markers than housekeeping genes.
The second possibility for intrafamilial spread is by parent-to-child transmission. Several serology-based studies have shown that infected parents, in particular mothers, have a key role (Brenner et al., 1998) and that there is a twofold increase in risk when both parents are infected (Dominici et al., 1999). Transmission from mother to child has been demonstrated by strain genotyping in Japan (Malaty et al., 2000; Taneike et al., 2001) and in Germany (Han et al., 2000). However, it was reported that even when both parents in one family in Germany had an identical strain, the child isolate was different (Suerbaum et al., 1998). In the present study, one subject (a 14-year-old boy) was infected with a strain that had the same amino acid profile as the strain infecting the father (Belfast family H). However, the mother in that family was not infected with H. pylori, so either father-to-son transmission or the converse, son-to-father transmission, might have occurred. The latter would seem more likely, as it would be probable that the father had been colonized since early childhood (< 10 years of age). A Japanese family study (Taneike et al., 2001) showed that a son was probably infected originally from the father, but became reinfected after eradication with a strain from the mother. In a German family study (Suerbaum et al., 1998), DNA sequence data showed that in three of four families, mother and child had related strains, whereas only one family provided direct evidence of father–child strain relatedness. The extended Coventry family provided additional opportunities of possible parent-to-child transmission. However, the grandmother (subject 5) and her son (subject 6), both members of a separate branch of the family, were infected by strains that lacked matching alleles, so transmission was unlikely to have taken place. By contrast, the grandfather (subject 2) shared ureI allele 34 with two of his sons (subjects 3 and 4) and also ahpC allele 34 with one son, indicating transmission of a related strain. There was also evidence of transmission between father (subject 3) and son (subject 1) in generations 2 and 3, as these isolates had one shared allele. Interestingly, there was a close association between the grandfather and grandson (subject 1), indicating conservation of two alleles over three generations and a possible direct infection, as the father had a different atpA allele. These data indicate that the alleles could have remained stable for at least 70 years in the family. Comparisons of the cagA and vacA s1 genotypes for the family isolates indicated that they were generally conserved, but the m-type could vary from m1 to m2 in passing between some subjects, such as between Coventry subjects 2 and 3, and as noted above in Belfast family K.
Other possible H. pylori transmission routes for which there are limited data include sibling-to-sibling infections (Goodman & Correa, 2000; Rothenbacher et al., 2000). Isolates from the two brothers in generation 2 of the Coventry family (subjects 3 and 4) were found to share ureI allele 34, which suggests that the isolates may have been derived from a common ancestral strain. DNA fingerprinting of isolates from two parents and six daughters in the Netherlands showed that all were closely related, but not identical (van der Ende et al., 1996), whereas identical strains were found in only two of six Lithuanian families (Chalkaukas et al., 1998). It is possible that some family infections (particularly in developing countries) arise from common environmental sources, but there are no genotyping data available to confirm the involvement of water, food or domestic pets (Brown, 2000). However, as the evidence to date is limited to a relatively small number of families worldwide, it remains difficult to reach firm conclusions about any one predominant intrafamilial route of transmission for H. pylori.
Assessment of interfamilial transmission is further complicated by the possibility of mixed H. pylori infections in individual subjects. The present study of family subjects was based on cultures originating from antral biopsies, for which there was no evidence of simultaneous infections with more than one strain or strain subtype. The presence of different strains at other gastric sites cannot be excluded, but investigations of paired antrum and corpus biopsies in unrelated subjects in this study, and in previous investigations of colonization patterns before treatment, showed that infections of UK individuals were typically by identical or closely related subtypes (Owen, 1993; Owen et al., 1999). Studies on individuals in other countries suggest a spectrum of diversity in different individuals; for instance, analysis of the parent strain J99 and its derivatives after 6 years indicated continuous microevolution, attributed to loss and acquisition of exogenous DNA over time (Israel et al., 2001).
The introduction of DNA sequence typing to investigate clonal descent and microdiversity of H. pylori within individuals and in families (Suerbaum et al., 1998; Falush et al., 2001; Israel et al., 2001) is a significant development because it provides an unambiguous approach, avoiding the problems of interpreting conventional genotyping data based on DNA fragment fingerprinting by ribotyping, RAPD, PCR-RFLP and PFGE. To date, direct sequencing has used both core fragments of pathogenicity-associated genes (Suerbaum et al., 1998) and of housekeeping genes (Achtman et al., 1999; Falush et al., 2001; Maggi Solcà et al., 2001). We have extended this approach to designate STs, albeit in a restricted form based on three housekeeping genes, to define clonal descent or evidence of residual clonality within families, as interpretation was equivocal using ribotyping and restriction digest analysis (Nwokolo et al., 1992; Bamford et al., 1993). Thus, in the Coventry family, the ureI allele 34 was identified as unique and conserved in isolates from three generations, and the atpA allele 32 was conserved in the child and the grandfather. These findings suggest that some alleles may have been present for decades in strains causing infections within some families. Strains within families may have different STs due to differences in single alleles; even so, it was evident from the gene trees that such strains were more similar to other strains of the same family than to strains from unrelated individuals – a finding supported by analyses including additional ureI and atpA sequence data on some 46 strains from GenBank (R. J. Owen, unpublished results). We also found that analysis of amino acid sequences provides evidence of common descent in some loci, after removal of differences due to synonymous mutations. However, as certain amino acid sequences (such as AtpA type 1) were highly conserved amongst strains from unrelated individuals, their use as markers is less precise for defining unique family lineages. Overall, our study has shown that sequence typing provides unambiguous information about common descent, based on unique shared alleles of housekeeping genes that are not present in other members of the population. These alleles can be used to identify genetic drift, as H. pylori strains are transmitted between individuals in the same or different generations of a family.
In conclusion, related strains of H. pylori can be transmitted amongst different family members. Over time, the species genotype may change as populations continually adapt to different human hosts by the formation of successful recombinants. Some alleles of housekeeping loci are sufficiently stable to provide family-specific markers, so sequence typing provides a precise means of identifying and monitoring their distribution and evolution. Sequence databases using a combination of data from housekeeping and pathogenicity-associated loci will facilitate development of a global typing strategy for future studies on H. pylori transmission.
|
Acknowledgments |
|---|
|
REFERENCES |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. H. Argent, R. J. Thomas, F. Aviles-Jimenez, D. P. Letley, M. C. Limb, E. M. El-Omar, and J. C. Atherton Toxigenic Helicobacter pylori Infection Precedes Gastric Hypochlorhydria in Cancer Relatives, and H. pylori Virulence Evolves in These Families Clin. Cancer Res., April 1, 2008; 14(7): 2227 - 2235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-H. Chuang, M.-S. Wu, W.-L. Lo, J.-T. Lin, C.-H. Wong, and S.-H. Chiou The antioxidant protein alkylhydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function PNAS, February 21, 2006; 103(8): 2552 - 2557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lundin, B. Bjorkholm, I. Kupershmidt, M. Unemo, P. Nilsson, D. I. Andersson, and L. Engstrand Slow Genetic Divergence of Helicobacter pylori Strains during Long-Term Colonization Infect. Immun., August 1, 2005; 73(8): 4818 - 4822. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Kersulyte, A. Kalia, M. Zhang, H.-K. Lee, D. Subramaniam, L. Kiuduliene, H. Chalkauskas, and D. E. Berg Sequence Organization and Insertion Specificity of the Novel Chimeric ISHp609 Transposable Element of Helicobacter pylori J. Bacteriol., November 15, 2004; 186(22): 7521 - 7528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. M. Carroll, N. Ahmed, S. M. Beesley, A. A. Khan, S. Ghousunnissa, C. A.O Morain, C. M. Habibullah, and C. J. Smyth Microevolution between paired antral and paired antrum and corpus Helicobacter pylori isolates recovered from individual patients J. Med. Microbiol., July 1, 2004; 53(7): 669 - 677. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Chisholm and R. J. Owen Frameshift mutations in frxA occur frequently and do not provide a reliable marker for metronidazole resistance in UK isolates of Helicobacter pylori J. Med. Microbiol., February 1, 2004; 53(2): 135 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Dailidiene, G. Dailide, K. Ogura, M. Zhang, A. K. Mukhopadhyay, K. A. Eaton, G. Cattoli, J. G. Kusters, and D. E. Berg Helicobacter acinonychis: Genetic and Rodent Infection Studies of a Helicobacter pylori-Like Gastric Pathogen of Cheetahs and Other Big Cats J. Bacteriol., January 15, 2004; 186(2): 356 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Owen, J. Xerry, T. Gotada, G. Naylor, and D. Tompkins Analysis of geospecific markers for Helicobacter pylori variants in patients from Japan and Nigeria by triple-locus nucleotide sequence typing Microbiology, January 1, 2004; 150(1): 151 - 161. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
