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Journal of Clinical Microbiology, October 2000, p. 3646-3651, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Helicobacter pylori: Clonal Population
Structure and Restricted Transmission within Families Revealed by
Molecular Typing
Shan-Rui
Han,1
Hans-Christoph E.
Zschausch,1
Heinz-Georg W.
Meyer,1
Thomas
Schneider,2
Michael
Loos,1
Sucharit
Bhakdi,1 and
Markus J.
Maeurer1,*
Department of Medical
Microbiology1 and Department of
Paediatrics,2 Johannes Gutenberg University,
D-55101 Mainz, Germany
Received 3 April 2000/Returned for modification 11 May
2000/Accepted 31 July 2000
 |
ABSTRACT |
Helicobacter pylori infects up to 50% of the human
population worldwide. The infection occurs predominantly in childhood
and persists for decades or a lifetime. H. pylori is
believed to be transmitted from person to person. However, tremendous
genetic diversity has been reported for these bacteria. In order to
gain insight into the epidemiological basis of this phenomenon, we performed molecular typing of H. pylori isolates from
different families. Fifty-nine H. pylori isolates from
27 members of nine families were characterized by using restriction
fragment length polymorphism analysis of five PCR-amplified genes, by
pulsed-field gel electrophoresis (PFGE) of chromosomal DNA, and by
vacA and cagA genotyping. The 16S rRNA gene
exhibited little allelic variation, as expected for a unique bacterial
species. In contrast, the vacA, flaA,
ureAB, and lspA-glmM genes were highly
polymorphic, with a mean genetic diversity of 0.83, which exceeds the
levels recorded for all other bacterial species. In conjunction with
PFGE, 59 H. pylori isolates could be differentiated
into 21 clonal types. Each individual harbored only one clone,
occasionally with a clonal variant. Identical strains were always found
either between siblings or between a mother and her children.
Statistical analysis revealed clonality of population structure in all
isolates. The results of this study suggest the possible coexistence of
a large array of clonal lineages that are evolving in each individual
in isolation from one another. Transmission appears to occur primarily
from mother to child and perhaps between siblings.
| |
INTRODUCTION |
Helicobacter
pylori-associated gastritis is today recognized as the major cause
of duodenal and gastric ulcers, gastric adenocarcinoma and
mucosa-associated lymphoid tissue lymphoma (9).
H. pylori infects up to 50% of the human population
worldwide. The infection occurs predominantly in childhood. Once the
stomach is colonized, the organism persists for decades, if not for a
lifetime. Many lines of evidence suggest person-to-person transmission
of H. pylori, possibly by an oral-oral, fecal-oral, or
gastric-oral route (4, 8, 9, 32-35, 44, 48). However,
tremendous genetic diversity has been reported for these bacteria
(23, 26). The molecular and epidemiological bases for this
phenomenon are not understood.
Limited data suggest that the H. pylori population
exhibits a panmictic or recombinational structure (1, 19, 24, 37, 39), and horizontal gene transfer followed by frequent DNA
recombination has been considered to be an important source of the
immense genetic diversity. However, clonal groupings of H. pylori have also been detected from different ethnic groups in New
Zealand, China, The Netherlands, and other countries (1, 7, 25,
43, 45, 46). Moreoveor, comparison of the complete genomic
sequences of two unrelated H. pylori isolates (2,
42) reveals that the overall genomic organizations, gene orders,
and predicted proteomes are quite similar, with the majority of
nucleotide changes representing synonymous substitutions. Based on
these observations, Wang et al. (47) pointed out that
mutation must be of major importance in generating the genetic
variation of H. pylori.
In the present study, 59 H. pylori isolates from 27 different family members of nine families were characterized in order to define the H. pylori population structure. The
results are most compatible with the concept that a large array of
clonal lineages coexist and are evolving in isolation from one another. The evidence also indicates that transmission of H. pylori occurs predominantly and perhaps even exclusively within
families. If this is correct, H. pylori would represent
the first human pathogen recognized to display this remarkably
restricted mode of transmission.
(Part of this work was performed in fulfillment of the requirements for
a doctoral thesis by H.-C. E. Zschausch.)
| |
MATERIALS AND METHODS |
Subjects.
Nine index children from different families,
designated families A through I, were referred to the Department of
Pediatrics, Johannes Gutenberg University, Mainz, Germany, due to
recurrent abdominal pain. One or more gastric (antral and corpus) and
duodenal biopsy specimens were obtained from each of these patients and from at least one additional family member (see Fig. 2). In total, 27 members from nine families were investigated, represented by 2 to 6 members in each family. These patients were 23 children (11 boys and 12 girls with an age range of 2 to 18 years) and 4 adults from three
families (2 women and 2 men from 29 to 44 years of age). The other
parents were not sampled. They lived either within or in close
proximity to Mainz.
Culture and PCR.
Biopsy specimens were cultured on Columbia
agar with 7% human erythrocytes and an H. pylori-selective supplement at 37°C under microaerobic
conditions. H. pylori was identified and bacterial genomic DNA was prepared as previously described (23). PCR
amplification of the vacA, flaA,
ureAB, lspA-glmM (formerly ureCD), or
cagA gene fragments was performed using the primer pairs
described previously (23). The cagA gene status
was determined by the presence or absence of cagA amplicons.
The vacA genotypes, including signal(s) sequences and
midregion (m) types, were characterized by a one-step PCR method
(22). For ribotyping, a 1.5-kb fragment was amplified with
broad-specificity 16S ribosomal DNA (rDNA) primer pairs pA-f
(5'-AGAGTTTGATCCTGGCTCAG-3') and pH-b
(5'-AAGGAGGTGATCCAGCCGCA-3') (15), using an
initial denaturation step at 94°C for 2 min, followed by 35 cycles of
denaturation at 94°C, annealing at 55°C, and extension at 72°C
(each for 1 min), and a final extension at 72°C for 10 min. In order
to eliminate contaminating DNA within PCR reagents, UV light and
8-methoxypsoralen (Sigma, Munich, Germany) were used (31).
Genomic DNA was added after irradiation of the PCR mixture with UV
light (366 nm) at room temperature for 20 min.
PCR-RFLP analysis.
PCR-amplified vacA,
flaA, ureAB, lspA-glmM, or 16S rDNA
fragments were digested with HaeIII (for ureAB or
16S rDNA), HhaI (for vacA, flaA, or
lspA-glmM), HinfI (for 16S rDNA), HphI
(for vacA), or Sau3AI (for flaA,
ureAB, or lspA-glmM) for 3 h at 37°C in
the appropriate buffer recommended by the supplier (New England Biolabs, Schwalbach/Taunus, Germany), and the DNA digests were analyzed
on ethidium bromide-stained 2% agarose gels. Each isolate was thus
characterized by 10 individual gene-enzyme patterns. A combination of
these 10 patterns was designated a PCR-based locus-specific restriction
fragment length polymorphism (PCR-RFLP) type, which defined the clonal
type of an H. pylori isolate. Additionally, a
combination of two restriction patterns of 16S rDNA was defined as a
ribotype. The cagA gene fragments were not analyzed by
PCR-RFLP, since the cagA gene could be detected in only 34 of 59 H. pylori isolates.
PFGE.
The methods previously described by Taylor et al.
(41) were modified. Fresh 2-day cultures of H. pylori were harvested and washed three times using normal saline.
Alternatively, in order to inhibit DNase activity, 0.9-ml portions of
the cell suspensions were incubated with 0.1 ml of 37% formaldehyde
solution for 1 h at room temperature, and then washed
(18). Approximately 1 ml of cell suspension with an optical
density at 600 nm of 0.9 was needed for the preparation of two agarose
plugs. The required volume of cell suspension was centrifuged, and
bacteria were resuspended in 10 mM Tris-HCl-10 mM EDTA (pH 8.0) (0.1 ml for 2 plugs). This suspension was mixed with an equal volume of
1.2% agarose equilibrated to 56°C to prepare 0.6% agarose plugs.
The solidified plugs were incubated overnight in ESP lysis buffer (0.25 M EDTA [pH 9.0], 1% lauroyl sarcosine, 1 mg of proteinase K per ml)
(Sigma, St. Louis, Mo.) at 56°C. The plugs were washed two times in
15 ml of the Tris-EDTA buffer described above for 2 h each at
4°C. For the subsequent enzyme reactions, the plugs were washed once
in 15 ml of 10 mM Tris-HCl-0.5 mM EDTA (pH 8.0) for 30 min at 4°C. The plugs could be stored in 10 mM Tris-HCl-50 mM EDTA (pH 8.0) at
4°C for 3 months.
For restriction enzyme digestion, plugs were incubated with 1 ml of the
appropriate 1× enzyme buffer for 30 to 60 min at room temperature. The
enzyme buffer was replaced with 100 µl of fresh enzyme buffer
containing 25 U of NotI, NruI, or SmaI
(New England Biolabs) and incubated overnight at 37°C (25°C for
SmaI). The plugs were chilled on ice for 15 min, and the
enzyme buffer was replaced with 10 mM Tris-HCl-10 mM EDTA (pH 8.0),
followed by loading of the plugs into the 1% running gel (Pulsed field
certified agarose; Bio-Rad Laboratories, Hercules, Calif.). A lambda
ladder PFG marker (New England Biolabs) served as a size standard. For pulsed-field gel electrophoresis (PFGE), CHEF-DR III PFGE systems (Bio-Rad Laboratories) were used. An agarose gel prepared in 0.5× Tris-borate-EDTA running buffer (Bio-Rad Laboratories) was subjected to
electrophoresis for 20.3 h at 200 V and a 0.5- to 60.4-s switch time at a constant temperature of 14°C. Agarose gels were stained with ethidium bromide and photographed. A combination of three restriction patterns in an isolate was designated a PFGE type.
Statistical analysis.
For the RFLP data, different patterns
of each of the analyzed genes were treated as different alleles. A
distinct combination of 10 individual patterns in an H. pylori isolate was designated an RFLP type (clonal type) and
equated with an allelic combination. Genetic diversity (h)
for a locus j is calculated as hj = 1 
i2[n/(n 1)], where i is the frequency of
the ith allele at the locus, n is the number of
isolates in the sample, and n/(n 1) is a correction
for bias in small samples (38). Genotypic diversity was
calculated by the same formula, in which i is
the frequency of the ith RFLP type and n is the
number of RFLP types. Genetic diversity and genotypic diversity have a
range of between 0 and 1. The mean diversity per locus is the
arithmetic average of h over all loci examined. The genetic
distance between pairs of isolates was calculated as the proportion of
loci at which dissimilar alleles occur, i.e., the proportion of
mismatches (38). The dendrogram was constructed from
PCR-RFLP typing data by the unweighted pair group cluster method with
arithmetic means in the computer program MEGA (28).
The index of association between loci (
IA) was
calculated as described previously (
30). The
IA value is a measure of the
degree of
association between loci and has an expected value of
zero for a large
random-mating (panmictic) bacterial population.
In contrast, for
bacterial species that are clonal at all levels
as a result of
geographic isolation or infrequent genetic recombination,
the expected
value of
IA should deviate significantly from
zero.
To determine whether
IA is significantly
greater than zero, a
Monte Carlo procedure can be performed. Computer
programs written
by J. G. Lorén (
17) were used to
calculate locus and genotypic
diversity, to calculate
IA, and to perform the Monte Carlo
simulations.
| |
RESULTS |
Figure 1 illustrates the RFLP and
PFGE patterns obtained with nine isolates from the mother, the father,
and two children of family A. These results are representative.
Whenever possible, analyses were performed on isolates obtained from
the antrum (a), corpus (c), and duodenum (d). Most patients harbored
only a single H. pylori isolate. If a second isolate
was detected, it differed only in a single restriction pattern, except
for one isolate (H4a). An example is the isolate from the antrum of the
mother (A1a), which varied from the corpus isolate A1c exclusively in
the flaA-HhaI patterns. We considered isolate A1a to be a
mutational variant of the parent isolate A1c.
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FIG. 1.
Representative molecular typing results for
H. pylori isolates from family A. PCR-RFLP analyses of
16S rDNA fragments digested with HaeIII (A), 16S rDNA
fragments digested with HinfI (B), flaA fragments
digested with HhaI (C), flaA fragments digested
with Sau3AI (D), ureAB fragments digested with
HaeIII (E), lspA-glmA fragments digested with
HhaI (F), and vacA fragments digested with
HphI (G) and PFGE of genomic DNA digested with
NotI (H) are shown. Lanes: M, size markers; A1a to A4c,
H. pylori isolates, designated according to the origins
of the isolates (A, family A; 1 to 4, family members; a, c, and d,
antrum, corpus, and duodenum, respectively).
|
|
Conspicuously, apparently identical isolates were very frequently found
within families, where they were identified between siblings or between
mother and child. The collective results of molecular typing are
summarized in Fig. 2. For 59 H. pylori isolates, analysis of the 16S rRNA gene
demonstrated an average of 2.5 RFLP patterns, a mean genetic diversity
of 0.08, and a mean genotypic diversity of 0.14. The sparse allelic
variation in the 16S rRNA gene was as expected for a unique bacterial
species (14). In contrast, the vacA,
flaA, ureAB, and lspA-glmM genes were
highly polymorphic, with an average of 11.25 RFLP patterns per gene, a
mean genetic diversity of 0.83, and a mean genotypic diversity of 0.91 for all of 59 H. pylori isolates. These values exceed the level of diversity recorded for all other bacterial species (19). The results of PFGE typing were concordant with the
PCR-RFLP analysis in 46 of 52 cases. Three variants (A1a, D2a, and I2c) were distinct from their parent strains by PCR-RFLP analysis but showed
PFGE patterns identical to those of the parent strains. Vice versa,
three variants (B1c, H4a, and H4c) were distinguishable from their
parent strains by PFGE but not by PCR-RFLP analysis. Hence, detection
of strain variants is sometimes possible only by implementing both
PCR-RFLP and PFGE analysis.
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FIG. 2.
Genetic relationships among 59 H. pylori isolates from nine different families. The dendrogram was
constructed from PCR-RFLP typing data by the unweighted pair group
cluster method with arithmetic means. Each distinct combination of 10 individual gene and enzyme patterns was designated a PCR-RFLP type,
corresponding to a clonal type. Ten major lineages, termed I through X,
separating at a genetic distance of 0.3 are indicated to the left. The
columns to the right indicate the designation of H. pylori isolates, the origin of the isolates, and the molecular
typing results. n.d., not determined; n.t., not typeable.
|
|
The genetic relationship among the 59 H. pylori
isolates exhibited a tree-like phylogenetic structure with a deep but
limited branching pattern (Fig. 2). Twenty-one clonal types clustered into 10 divisions (designated I to X). Isolates from one family tended
to cluster into the same division. Isolates from different families
were sometimes also found to be related, as evident in divisions II and IV.
Siblings tended to harbor identical isolates (families A, C, D, G, and
H), but distinct H. pylori isolates could also be found between siblings (families D, E, F, and I). We were able to analyze H. pylori isolates from only one pair of parents
(family A). The mother harbored the same isolate as her children,
whereas the father harbored an unrelated strain. Intriguingly, the
mother in family B also had the same isolate as her son. In contrast, the H. pylori strain of the father in family F was
distinct from the isolates recovered in either of his children. These
results indicate that H. pylori can be transmitted
within families and possibly from parent (mother) to child or between
siblings. However, a common source of exposure for H. pylori infection could not be completely ruled out.
The complete set of H. pylori isolates and subsets of
the population were analyzed for multilocus linkage disequilibrium
(Table 1). The 59 isolates differed on
average at 6.80 of the 10 loci examined, and the
IA was 0.43. The Monte Carlo procedure indicated that the IA was significantly different from
zero. Moreover, calculation of IA for the 21 PCR-RFLP types resulted in a value of 1.42, which was significantly
different from zero (P < 0.001) based on the Monte
Carlo procedure. These results, analyzed at the level of all isolates
or RFLP types, demonstrated a significant degree of linkage
disequilibrium among the H. pylori isolates from
different families, consistent with the clonal structure described by
Maynard Smith et al. (30).
| |
DISCUSSION |
Previous studies have suggested that H. pylori is
panmictic and that the immense genetic diversity among H. pylori strains arises by frequent horizontal DNA transfer and
recombination (1, 19, 24, 37, 39). Indeed, direct evidence
for recombination between different H. pylori strains
has been observed in humans (27) and in mice
(10). However, several lines of evidence indicate that
mutations may also play an important role in generating the observed
diversity in H. pylori (47). We show in this
report that the bacterium H. pylori does in fact
exhibit a clonal structure. According to these data and previous
studies (23), each individual will harbor very few
H. pylori strains (usually only one). This hypothesis
was substantiated by our data (Fig. 2) that a large array of clonal
lineages coexist, which evolve in isolation, i.e., in each individual.
This would account for the finding that separate lineages of
H. pylori have been detected from different ethnic groups in New Zealand (Polynesians and Europeans), in China, in The
Netherlands, and in other countries (1, 7, 25, 43, 45, 46).
Identical flaA, flaB, or vacA alleles
were also found in unrelated H. pylori strains from a
population in Capetown, South Africa (39). These results are
consistent with the existence of divergent H. pylori
clonal lineages at the level of different ethnic groups or different
geographic regions.
The results presented in this report strongly suggest that mutations
which occur within the individual host are capable of generating
variant H. pylori strains. Recombination, although possible, will be a rather rare event in the absence of multiple infections. In fact, infection with multiple H. pylori
strains appears to be infrequent in the developed world. The
"different" strains recovered from the same patient often seemed
more closely related by fingerprinting than other strains recovered
from different patients. In experimentally infected rhesus monkeys, a
mixed infection tends to resolve quickly, with just one strain
generally predominating within a year (8). If the working
hypothesis is correct, the clonal population structure of H. pylori would only slowly be eroded by recombination. The fact that
the mode of H. pylori transmission is probably very
restricted gains particular importance in this context. Although the
number of subjects included here was still relatively small, all of the
results would be in line with the contention that H. pylori transmission occurs mainly, if not exclusively, within
families. Thereby, transmission from parent (mother) to child and
perhaps from child to child predominates, occurring conceivably via the
gastric-oral, oral-oral, or fecal-oral route. This contention would be
consistent with many studies that have utilized epidemic (5,
11-13, 16, 35) or molecular typing (4, 8, 32, 34, 44,
48) methods. A small number of epidemic studies have concluded
otherwise (3, 6, 20, 21), but this may have been due to
factors affecting H. pylori transmission, e.g., the
general prevalence of H. pylori in different age
groups, the timing of acquisition, and the socioeconomy and ethnic
composition of the studied population. An investigation by Goodman and
Correa (21) with rural Colombian children appeared to reveal
transmission of H. pylori from older to younger
siblings. However, Rowland (36) has commented on the
problems surrounding the interpretation of those data. The hypothesis
that H. pylori transmission occurs predominantly from
parents to child, and not vice versa, is supported by the observations
that (i) H. pylori acquisition occurs predominantly in
childhood and (ii) the prevalence of H. pylori
infection in parents is much higher than that in children.
Additionally, interspousal transmission of H. pylori
occurs rarely (29, 40). Our molecular analysis of
H. pylori suggests the vertical spread from parent (mother) to child, perhaps combined with the horizontal transmission between siblings, as the pathway for H. pylori
transmission. If this proves to be correct, H. pylori
would represent the first human pathogen recognized to display this
remarkably restricted mode of transmission.
| |
ACKNOWLEDGMENTS |
We are indebted to J. G. Lorén, Departament de
Microbiologia i Parasitologia Sanitàries, Facultat de
Farmàcia, Divisió de Ciències, de la Salut,
Universitat de Barcelona, Barcelona, Spain, for providing computer
programs of linkage disequilibrium and helpful discussions. We
acknowledge G. Rippin, Department of Medical Statistics and
Documentation, University of Mainz, for helpful discussions on
statistical methods. We thank D. E. Taylor and Q. Jiang,
Department of Medical Microbiology and Immunology, University of
Alberta, Edmonton, Alberta, Canada, and B. Geilhausen, Department of Medical Microbiology and Hygiene, University of Cologne,
Cologne, Germany, for helpful communications concerning the PFGE method.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology, Johannes Gutenberg University, Hochhaus am
Augustusplatz, D-55101 Mainz, Federal Republic of Germany.
Phone: (49)-6131-393-3645. Fax: (49)-6131-393-5580. E-mail:
maeurer{at}mail.uni-mainz.de.
| |
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Journal of Clinical Microbiology, October 2000, p. 3646-3651, Vol. 38, No. 10
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Abstract
Introduction
