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Previous Article | Next Article 
Journal of Bacteriology, June 2000, p. 3219-3227, Vol. 182, No. 11
Department of Molecular Microbiology,
Washington University Medical School, St. Louis, Missouri
63110,1 National Institute of
Cholera and Enteric Disease,2 and
Department of Gastroenterology, Institute of Post Graduate
Medical Education and Research,4 Calcutta,
India, and Second Department of Internal Medicine, Fukui
Medical University, Fukui, Japan3
Received 31 January 2000/Accepted 15 March 2000
The genotypes of 78 strains of Helicobacter pylori from
Calcutta, India (55 from ulcer patients and 23 from more-benign
infections), were studied, with a focus on putative virulence genes and
neutral DNA markers that were likely to be phylogenetically
informative. PCR tests indicated that 80 to 90% of Calcutta strains
carried the cag pathogenicity island (PAI) and potentially
toxigenic vacAs1 alleles of the vacuolating cytotoxin
gene (vacA), independent of disease status. This was higher
than in the West (where cag PAI+
vacAs1 genotypes are disease associated) but lower than
in east Asia. The iceA2 gene was weakly disease associated
in Calcutta, whereas in the West the alternative but unrelated
iceA1 gene at the same locus is weakly disease associated.
DNA sequence motifs of vacAm1 (middle region) alleles
formed a cluster that was distinct from those of east Asia and the
West, whereas the cagA sequences of Calcutta and Western
strains were closely related. An internal deletion found in 20% of
Calcutta iceA1 genes was not seen in any of ~200 strains
studied from other geographic regions and thus seemed to be unique to
this H. pylori population. Two mobile DNAs that were rare
in east Asian strains were also common in Calcutta. About 90% of
Calcutta strains were metronidazole resistant. These findings support
the idea that H. pylori gene pools differ regionally and
emphasize the potential importance of studies of Indian and other
non-Western H. pylori populations in developing a global
understanding of this gastric pathogen and associated disease.
Helicobacter pylori is a
gastric pathogen that chronically infects more than half of all people
worldwide (for reviews see references 48 and
64) and constitutes a major cause of peptic ulcer
disease and an early risk factor for gastric cancer. It may also
contribute to childhood malnutrition and increase the risk or severity
of infection by other gastrointestinal pathogens such as Vibrio
cholerae, especially in developing countries (18, 19).
It appears to be one of the most genetically diverse of bacterial
species, because DNA fingerprinting can distinguish any given isolate
from most others (5, 56) and because of the ~3 to 5% DNA
sequence divergence typically found in essential genes from unrelated
strains (3, 29). This mutational diversity is enhanced by a
rich history of interstrain recombination (29, 40, 54). In
contrast, most other well-studied bacterial species characterized to
date are much more strongly clonal (see, e.g., references 28,
31, and 53).
The observed genetic diversity implies a lack of population-wide
selection for just one or a few universally most fit H. pylori genotypes. Some of this may reflect preferential
transmission within families and among people in close contact, not in
large epidemics (11, 24, 50). Such a pattern means that no
individual strain would compete simultaneously against many others
(12, 38, 55). H. pylori diversity would also be
enhanced if humans differ in traits that are important to individual
strains (e.g., specificity or strength of immune and inflammatory
responses or availability of receptors used for H. pylori
adherence [26, 33]).
There are also indications of significant geographic differences among
strains. For example, only one-half to two-thirds of U.S. and European
strains carry the cag pathogenicity island (PAI), a 40-kb
DNA segment many of whose genes seem to help induce interleukin 8 and
thereby a strong and potentially damaging inflammatory response; such
strains are recovered preferentially from persons with overt disease
(4, 8, 9, 17). In contrast, nearly all east Asian strains
carry the cag PAI independent of disease status (34,
47). Similarly, somewhat more than half of U.S. and European strains carry toxigenic (vacAs1) alleles of the
vacuolating cytoxin gene, with other strains carrying nontoxigenic
(vacAs2) alleles (in general, vacAs1
strains carry the cag PAI (65); nearly all east Asian strains carry vacAs1 alleles. Potentially
more significant in terms of host interaction and evolution were
findings that east Asian and Western strains differ markedly in
DNA sequence motifs in the vacA and cagA genes
(3, 34, 35, 46, 58, 62), since the proteins these genes
encode probably each interact directly with host factors (CagA protein
is translocated to host cells and is tyrosine phosphorylated in them
but is not needed for interleukin 8 induction) (7, 45, 51).
Even though less geographic partitioning was found in a sampling of
housekeeping genes (3, 58), it is not clear whether the
regional differences in cagA and vacA alleles
reflect natural selection, random genetic drift including founder
effects, or both during H. pylori evolution. It is
noteworthy in this context that the two strains whose genome sequences
have been determined (6, 57) and compared to better understand H. pylori genetic diversity (23) are
from ethnic European patients (26695 from the United Kingdom
[6]; J99 from a Caucasian in Pulaski, Tenn.
[57; T. L. Cover, personal communication]) and thus may not be fully representative of H. pylori worldwide.
We began studies of genotypes of H. pylori strains of India,
motivated by the differences between strains of east Asia and the West
found to date and a sense that the peoples of the vast Indian
subcontinent (some one-fifth of all humanity) may have been
sufficiently isolated during much of human history to have allowed the
emergence of a distinct H. pylori gene pool (37, 42,
63). It is well established that H. pylori infection
and peptic (especially duodenal) ulcer disease are very common in India
(1, 36, 44, 52) and that a large fraction of strains may be
resistant to metronidazole (Mtzr) (2), but to
our knowledge there has been very little analysis to date of the
genotypes of the underlying H. pylori strains
(20). Here we identify several markers that help distinguish
H. pylori strains from Calcutta from those of east Asia and
the West.
Patient samples.
Adult ethnic Bengali patients of both
sexes, age 21 to 65 years, who presented with gastric complaints were
endoscoped at the Hospital of the Institute of Post Graduate Medical
Education and Research in Calcutta, India, using well-washed and
sterilized fiber optic endoscopes. Biopsies and gastric juice samples
used in the present study were obtained during endoscopy with informed consent, under protocols approved by the institutional review boards of
the Institute of Post Graduate Medical Education and Research and the
National Institute of Cholera and Enteric Disease (Calcutta, India).
Two biopsies were taken for culture, one from the gastric antrum and
one from the corpus, and were stored at H. pylori culture.
Cultures were prepared by
smearing single biopsy specimens on petri plates containing brain heart
infusion (BHI) agar (Difco) supplemented with 7% horse blood, 0.4%
IsoVitaleX, amphotericin B (8 µg/ml), trimethoprim (5 µg/ml), and
vancomycin (6 µg/ml) and were incubated at 37°C in an atmosphere of
5% O2-10% CO2-85% N2 for 3 to
6 days. H. pylori colonies were identified based on their
typical morphology, characteristic appearance on Gram staining, a
positive urease test, and subsequent gene-specific PCR tests. The
H. pylori cells that grew out from one biopsy on the primary culture plate were collected as a pooled population, and preserved in
sterile BHI broth with 15% glycerol at
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Distinctiveness of Genotypes of Helicobacter pylori in
Calcutta, India
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C in 0.5 ml of brucella
broth (Difco) with 15% glycerol until culture. Two milliliters of
gastric juice was also collected during endoscopy in some cases and
stored frozen at 70°C until use. Diagnoses of peptic ulcer disease
were based on visual examination of the stomach and duodenum during
endoscopy and also on any patient history of earlier peptic ulcer. If
no evidence of peptic ulcer disease was found, the patient was
considered to have a more-benign infection (nonulcer dyspepsia,
gastritis only). Fifty-five of the patients had peptic ulcer disease,
and 23 had gastritis only.
70°C. In general, only one
such culture was analyzed per patient.
DNA methods. Chromosomal DNA was prepared by the CTAB (hexadecyltrimethyl ammonium bromide) extraction method (10) from confluent BHI agar plate cultures. DNA was also extracted from samples of gastric juice using a QIAamp DNA minikit (Qiagen Corporation, Chatsworth, Calif.).
Specific PCR was carried out in 20-µl volumes using 10 ng of DNA, 1 U of Taq polymerase (Promega, Madison, Wis.), 10 pmol of each primer per reaction, 0.25 mM (each) deoxynucleoside triphosphate, and 2 to 3 mM MgCl2 in standard PCR buffer for 30 cycles generally under the following conditions: 94°C for 40 s, 55°C for 40 s, and 72°C for a time chosen based on the size of the expected fragment (1 min/kb). Arbitrarily primed PCR (randomly amplified polymorphic DNA [RAPD]) DNA fingerprinting was carried out using buffer with 4 mM MgCl2 for 45 cycles of 94°C for 1 min, 36°C for 1 min, and 72°C for 2 min. PCR primers are listed in Table 1.
|
Nucleotide sequence accession numbers. Sequences obtained during this study have been assigned the following GenBank accession numbers: AF217727 to AF217735 (vacAs region), AF220110 to AF220120 (vacAm region), AF202219 to AF202225 (cagA [5' end]), AF222807 to AF222809 (cagA [3' end]), and AF239991 to AF239994 (iceA1).
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RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Genetic diversity and drug resistance of H. pylori
strains in Calcutta.
Arbitrarily primed PCR (RAPD) fingerprinting
was carried out on DNAs from single-colony isolates of H. pylori from 14 patients with peptic ulcers to assess the overall
diversity of strains in Calcutta. A different profile was obtained
reproducibly from each isolate with each of several primers tested
(Fig. 1), indicating that each isolate
was unique in overall genotype. This genetic diversity was in accord
with that seen with clinical isolates from other parts of the world.
|
|
cag PAI in H. pylori from Calcutta.
The presence or absence of the cag PAI was scored by PCR
with specific primers (4) using DNAs extracted from cultured
strains or gastric juice. Products indicative of the cag PAI
(Fig. 2A) were obtained with primers
specific for the cagA gene from the great majority of
strains: 53 of 55 cultures from patients with overt disease and 22 of
23 patients with benign infections. The three cultures from which no
cag PAI-specific PCR product was obtained yielded an
empty-site product of the expected 550-bp size (Fig. 2B), indicating
that they truly lacked the cag PAI (4). In
addition, products corresponding to the empty site (Fig. 2A and B,
lanes labeled mixed) were also obtained from 11 of the patients found
to be infected with strains carrying the cag PAI, indicating
that they had mixed infections, i.e., a mixture of strains with and
without the cag PAI.
|
|
vacA (vacuolating cytotoxin gene). The types of alleles at the 5' end of the vacuolating cytotoxin gene (vacA) (vacAs1, generally toxigenic; vacAs2, generally nontoxigenic) were assessed, based on sizes of PCR products generated with appropriate vacA-specific primers (Fig. 2C). Of the 55 cultures from ulcer patients tested, 49 yielded a 259-bp fragment, indicating vacAs1 alleles, two yielded a 286-bp fragment, indicating vacAs2 alleles, and the remaining four yielded both the 259- and 286-bp fragments, indicating mixed infections (Table 2). Each infection that seemed to be due solely to a strain carrying vacAs2 in this test had also been scored as due solely to strain lacking the cag PAI in tests above. Similarly, the single-colony isolates lacking the cag PAI obtained from three mixed infections also carried the vacAs2 allele.
In equivalent PCR tests of DNAs from cultured strains or gastric-juice samples from the 23 patients with benign infections, 18 yielded vacAs1 products, one yielded a vacAs2 product, and the other four yielded both vacAs1 and vacAs2 products, again indicating mixed infections. Thus, the associations of the cag PAI with vacAs1 and the absence of the cag PAI with vacAs2 observed here match those typically seen in the West. In contrast, the lack of association of the genotype in which vacAs2 is present and the cag PAI is absent with more-benign infection is distinct from what is typically seen in the West. PCR products containing vacAs1 alleles of seven representative strains were sequenced directly. Six of them were of the vacAs1a allele type, and one was of the vacAs1b type (Fig. 4A), which are each common in European and U.S. populations. None of them were of the vacAs1c type, which was found in more than three-fourths of east Asian strains (35, 60; Y. Ito and T. Azuma, unpublished data). Two vacAs2 products were also sequenced and were found to be closely related to those of European or U.S. H. pylori strains (GenBank accession no. AF217727 and AF217733).
|
Polymorphism for iceA1 and iceA2 genes. PCR was used to distinguish between iceA1, the restriction endonuclease NlaIII homolog that is associated with virulence in the West and whose expression is induced by gastric epithelial cell contact (49, 61), and the iceA2 gene that, although unrelated in sequence, occupies the same chromosomal locus in strains lacking iceA1 (Fig. 2E and F). The iceA1 gene was found alone in 32 of the 55 (58%) peptic ulcer patients and in 16 of 23 (70%) gastritis-only patients, whereas the iceA2 gene was found alone in cultures from 21 of 55 (38%) peptic ulcer patients and 5 of 23 (22%) gastritis-only patients. A few patients had mixture of iceA1 and iceA2 strains (Table 2). This distribution of iceA1 and iceA2 genes in relation to disease status in the Calcutta population differed significantly from that found in Tennessee (49) and The Netherlands (61) (Cochran-Mantel-Haenszel chi square test result, P = 0.001), with iceA2 being weakly disease associated in Calcutta, rather than iceA1, as in Europe and the United States.
In further studies, a characteristic 94-bp deletion was found by PCR near the 3' end of iceA1 (Fig. 2G) from 10 of the 48 Calcutta strains (8 of the 32 from peptic ulcer patients and 2 of the 16 from gastritis-only patients), with the same deletion end points in each case sequenced (Fig. 4C). This deletion was not found in iceA1 genes from any of 211 strains from other geographic regions (Japan, Hong Kong, South Africa, Spain, North Europe, Alaska, or Peru; Y. Ito, T. Azuma, and D. E. Berg, unpublished data), suggesting that it may be useful as an Indian ethnic group-specific marker.Markers that differ geographically but are not implicated in virulence. The prevalence of several mobile DNAs was also studied, because of their potential utility for detecting ancient phylogenetic lineages. Hybridization and PCR tests identified sequences from IS606, IS605, and the recently discovered IS.Inv element (N. Akopyants, A. Raudonikiene, and D. E. Berg, unpublished data) in some 17 to 25% of cultures (Table 2), with carriage of each element apparently being independent of that of each of the others (see legend). The IS606 and IS.Inv elements are unrelated in sequence, and it is striking that each is much less common in east Asian strains than in Calcutta strains (<2% in each case in east Asia [A. K. Mukhopadhyay, Z. J. Pan, D. E. Berg, et al., unpublished data] versus ~17% in Calcutta).
In other studies, we found that DNA sequence motifs at the right end of the cag PAI in Calcutta strains also differed markedly from those in other geographic regions (see Calcutta H. pylori strain GenBank accession no. AF190663, AF191015, AF191016, AF200689, AF201074, and AF201075) (41).| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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H. pylori strains from Calcutta were studied to better understand the global population genetic structure and evolution of this gastric pathogen, in particular, to test if the H. pylori gene pool in at least this part of India is distinct from that found in east Asia, Europe, or both and also to test putative virulence genes of H. pylori for disease associations in an Indian setting. We found that strains carrying the cag PAI and the potentially toxigenic vacAs1 alleles of the vacuolating cytotoxin gene (vacA) were more abundant in Calcutta than in the West (8) but that about 10 to 20% of Calcutta strains lacked the cag PAI entirely and contained vacAs2 (nontoxigenic) rather than vacAs1 (potentially toxigenic) alleles. In contrast, essentially all Chinese and Japanese strains studied to date carried cag PAI genes and vacAs1 alleles (34, 35, 46, 47). Both iceA2 and iceA1 were present in Calcutta populations, but iceA2 seemed to be somewhat disease associated, not iceA1, as in the West. An iceA1 DNA deletion motif found in about one-fifth of Calcutta strains also seemed to be a region-specific marker, and two mobile DNAs that were very rare in east Asian strains were each quite common in Calcutta strains. It will be of great interest to learn just how these and other polymorphic traits might be distributed in other parts of the Indian subcontinent, in peoples separated from those of Calcutta by distance, language, culture, ethnicity, and ancestry (13, 63).
Given the existence of some truly vacAs2 strains with the cag PAI in Calcutta, it is noteworthy that such strains were recovered at similar frequency from patients with ulcers and more-benign infections. In contrast, in the West vacAs2 strains lacking the cag PAI are recovered disproportionately from persons with benign infections (8). One explanation for strains lacking the cag PAI being equally associated with overt disease and benign infection assumes that infections by such strains in India are often mixed with infections by strains with the cag PAI, the latter strains being responsible for most of the observed pathology. Additional explanations include (i) host or environmental factors and (ii) additional bacterial genetic virulence determinants that might be specific to Indian strains and/or that might be more important than cag PAI and vacAs1 status as determinants of disease in the Indian setting. The second of these alternative explanations is supported by our finding of several genetic differences between Calcutta and other (non-Indian) H. pylori populations.
The evolutionary forces of natural selection and random genetic drift (which includes founder effects) may have each helped shape the gene pool of H. pylori in Calcutta and made it distinct from those in other parts of the world. The effect of natural selection may be best illustrated by the Mtzr of some 90% of H. pylori strains in Calcutta, in contrast to the much lower Mtzr frequencies (~10 to 30%) in Japan and in the West. We have found that in India, as in other societies, Mtzr results from mutation of the chromosomal rdxA nitroreductase gene (HP0954), not the acquisition of new "resistance genes" (e.g., in plasmids or transposons) (30; J.-Y. Jeong, A. K. Mukhopadhyay, and D. E. Berg, unpublished data) and that MTZ is mutagenic (G. Sisson, J.-Y. Jeong, D. E. Berg, and P. S. Hoffman, unpublished data). As noted above, MTZ is used frequently against a variety of illnesses in India. The present abundance of Mtzr in Indian H. pylori strains can be ascribed to this frequent use of MTZ, generally at doses that may induce and select for resistant mutants of resident H. pylori strains without eradicating them (59).
The unrelated IS606 and IS.Inv mobile DNA elements were found in about 17% of Calcutta strains (present results) but in <2% of strains from China and Japan analyzed in the same way (A. K. Mukhopadhyay, Z. J. Pan, W. W. Su, and D. E. Berg, unpublished data). We suggest that this reflects random genetic drift, not selection. This is based on an assumption that these elements do not affect the risk of H. pylori infection, persistence, or virulence in a manner specific to particular human ethnic groups or geographic regions. Also possibly indicating involvement of genetic drift is our finding, mentioned above, of different DNA sequence motifs at the right end of the cag PAI (41). A model invoking genetic drift in the divergence of H. pylori in different regions would be in accord with its transmission preferentially within family groups and local communities, rather than in worldwide epidemics (11, 24, 40), and the relative separation of peoples of different ethnicities (including Indians from east Asians and from Europeans) during thousands of years of human history (13, 16).
The high frequency of the cag PAI and vacAs1 types among Calcutta H. pylori strains would be in accord with the high overall risk of H. pylori infection in India and the evolutionary consideration that high rates of transmission favor the emergence of more-virulent strains of a pathogen (27). There is also speculation, however, that H. pylori might have jumped recently from various animal species to humans, perhaps in early agricultural communities (21, 22, 25, 41, 43) versus an alternative in which humans have always carried H. pylori (14, 15). In the first scenario, any distinctive adaptive features of Calcutta H. pylori (abundance of cag PAI vacAs1 genotypes and/or particular DNA sequence motifs that distinguish these strains from others) might be a legacy of divergent selection pressures in different putative ancestral animal hosts, rather than in humans. Many of these questions may be resolved with the development of new cell culture and animal models that give further insight into the biological activities of H. pylori virulence proteins and their contributions to bacterial fitness and through further population genetic analyses of H. pylori elsewhere in India and in expatriate Indians living abroad.
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ACKNOWLEDGMENTS |
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We are grateful to Bill Shannon, Division of Biostatistics in Medicine, Washington University Medical School, for statistical analyses, and to N. K. Ganguly, Indian Council of Medical Research, for his encouragement.
This work was supported in part by NIH grants AI38166 and DK53727 to D.E.B. and P30 DK52574 to Washington University.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Microbiology, Campus Box 8230, Washington University Medical School, 4566 Scott Ave., St. Louis, MO 63110. Phone: (314) 362-2772. Fax: (314) 362-1232 or -3203. E-mail: berg{at}borcim.wustl.edu.
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Discussion
References
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