distinctiveness of genotypes of helicobacter pylori in ... · internal deletion found in 20% of...

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

June 2000, p. 3219–3227 Vol. 182, No. 11

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Distinctiveness of Genotypes of Helicobacter pylori in Calcutta, IndiaASISH K. MUKHOPADHYAY,1 DANGERUTA KERSULYTE,1 JIN-YONG JEONG,1 SIMANTI DATTA,2

YOSHIYUKI ITO,1,3 ABHIJIT CHOWDHURY,4 SUJIT CHOWDHURY,4 AMAL SANTRA,4

SUJIT K. BHATTACHARYA,2 TAKESHI AZUMA,3 G. BALAKRISH NAIR,2

AND DOUGLAS E. BERG1*

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, Instituteof 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 frommore-benign infections), were studied, with a focus on putative virulence genes and neutral DNA markers thatwere likely to be phylogenetically informative. PCR tests indicated that 80 to 90% of Calcutta strains carriedthe 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 PAI1 vacAs1 genotypes aredisease 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 eastAsia and the West, whereas the cagA sequences of Calcutta and Western strains were closely related. Aninternal deletion found in 20% of Calcutta iceA1 genes was not seen in any of ;200 strains studied from othergeographic regions and thus seemed to be unique to this H. pylori population. Two mobile DNAs that were rarein east Asian strains were also common in Calcutta. About 90% of Calcutta strains were metronidazoleresistant. These findings support the idea that H. pylori gene pools differ regionally and emphasize the potentialimportance of studies of Indian and other non-Western H. pylori populations in developing a global under-standing of this gastric pathogen and associated disease.

Helicobacter pylori is a gastric pathogen that chronically in-fects more than half of all people worldwide (for reviews seereferences 48 and 64) and constitutes a major cause of pepticulcer disease and an early risk factor for gastric cancer. It mayalso contribute to childhood malnutrition and increase the riskor severity of infection by other gastrointestinal pathogenssuch as Vibrio cholerae, especially in developing countries (18,19). It appears to be one of the most genetically diverse ofbacterial species, because DNA fingerprinting can distinguishany given isolate from most others (5, 56) and because of the;3 to 5% DNA sequence divergence typically found in essen-tial genes from unrelated strains (3, 29). This mutational di-versity is enhanced by a rich history of interstrain recombina-tion (29, 40, 54). In contrast, most other well-studied bacterialspecies 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. pylorigenotypes. Some of this may reflect preferential transmissionwithin families and among people in close contact, not in largeepidemics (11, 24, 50). Such a pattern means that no individualstrain would compete simultaneously against many others (12,38, 55). H. pylori diversity would also be enhanced if humansdiffer in traits that are important to individual strains (e.g.,specificity or strength of immune and inflammatory responsesor availability of receptors used for H. pylori adherence [26,33]).

There are also indications of significant geographic differ-ences among strains. For example, only one-half to two-thirdsof U.S. and European strains carry the cag pathogenicity island(PAI), a 40-kb DNA segment many of whose genes seem tohelp induce interleukin 8 and thereby a strong and potentiallydamaging inflammatory response; such strains are recoveredpreferentially from persons with overt disease (4, 8, 9, 17). Incontrast, nearly all east Asian strains carry the cag PAI inde-pendent of disease status (34, 47). Similarly, somewhat morethan half of U.S. and European strains carry toxigenic (vacAs1)alleles of the vacuolating cytoxin gene, with other strains car-rying nontoxigenic (vacAs2) alleles (in general, vacAs1 strainscarry the cag PAI (65); nearly all east Asian strains carryvacAs1 alleles. Potentially more significant in terms of hostinteraction and evolution were findings that east Asian andWestern strains differ markedly in DNA sequence motifs in thevacA and cagA genes (3, 34, 35, 46, 58, 62), since the proteinsthese genes encode probably each interact directly with hostfactors (CagA protein is translocated to host cells and is ty-rosine phosphorylated in them but is not needed for interleu-kin 8 induction) (7, 45, 51). Even though less geographic par-titioning was found in a sampling of housekeeping genes (3,58), it is not clear whether the regional differences in cagA andvacA alleles reflect natural selection, random genetic drift in-cluding founder effects, or both during H. pylori evolution. It isnoteworthy in this context that the two strains whose genomesequences have been determined (6, 57) and compared tobetter understand H. pylori genetic diversity (23) are fromethnic 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 represen-tative of H. pylori worldwide.

We began studies of genotypes of H. pylori strains of India,

* Corresponding author. Mailing address: Department of MolecularMicrobiology, Campus Box 8230, Washington University MedicalSchool, 4566 Scott Ave., St. Louis, MO 63110. Phone: (314) 362-2772.Fax: (314) 362-1232 or -3203. E-mail: [email protected].

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motivated by the differences between strains of east Asia andthe West found to date and a sense that the peoples of the vastIndian subcontinent (some one-fifth of all humanity) may havebeen sufficiently isolated during much of human history to haveallowed 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 beresistant to metronidazole (Mtzr) (2), but to our knowledgethere has been very little analysis to date of the genotypes ofthe underlying H. pylori strains (20). Here we identify severalmarkers that help distinguish H. pylori strains from Calcuttafrom those of east Asia and the West.

MATERIALS AND METHODS

Patient samples. Adult ethnic Bengali patients of both sexes, age 21 to 65years, who presented with gastric complaints were endoscoped at the Hospital ofthe Institute of Post Graduate Medical Education and Research in Calcutta,India, using well-washed and sterilized fiber optic endoscopes. Biopsies andgastric juice samples used in the present study were obtained during endoscopywith informed consent, under protocols approved by the institutional reviewboards of the Institute of Post Graduate Medical Education and Research andthe National Institute of Cholera and Enteric Disease (Calcutta, India). Twobiopsies were taken for culture, one from the gastric antrum and one from thecorpus, and were stored at 270°C in 0.5 ml of brucella broth (Difco) with 15%glycerol until culture. Two milliliters of gastric juice was also collected duringendoscopy in some cases and stored frozen at 270°C until use. Diagnoses ofpeptic ulcer disease were based on visual examination of the stomach andduodenum during endoscopy and also on any patient history of earlier pepticulcer. If no evidence of peptic ulcer disease was found, the patient was consid-ered to have a more-benign infection (nonulcer dyspepsia, gastritis only). Fifty-five of the patients had peptic ulcer disease, and 23 had gastritis only.

H. pylori culture. Cultures were prepared by smearing single biopsy specimenson petri plates containing brain heart infusion (BHI) agar (Difco) supplementedwith 7% horse blood, 0.4% IsoVitaleX, amphotericin B (8 mg/ml), trimethoprim(5 mg/ml), and vancomycin (6 mg/ml) and were incubated at 37°C in an atmo-sphere of 5% O2–10% CO2–85% N2 for 3 to 6 days. H. pylori colonies wereidentified based on their typical morphology, characteristic appearance on Gramstaining, 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 werecollected as a pooled population, and preserved in sterile BHI broth with 15%glycerol at 270°C. In general, only one such culture was analyzed per patient.

Resistance and susceptibility to metronidazole (MTZ) were scored by spottingaliquots of ;107 exponentially growing H. pylori cells on BHI agar containingMTZ, typically at 8 mg/ml, and on MTZ-free control plates in parallel andmonitoring the amount of growth after incubation. For more-sensitive scoring,10-ml aliquots of a serially diluted suspension, ranging from 107 to 102 viable cellsper aliquot, were spotted on BHI agar containing various fixed concentrations ofMTZ, and survival (efficiency of colony formation) was scored after incubation asa function of MTZ dose.

DNA methods. Chromosomal DNA was prepared by the CTAB (hexadecyl-trimethyl ammonium bromide) extraction method (10) from confluent BHI agarplate cultures. DNA was also extracted from samples of gastric juice using aQIAamp DNA minikit (Qiagen Corporation, Chatsworth, Calif.).

Specific PCR was carried out in 20-ml volumes using 10 ng of DNA, 1 U of Taqpolymerase (Promega, Madison, Wis.), 10 pmol of each primer per reaction, 0.25mM (each) deoxynucleoside triphosphate, and 2 to 3 mM MgCl2 in standardPCR 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 expectedfragment (1 min/kb). Arbitrarily primed PCR (randomly amplified polymorphicDNA [RAPD]) DNA fingerprinting was carried out using buffer with 4 mMMgCl2 for 45 cycles of 94°C for 1 min, 36°C for 1 min, and 72°C for 2 min. PCRprimers are listed in Table 1.

PCR products were sequenced directly, after purification with the QIAquickgel extraction kit (Qiagen) using the BigDye Terminator cycle sequencing kit(Perkin-Elmer-Applied Biosystems, Foster City, Calif.) and an ABI automatedsequencer. DNA sequence editing and analysis were performed with programs inthe GCG package (Genetics Computer Group, Madison, Wis.), programs anddata in the TIGR H. pylori genome database (57) (http://www.tigr.org/tdb/mdb/hpdb.html), and the Phylip program of J. Felsenstein (http://evolution.genetics.washington.edu/phylip.html).

Dot blot hybridization was performed using Hybond-N1 nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) containing ;10- to 20-ngaliquots of genomic DNA per spot from each strain of interest and hybridizationprobes labeled using the enhanced chemiluminescence kit (ECL; AmershamPharmacia Biotech) according to the manufacturer’s instructions. Probes for theorfA and orfB segments of IS605 were generated by PCR from 26695 genomicDNA with primers ORF18F and ORF18R (370 bp) and ORF19F and ORF19R

(661 bp) (39). Similarly, a probe for IS606 was generated from strain 84-183genomic DNA with primers FB1 and FB8 (784 bp). A probe for the IS.Invelement region was generated from NCTC11637 genomic DNA using primersX14 and X24 (3.4 kb). PCR-amplified DNAs were purified using the Qiagen gelextraction kit prior to ECL labeling for use in hybridization.

Statistical analyses of iceA1 and iceA2 frequencies were kindly carried out byWilliam Shannon, Division of Biostatistics in Medicine, Washington UniversityMedical School, using the Cochran-Mantel-Haenszel test.

Nucleotide sequence accession numbers. Sequences obtained during this studyhave been assigned the following GenBank accession numbers: AF217727 toAF217735 (vacAs region), AF220110 to AF220120 (vacAm region), AF202219 toAF202225 (cagA [59 end]), AF222807 to AF222809 (cagA [39 end]), and AF239991to AF239994 (iceA1).

RESULTS

Genetic diversity and drug resistance of H. pylori strains inCalcutta. Arbitrarily primed PCR (RAPD) fingerprinting wascarried out on DNAs from single-colony isolates of H. pylorifrom 14 patients with peptic ulcers to assess the overall diver-sity of strains in Calcutta. A different profile was obtainedreproducibly from each isolate with each of several primerstested (Fig. 1), indicating that each isolate was unique in over-all genotype. This genetic diversity was in accord with that seenwith clinical isolates from other parts of the world.

Pools of H. pylori that had been cultured from individualbiopsies from 55 patients with ulcers (the 14 surveyed in Fig. 1plus another 41) and also from 18 patients with more-benign(gastritis-only) infections were tested for drug susceptibility(see Materials and Methods). None of these 73 cultures wereresistant to clarithromycin (0.5 mg/ml), which is in accord withmacrolides not being used very often in this Calcutta popula-tion. In contrast, 66 of them (90%) were resistant to at least an8-mg/ml concentration of MTZ (Table 2). Four of the sevennominally Mtzs cultures grew on medium with 3 mg of MTZ/ml, indicating a leaky resistance phenotype, whereas the otherthree cultures were killed on this medium and were as sensitiveas our standard Mtzs laboratory strains (J99, 26695). Thesedata are in accord with an earlier report (2) that the frequencyof Mtzr H. pylori is extremely high in India, although some trulyMtzs strains can still also be found.

cag PAI in H. pylori from Calcutta. The presence or absenceof 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 obtainedwith primers specific for the cagA gene from the great majorityof strains: 53 of 55 cultures from patients with overt diseaseand 22 of 23 patients with benign infections. The three culturesfrom which no cag PAI-specific PCR product was obtainedyielded an empty-site product of the expected 550-bp size (Fig.2B), indicating that they truly lacked the cag PAI (4). In addi-tion, products corresponding to the empty site (Fig. 2A and B,lanes labeled mixed) were also obtained from 11 of the patientsfound to be infected with strains carrying the cag PAI, indicat-ing that they had mixed infections, i.e., a mixture of strains withand without the cag PAI.

Twelve single-colony isolates from each of five such mixedinfections were tested further, and at least one isolate lackingthe cag PAI was obtained from each of 3 of them. Pairedisolates with and without the cag PAI from these three caseswere DNA fingerprinted, and a different RAPD profile wasfound in each case (data not shown). This indicated that thesecoexisting strains with and without the cag PAI were not re-lated to one another and had in each case probably resultedfrom separate infections by strains with and without the cagPAI, not by a single infection with a strain carrying the cag PAIand then cag PAI excision or a reciprocal cag PAI acquisitionby an infecting strain lacking the cag PAI.

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To assess the phylogenetic relationship between Indian cagPAIs and those from other regions, we focused first on asegment near the 59 end of the cagA gene that had been usedto distinguish east Asian and U.S. and European strains (58).This segment was amplified by PCR from seven Indian strainsand sequenced directly. The sequences obtained were closelyrelated to one another and also to those from Western strains,but not to those from Chinese and Japanese strains (Fig. 3A).A 1-kb segment near the 39 end of cagA that was similarlyuseful for distinguishing east Asian from Western strains (66)was sequenced from three other strains (GenBank accessionno. AF222807 to AF222809). These sequences also clusteredwith those from ethnic European strains (94 to 96% DNAsequence match), not with east Asian sequences (79 to 80%).

vacA (vacuolating cytotoxin gene). The types of alleles at the59 end of the vacuolating cytotoxin gene (vacA) (vacAs1, gen-erally toxigenic; vacAs2, generally nontoxigenic) were assessed,based on sizes of PCR products generated with appropriatevacA-specific primers (Fig. 2C). Of the 55 cultures from ulcerpatients tested, 49 yielded a 259-bp fragment, indicating vacAs1

alleles, two yielded a 286-bp fragment, indicating vacAs2 al-leles, and the remaining four yielded both the 259- and 286-bpfragments, indicating mixed infections (Table 2). Each infec-tion that seemed to be due solely to a strain carrying vacAs2 inthis test had also been scored as due solely to strain lacking thecag PAI in tests above. Similarly, the single-colony isolateslacking the cag PAI obtained from three mixed infections alsocarried the vacAs2 allele.

In equivalent PCR tests of DNAs from cultured strains orgastric-juice samples from the 23 patients with benign infec-tions, 18 yielded vacAs1 products, one yielded a vacAs2 prod-uct, and the other four yielded both vacAs1 and vacAs2 prod-ucts, again indicating mixed infections. Thus, the associationsof the cag PAI with vacAs1 and the absence of the cag PAI withvacAs2 observed here match those typically seen in the West.In contrast, the lack of association of the genotype in whichvacAs2 is present and the cag PAI is absent with more-benigninfection is distinct from what is typically seen in the West.

PCR products containing vacAs1 alleles of seven represen-tative strains were sequenced directly. Six of them were of the

TABLE 1. Primers used in this study

Region Primer Nucleotide sequence

vacAs1 or vacAs2 VA1-F 59-ATG GAA ATA CAA CAA ACA CACVA1-R 59-CTG CTT GAA TGC GCC AAA C

vacAm1a VA3-F 59-GGT CAA AAT GCG GTC ATG GVA3-R 59-CCA TTG GTA CCT GTA GAA AC

vacAm1b VAm-F3 59-GGC CCC AAT GCA GTC ATG GATVAm-R3 59-GCT GTT AGT GCC TAA AGA AGC AT

vacAm2 VA4-F 59-GGA GCC CCA GGA AAC ATT GVA4-R 59-CAT AAC TAG CGC CTT GCA C

vacA, 0.7-kb middle VAm-F 59-GCT CAT TAC GGC TTC CAC TAA TGTVAm-R 59-GCG GTT ATT GTT GTT ATA AAG GGC TA

cagA (59 end) cagA5 59-GGC AAT GGT GGT CCT GGA GCT AGG CcagA2 59-GGA AAT CTT TAA TCT CAG TTC GG

cagA (39 end) cagA-F40481 59-AGG ATT TCA GCA AGG TAA CGC AAG CCagA-R41660 59-TAA GAT TTT TGG AAA CCA CCT TTT GTA T

cag PAI empty site Luni1 59-ACA TTT TGG CTA AAT AAA CGC TGR5280 59-GGT TGC ACG CAT TTT CCC TTA ATC

iceA1 iceA1F 59-TAT TTC TGG AAC TTG CGC AAC CTG ATM.Hpy1R 59-GGC CTA CAA CCG CAT GGA TAT

iceA2 cysSF 59-CGG CTG TAG GCA CTA AAG CTAiceA2R 59-TCA ATC CTA TGT GAA ACA ATG ATC GTT

iceA1 D94 bp A1F673 59-GGT GAG TCG TTG GGT AAG CGT TAC AGA ATTA1R1174 59-CAC AAC CAT CAT ATT CAG CCT CCC CCT CAT A

IS605 orfA ORF18F 59-CGC CTT GAT CGT TTC AGG ATT AGCORF18R 59-CAA CCA ACC GAA GCA AGC ATA ATC

IS605 orfB ORF19F 59-GGC TGT TCT AGG GTC GTG TAT AACORF19R 59-CAA GCT AGA TGC AAT CTA GCT ACC

IS606 FB1 59-GAA TGT AAT TCT ACC TAA TCC ATT CRB8 59-GAG AAA CCT TGA TTG TTC CAT G

IS.Inv X14 59-GAA TTA TCG CAA GTT ACA GAG ATT GX24 59-GCA ATA TAA TCC GTT CTA AGA AAC

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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 inmore than three-fourths of east Asian strains (35, 60; Y. Itoand T. Azuma, unpublished data). Two vacAs2 products werealso sequenced and were found to be closely related to those ofEuropean or U.S. H. pylori strains (GenBank accession no.AF217727 and AF217733).

The alleles of the middle region of vacA, a part that seemsto affect host cell specificity (45), were also scored by PCR(Fig. 2D) and by sequencing several representatives. Productswere obtained with only vacAm1b primers from culture orgastric-juice samples from 43 of the 78 patients; conversely,products were obtained only with vacAm2 primers from 27 ofthe 78 patient samples; and products were obtained using bothprimer sets from another 7 of the patient samples, indicatingmixed infections, (4 of these 7 were from patients with infec-tions that were mixed in terms of vacAs1 and -s2 alleles,whereas the other 3 were not). Just one sample was exceptionalin yielding an amplification product with the vacAm1b forwardprimer and the vacAm2 reverse primer, suggesting that it wasa vacAm1-vacAm2 hybrid.

DNA sequencing of a 0.7-kb PCR fragment containing thevacA middle region from eight strains that had yielded prod-ucts with vacAm1b-specific primers (five ulcer and three gas-tritis) showed each to be closely related to one another butdistinct from both canonical vacAm1b alleles of east Asianstrains and vacAm1a alleles of ethnic European strains (Fig.3B and 4B). These Calcutta alleles, termed vacAm1c, were alsoclosely related to an allele from an unusual isolate from Ger-many (Fig. 3B, no. 7) (32), but for which patient ethnicity,community contacts, and travel history are not known. Themiddle regions of two strains that had yielded products withvacAm2-specific primers were each closely related to thoseof canonical vacAm2 strains from other societies (GenBank

accession no. AF220118 and AF220119), and the middle re-gion of the strain that had yielded a PCR product only withvacAm1b forward and vacAm2 reverse primers was found in-deed to contain a hybrid vacAm1c-vacAm2 allele (96 to 98%match with Indian vacAm1c type sequences proximally, 92 to94% match with vacAm2 sequences distally; GenBank acces-sion no. AF220120), reminiscent of the vacAm1b-vacAm2 re-combinant allele found among H. pylori strains from Shanghai(where vacAm1b and -m2 alleles are common) (46).

Polymorphism for iceA1 and iceA2 genes. PCR was used todistinguish between iceA1, the restriction endonuclease NlaIIIhomolog that is associated with virulence in the West andwhose expression is induced by gastric epithelial cell contact(49, 61), and the iceA2 gene that, although unrelated in se-quence, occupies the same chromosomal locus in strains lack-ing iceA1 (Fig. 2E and F). The iceA1 gene was found alone in32 of the 55 (58%) peptic ulcer patients and in 16 of 23 (70%)gastritis-only patients, whereas the iceA2 gene was found alonein cultures from 21 of 55 (38%) peptic ulcer patients and 5 of23 (22%) gastritis-only patients. A few patients had mixture oficeA1 and iceA2 strains (Table 2). This distribution of iceA1

FIG. 1. Arbitrarily primed PCR (RAPD) fingerprint patterns from 14 inde-pendent H. pylori isolates, each from a different Calcutta resident with pepticulcer disease.

TABLE 2. Distribution of genetic or phenotypic characteristics ofH. pylori from Calcutta in relation to disease status

Trait or markerbPatient disease statusa

Peptic ulcer Gastritis only

Mtzrc 49/55 17/18cag PAI1 onlyd 46/55 18/23cag PAI1,2 mixedd 7/55 4/23cag PAI2 onlyd 2/55 1/23vacAs1 only 49/55 18/23vacAs2 only 2/55 1/23vacAs1, -s2 mixed 4/55 4/23vacAm1c only 31/55 12/23vacAm2 only 19/55 8/23vacAm1c, -m2 mixed 4/55 3/23iceA1e 32/55 16/23iceA2 21/55 5/23iceA1, iceA2 mixed 1/55 2/23IS605f 16/55 3/18IS606 11/55 2/18IS.Inv 10/55 4/18

a Number with trait/number scored.b Distribution of DNA markers was determined by PCR or hybridization, as

illustrated in the figures.c Resistant to 8 mg of MTZ/ml in agar dilution test. Four of the seven strains

that were unable to grow on medium with 8 mg of MTZ/ml grew on medium withjust 3 mg of MTZ/ml, indicating leaky resistance phenotypes (see text). Thepossibility that many of these Mtzr infections were mixed and contained only asmall fraction of Mtzr cells and many Mtzs cells (as was common in a Peruvianpopulation) (12) was tested by streaking representative Mtzr cultures on MTZ-free medium: Two single colonies from each of 10 such cultures were found tobe Mtzr, whereas each of the two single colonies from an 11th Mtzr culture wasMtzs. This suggests that, despite occasional Mtzs Mtzr mixed infections, themajority of cells in most nominally Mtzr infections in Calcutta are indeed Mtzr.

d cag PAI1 only, infection with strains carrying cag PAI only; cag PAI1,2

mixed, mixed infection with strains carrying the cag PAI and strains lacking thecag PAI; cag PAI2 only, infection with strains lacking the cag PAI only.

e Just one culture failed to give iceA1 or iceA2 amplification. Of the 10 iceA1genes (of 44 tested) that contained the 94-bp deletion (Fig. 6) not found in H.pylori from any other region studied to date, 8 were from ulcer patients and 2were from gastritis-only patients.

f Thirty-three of the 73 cultures contained just one type of mobile element,5 cultures contained two of them, 1 culture contained all three elements, and34 cultures lacked all three elements. Tests of 8 single colonies from each of11 cultures carrying IS606 and each of 10 carrying IS.Inv (in total, 88 and 80single colonies, respectively) identified these elements in every case, indicatingthat these estimates, based on hybridization to pools of H. pylori cultured fromindividual biopsies, are probably accurate.



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and iceA2 genes in relation to disease status in the Calcuttapopulation differed significantly from that found in Tennessee(49) and The Netherlands (61) (Cochran-Mantel-Haenszel chisquare test result, P 5 0.001), with iceA2 being weakly diseaseassociated in Calcutta, rather than iceA1, as in Europe and theUnited States.

In further studies, a characteristic 94-bp deletion was foundby PCR near the 39 end of iceA1 (Fig. 2G) from 10 of the 48Calcutta strains (8 of the 32 from peptic ulcer patients and 2 ofthe 16 from gastritis-only patients), with the same deletion endpoints in each case sequenced (Fig. 4C). This deletion was notfound in iceA1 genes from any of 211 strains from other geo-graphic 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 anIndian ethnic group-specific marker.

Markers that differ geographically but are not implicated invirulence. The prevalence of several mobile DNAs was alsostudied, because of their potential utility for detecting ancient

phylogenetic lineages. Hybridization and PCR tests identifiedsequences from IS606, IS605, and the recently discoveredIS.Inv element (N. Akopyants, A. Raudonikiene, and D. E.Berg, unpublished data) in some 17 to 25% of cultures (Table2), with carriage of each element apparently being indepen-dent of that of each of the others (see legend). The IS606 andIS.Inv elements are unrelated in sequence, and it is strikingthat each is much less common in east Asian strains than inCalcutta strains (,2% in each case in east Asia [A. K. Mu-khopadhyay, Z. J. Pan, D. E. Berg, et al., unpublished data]versus ;17% in Calcutta).

In other studies, we found that DNA sequence motifs at theright end of the cag PAI in Calcutta strains also differed mark-edly from those in other geographic regions (see CalcuttaH. pylori strain GenBank accession no. AF190663, AF191015,AF191016, AF200689, AF201074, and AF201075) (41).

DISCUSSION

H. pylori strains from Calcutta were studied to better under-stand the global population genetic structure and evolution ofthis gastric pathogen, in particular, to test if the H. pylori genepool in at least this part of India is distinct from that found ineast Asia, Europe, or both and also to test putative virulencegenes of H. pylori for disease associations in an Indian setting.We found that strains carrying the cag PAI and the potentiallytoxigenic 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 PAIentirely and contained vacAs2 (nontoxigenic) rather thanvacAs1 (potentially toxigenic) alleles. In contrast, essentially allChinese and Japanese strains studied to date carried cag PAIgenes and vacAs1 alleles (34, 35, 46, 47). Both iceA2 and iceA1were present in Calcutta populations, but iceA2 seemed to besomewhat disease associated, not iceA1, as in the West. AniceA1 DNA deletion motif found in about one-fifth of Calcuttastrains also seemed to be a region-specific marker, and twomobile DNAs that were very rare in east Asian strains wereeach quite common in Calcutta strains. It will be of greatinterest to learn just how these and other polymorphic traitsmight be distributed in other parts of the Indian subcontinent,in peoples separated from those of Calcutta by distance, lan-guage, culture, ethnicity, and ancestry (13, 63).

Given the existence of some truly vacAs2 strains with the cagPAI in Calcutta, it is noteworthy that such strains were recov-ered at similar frequency from patients with ulcers and more-benign infections. In contrast, in the West vacAs2 strains lack-ing the cag PAI are recovered disproportionately from personswith benign infections (8). One explanation for strains lackingthe cag PAI being equally associated with overt disease andbenign infection assumes that infections by such strains inIndia are often mixed with infections by strains with the cagPAI, the latter strains being responsible for most of the ob-served pathology. Additional explanations include (i) host orenvironmental factors and (ii) additional bacterial genetic vir-ulence determinants that might be specific to Indian strainsand/or that might be more important than cag PAI and vacAs1status as determinants of disease in the Indian setting. Thesecond of these alternative explanations is supported by ourfinding of several genetic differences between Calcutta andother (non-Indian) H. pylori populations.

The evolutionary forces of natural selection and randomgenetic drift (which includes founder effects) may have eachhelped shape the gene pool of H. pylori in Calcutta and madeit distinct from those in other parts of the world. The effect ofnatural selection may be best illustrated by the Mtzr of some

FIG. 2. Representative sequence-specific PCR tests. (A) Test for the pres-ence of cagA gene using primers cagA5 and cagA2. (B) Test for the presence ofthe cag PAI empty site using primers Luni1 and R5280. (C) Test for the presenceof vacAs1 versus vacAs2 alleles using primers VA1-F and VA1-R (generating259- and 286-bp products from vacAs1 and -s2 alleles, respectively). (D) Test forthe presence of vacAm1 using primers VAm-F and VAm-R and vacAm2 allelesusing primers VA4-F and VA4-R. (E) Test for iceA1 using primers iceA1F andM.Hpy1R. (F) Test for iceA2 using primers cysF and iceA2R. (G) Test for thepresence or absence of the 94-bp deletion in iceA1 using primers A1F673 andA1R1174.



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FIG. 3. Phylogenetic trees of sequences within the cagA gene and vacAm1 alleles. Sequences from non-Indian strains that were used here were from publicdatabases, as indicated. (A) Phylogenetic tree based on an informative 220-bp segment of cagA (62) of H. pylori strains determined in this study (Calcutta strains 5 to7, 16 to 18, and 23) or reported by others. The tree was generated using PHYLIP (Phylogeny Inference Package), version 3.5c, of J. Felsenstein (see Materials andMethods). The strains used are as follows (GenBank accession numbers are in parentheses): 1, Dutch161A (AJ252965); 2, 26695 (AE000569); 3, Peru8C (AF198478);4, Lith5-1 (AJ239734); 5, India10 (AF202222); 6, India19 (AF202225); 7, India3 (AF202219); 8, Peru24C (AF198473); 9, Peru2B (AF198474); 10, Dutch292(AJ252971); 11, Peru35B (AF198476); 12, Dutch79 (AJ252970); 13, Gambia4659 (AF198468); 14, Peru4A (AF198477); 15, Gambia4797 (AF198469); 16, India18(AF202224); 17, India9 (AF202221); 18, India7 (AF202220); 19, Dutch25 (AJ252968); 20, Dutch419 (AJ252974); 21, Guatemala88 (AF198472); 22, South Africa19(AF198470); 23, India17 (AF202223); 24, Dutch107 (AJ252963); 25, Peru34B (AF198475); 26, ChinaR47 (AJ252985); 27, HongKong77 (AF198485); 28, Thailand88-28(AJ239722); 29, ChinaR27 (AJ252979); 30, HongKong97-42 (AJ239733); 31, HongKong81 (AF198486); 32, ChinaR40 (AJ252982); 33, ChinaR59 (AJ252986); 34,ChinaR29 (AJ252980); 35, JapanF32 (AJ239726); 36, JapanGC4 (AF198484); 37, ChinaR48 (AJ252983). (B) Tree based on informative 650-bp segment of vacA genecontaining vacAm1 alleles of Calcutta H. pylori strains determined in this study (strains 1 to 9 except strain 7) or reported by others. The tree was generated usingPHYLIP (Phylogeny Inference Package), version 3.5c, of J. Felsenstein (see Materials and Methods). The sequences of east Asian and ethnic European vacAm1 alleleswere taken from GenBank. Each number in this figure indicates the vacAm1 sequence from a given strain, as follows (GenBank accession numbers in parentheses):1, India19 (AF220111); 2, India226 (AF220115); 3, India89 (AF220114); 4, India48 (AF220112); 5, India18 (AF220110); 6, India230 (AF220117); 7, GermanyMz19(AJ006967); 8, India66 (AF220113); 9, India227 (AF220116); 10, JapanF52 (AF049631); 11, JapanF55 (AF049632); 12, ChinaR59 (AF035611); 13, JapanF63(AF049635); 14, ChinaR13 (AF035610); 15, JapanF42 (AF049626); 16, Japan94 (AF049640); 17, JapanF72 (AF049651); 18, JapanF73 (AF049652); 19, JapanF47(AF049629); 20, JapanF57 (AF049634); 21, JapanF35 (AF049625); 22, JapanF61 (AF049645); 23, JapanF36 (AF049462); 24, JapanF64 (AF049647); 25, JapanF45(AF0496628); 26, Poland1492 (AF097570); 27, Poland278 (AF097571); 28, NCTC11637 (AF049653); 29, J99 (AE001511); 30, NCTC11638 (U07145); 31, 26695(AE000598).



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90% of H. pylori strains in Calcutta, in contrast to the muchlower Mtzr frequencies (;10 to 30%) in Japan and in theWest. We have found that in India, as in other societies, Mtzr

results from mutation of the chromosomal rdxA nitroreductasegene (HP0954), not the acquisition of new “resistance genes”(e.g., in plasmids or transposons) (30; J.-Y. Jeong, A. K. Mu-khopadhyay, and D. E. Berg, unpublished data) and that MTZis mutagenic (G. Sisson, J.-Y. Jeong, D. E. Berg, and P. S. Hoff-man, unpublished data). As noted above, MTZ is used fre-

quently against a variety of illnesses in India. The presentabundance of Mtzr in Indian H. pylori strains can be ascribed tothis frequent use of MTZ, generally at doses that may induceand select for resistant mutants of resident H. pylori strainswithout eradicating them (59).

The unrelated IS606 and IS.Inv mobile DNA elements werefound in about 17% of Calcutta strains (present results) but in,2% of strains from China and Japan analyzed in the sameway (A. K. Mukhopadhyay, Z. J. Pan, W. W. Su, and D. E.

FIG. 4. DNA sequence alignments, illustrating motifs found in H. pylori strains from Calcutta. Sequences from non-Indian strains that were used here were frompublic databases, as indicated. (A) vacAs region sequences, showing differences between vacAs1a, -s1b, and -s1c type alleles. vacAs1a alleles are common in the CalcuttaH. pylori strains, whereas vacAs1c alleles are most common elsewhere in Asia. Each of the 97-bp sequences presented here starts at position 27 of a reference vacAopen reading frame (GenBank accession no. U07145). Identical nucleotides are indicated by hyphens. GenBank accession numbers for the sequences depicted hereare U07145 (NCTC11638), AF217728 (India9), AF217729 (India10), AF217730 (India17), AF217731 (India18), AF217734 (India29), AF217735 (India67), AE001511(J99), AF217732 (India21), AF091830 (Taiwan34), AF049632 (Japan55), and AF049638 (Japan78). (B) vacAm1 middle region sequences, showing differences amongvacAm1a, vacAm1b, and two of the representative vacAm1c allele types that we characterized. The vacAm1c alleles, which were common in Calcutta strains, were rarein populations from outside India that have been studied to date. Each of the 650-bp sequences presented here corresponds to nucleotides (nt) 2060 to 2709 of thevacA gene of reference strain NCTC11638 (Genbank accession no. U07145). GenBank accession numbers for the sequences depicted here are U07145 (NCTC11638),AF035610 (ChinaR13), AF220110 (India18; strain 5 in Fig. 3) and AF220117 (India230; strain 6 in Fig. 3). (C) Alignment of four iceA1 genes of Calcutta isolates, alongwith reference strain 60190 (GenBank accession no. U43917). The sequences presented here correspond to nt 426 to 558 in India227 (accession no. AF239991), nt 446to 558 in India34A (accession no. AF239992), nt 430 to 478 in India18A (accession no. AF239993), and nt 423 to 471 in India44A (accession no. AF239994).



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Berg, unpublished data). We suggest that this reflects randomgenetic drift, not selection. This is based on an assumption thatthese elements do not affect the risk of H. pylori infection,persistence, or virulence in a manner specific to particularhuman ethnic groups or geographic regions. Also possibly in-dicating involvement of genetic drift is our finding, mentionedabove, of different DNA sequence motifs at the right end ofthe cag PAI (41). A model invoking genetic drift in the diver-gence of H. pylori in different regions would be in accord withits transmission preferentially within family groups and localcommunities, 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) dur-ing thousands of years of human history (13, 16).

The high frequency of the cag PAI and vacAs1 types amongCalcutta H. pylori strains would be in accord with the highoverall risk of H. pylori infection in India and the evolutionaryconsideration that high rates of transmission favor the emer-gence of more-virulent strains of a pathogen (27). There is alsospeculation, however, that H. pylori might have jumped re-cently from various animal species to humans, perhaps in earlyagricultural communities (21, 22, 25, 41, 43) versus an alterna-tive in which humans have always carried H. pylori (14, 15). Inthe first scenario, any distinctive adaptive features of CalcuttaH. pylori (abundance of cag PAI vacAs1 genotypes and/or par-ticular DNA sequence motifs that distinguish these strainsfrom others) might be a legacy of divergent selection pressuresin different putative ancestral animal hosts, rather than inhumans. Many of these questions may be resolved with thedevelopment of new cell culture and animal models that givefurther insight into the biological activities of H. pylori viru-lence proteins and their contributions to bacterial fitness andthrough further population genetic analyses of H. pylori else-where in India and in expatriate Indians living abroad.

ACKNOWLEDGMENTS

We are grateful to Bill Shannon, Division of Biostatistics in Medi-cine, Washington University Medical School, for statistical analyses,and to N. K. Ganguly, Indian Council of Medical Research, for hisencouragement.

This work was supported in part by NIH grants AI38166 andDK53727 to D.E.B. and P30 DK52574 to Washington University.

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