DNA-Level Diversity and Relatedness of Helicobacter pylori Strains in Shantytown Families in Peru and Transmission in a Developing-Country Setting

The efficiency of transmission of a pathogen within familiescompared with that between unrelated persons can affect boththe strategies needed to control or eradicate infection andhow the pathogen evolves. In industrialized countries, mostcases of transmission of the gastric pathogen Helicobacter pyloriseems to be from mother to child. An alternative model, potentiallyapplicable among the very poor in developing countries, whereinfection is more common and the sanitary infrastructure isoften deficient, invokes frequent transmission among unrelatedpersons, often via environmental sources. In the present study,we compared the genotypes of H. pylori from members of shantytownhouseholds in Peru to better understand the transmission ofH. pylori in developing-country settings. H. pylori culturesand/or DNAs were obtained with informed consent by the stringtest (a minimally invasive alternative to endoscopy) from atleast one child and one parent from each of 62 families. Therandom amplified polymorphic DNA fingerprints of 57 of 81 (70%)child-mother strain pairs did not match, nor did the diagnosticgene sequences (>1% DNA sequence difference), independentof the child's age (range, 1 to 39 years). Most strains fromsiblings or other paired family members were also unrelated.These results suggest that H. pylori infections are often communityacquired in the society studied. Transmission between unrelatedpersons should facilitate the formation of novel recombinantgenotypes by interstrain DNA transfer and selection for genotypesthat are well suited for individual hosts. It also implies thatthe effective prevention of H. pylori infection and associatedgastroduodenal disease will require anti-H. pylori measuresto be applied communitywide.

INTRODUCTION

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INTRODUCTION

How a pathogen is transmitted in host populations can affectthe strategies needed to prevent or eradicate infection anddisease. It can also affect the relative contributions to genomeevolution of interstrain recombination versus mutation and ofthe selection for increased fitness versus random genetic drift.At one extreme are the epidemic strains of Vibrio cholerae,which thrive in South Asian aquatic ecosystems and which causedebilitating, potentially lethal diarrheal disease in many thousandsof people annually (20). Several new strains swept through at-riskhuman populations following the transfer of genes that increasedtheir infectivity in partially immune hosts or their proliferationor persistence in environmental reservoirs or that conferredresistance to commonly used antibiotics (21, 39). At anotherextreme are obligate endosymbionts, such as those of nematodesand insects (35, 36), which evolve solely by mutation and selectionfor phenotypes that may benefit their hosts or that are at leastbenign. Other microbes, including Helicobacter pylori (7, 8,19), which was examined in this study have lifestyles intermediatebetween the extremes of an epidemic, highly virulent pathogenand a benign endosymbiont.

H. pylori chronically infects the gastric mucosa of billionsof people worldwide, typically for decades, despite inflammationand other host defenses. Such chronic H. pylori infections constitutemajor risk factors for duodenal and gastric ulcers and for gastriccancer, one of the most frequently lethal malignancies (3, 13,33). Especially among the poor in developing countries, H. pyloriinfection also increases susceptibility to other food- and waterbornepathogens and contributes to iron deficiency anemia and malnutrition(10, 37, 46). This said, most infections seem to be benign;and some, it has been argued, can even be beneficial (7), althoughthe latter view is controversial (25).

Most people in developing countries become chronically infectedby H. pylori in infancy (8, 19) and may be exposed to it repeatedlythereafter. For example, in the shantytown in Peru evaluatedfor the present study, we had estimated that adults are exposedto infectious doses (doses sufficient to reestablish chronicinfection after therapeutic eradication of prior infection)at rates of about 20% per person per year. This reinfectionrate is far higher than that seen in equivalent clinical trialsin industrialized nations (<1% per person per year) (44).Although it is not feasible to monitor transmission directly,it is often ascribed to fecal or vomitus contamination of foodor water or perhaps utensils, fingers, etc. (8, 9, 19, 38).The fact that the prevalence of H. pylori infection has declinedmarkedly in recent decades in industrialized countries but notamong the poor in developing countries can be ascribed to bettersanitation and hygiene in industrialized countries and a negligiblerisk of reinfection among individuals in such countries oncea prior infection has been cured (1, 8, 19, 38).

A consensus "all-in-the-family" model (12, 27), involving mostlymother-to-child transmission, was proposed to explain new infectionsin industrialized countries. This was based on serologic andurea breath test survey data showing a statistical associationbetween infection in children and in their parents (22, 24,48) and DNA fingerprint data that revealed identical or nearlyidentical strains in different members of individual families(15, 28, 32, 41). The DNA analyses are particularly compellingbecause H. pylori is genetically diverse, and isolates fromunrelated persons are usually readily distinguished by randomamplified polymorphic DNA (RAPD) fingerprinting or the sequencingof one or a few genes (typical divergence in DNA sequence, $≥$ 3%,on average). As a consequence, any finding of closely matchedstrains indicates transmission within families or from a commonsource.

Although H. pylori is typically spoken of as human specific,it was not clear a priori if most established infections indeveloping countries would also stem from direct transmissionfrom mothers to their infants rather than from other sources.An early finding that the risk of infection in shantytowns inPeru is inversely related to household water quality (30) isconsistent with either explanation. However, a model involvingat least transient environmental niches for H. pylori that allowsits spread between unrelated persons gained plausibility withlaboratory findings of H. pylori survival for a few days inunchlorinated mineral water or seawater (31, 40), biofilm formationon inanimate surfaces (11, 50), and proliferation when it wascultured with amoebae (51).

We studied the genotypes of H. pylori strains from members ofhouseholds in a shantytown in Peru. We found that only $~$ 30% ofstrains from children were closely matched to those from theirmothers and that the frequency of matched strains did not dependon the child's age. This outcome suggests that H. pylori transmissionamong unrelated persons is particularly important in developingcountries, an outcome with important evolutionary and diseaseprevention implications, as will be discussed below.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Setting. Our study focused on H. pylori strains from residents of LasPampas de San Juan de Miraflores (PSJM), a periurban shantytownof 40,000 persons of low income ( $≤$ $1,500 per family per year,on average) in Lima, Peru, that is representative of many developing-countrysocieties. The health of the residents of PJSM has been undersurveillance for more than 20 years by physicians and healthcare and social workers of the Peruvian nongovernmental organizationProyectos en Inform

ica, Salud, Medicina y Agricultura (PRISMA; http://www.prisma.org.pe/nwWeb/Portal/)(23, 29, 30, 37). Most residents of PJSM become chronicallyinfected with H. pylori in early childhood (30), as is typicalin developing countries worldwide (8, 19). All strains and DNAsanalyzed here were from persons specifically recruited for thisstudy with informed consent or assent, as appropriate. Nonewere samples of convenience that might have initially been obtainedfor other purposes.

One hundred eighty-one families in PJSM were chosen at randomfor possible inclusion from among families found in a recentcensus to contain at least one parent and one child in the samehousehold. Ninety-eight families met the criteria of agreementto participate and the availability of at least one offspringand one parent. The participants were scheduled for minimallyinvasive string tests (42, 49) to obtain culturable H. pylorior DNA for PCR, when culture was unsuccessful. Families in whichnone of the children or either parent tested seemed to be H.pylori positive were excluded (36 families). Sixty-two familiesmet our final inclusion criteria.

Participant characteristics. For each of the 62 participating families, each family memberincluded in the study lived in the same house. The age rangeof the participating children was 1 to 39 years, with a medianof 9.5 years and a median interquartile range (between the firstand the fourth quartiles) of 12 years. The age range of theparticipating parents was 19 to 59 years, with a median of 37years and an interquartile range of 12.75 years. Among the parents,34% had $≤$ 6 years of education. Seventy-nine percent of the familieshad domestic animals in and about their houses; 74% of the houseswere connected to the Lima municipal water system and had asewage connection, and all but one family lived in a houseswith electricity. This distribution of features is typical forresidents of this shantytown (Table 1 and Table 2).

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TABLE 1. Sociodemographic characteristics of participating shantytown families

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TABLE 2. Housing characteristics of participating shantytown families

Ethical considerations. Our study protocol, including the use of Spanish-language informedconsent and assent forms, was approved by three human studiesor ethics committees: those of the Universidad Peruana CayetanoHeredia and the Asociacion Benefica PRISMA (both of Lima, Peru)and The Johns Hopkins Bloomberg School of Hygiene and PublicHealth (Baltimore, MD).

Sociodemographic assessment. Standard demographic information, including sex, age, maritalstatus, employment, and educational level, was obtained in initialinterviews (Table 1). Housing characteristics were also evaluated(Table 2).

String test procedure. All consenting subjects were screened for H. pylori by usingthe minimally invasive string test procedure (42, 49), whichhas been approved for use with adults and children who are atleast 1 year old by the three institutional ethics committeeslisted above. String test participants swallow a gelatin capsulecontaining an absorbent cotton string with a protruding end,which is taped to the subject's cheek. The string is gentlywithdrawn 90 min later, and the distal 20 to 30 cm, which hadbeen in the stomach, is put in 500 µl of transport mediumat 4°C and brought to the microbiology laboratory within4 h of collection. Our earlier studies had confirmed that theH. pylori strains cultured from strings match those from traditionalbiopsy specimens (49).

H. pylori culture. Tubes containing string samples in liquid were vortexed for3 min, and 150-µl aliquots were spread on brain heartinfusion agar containing 5% defibrinated sheep blood and Skirrowsupplement (Oxoid) and also on Columbia-colistin-nalidixic acidagar with Dent supplement (Oxoid). The plates were incubatedunder microaerobic conditions (10% CO₂, 5% O₂) at 37°C for4 to 10 days. H. pylori colonies were identified by characteristicmorphology, a positive urease test, and Gram staining (49).

ureB PCR. PCR with primers specific for a 463-bp ureB (urease) gene segmentwas used to detect H. pylori (44) in string samples and wasperformed in parallel with H. pylori culturing. To prepare DNAfrom the strings for PCR tests, $~$ 300 µl of phosphate-bufferedsaline was added to the remaining liquid in the sample tube,which was then centrifuged at 13,000 x g for 10 min. DNA wasextracted from the pellets with a QIAamp DNA minikit (Qiagen).PCR was carried out under standard conditions with the primersshown in Table 3; product formation was assayed by electrophoresisin agarose gels.

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TABLE 3. Primers used for PCR-based sequencing

H. pylori strain typing by fingerprinting. RAPD fingerprinting of H. pylori genomic DNAs was used to assessthe relatedness of strains from household members whenever purecultures were available by using at least two of the followingprimers in separate reactions: primer 1254 (5'-CCGCAGCCAA),primer 1281 (5'-AACGCGCAAC), primer 1283 (5'-GCGATCCCCA), andprimer 1290 (5'-GTGGATGCGA) (14, 49). RAPD fingerprinting resultedin informative profiles, such as those illustrated in Fig. 1.RAPD fingerprinting was not attempted when only DNAs from stringtests were available because the abundance of other DNAs (theDNAs of the human host and other microbes) results in RAPD profilesthat do not reliably indicate which H. pylori strains are relatedand which are not related to one another.

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FIG. 1. Representative RAPD profiles from pools of H. pylori colonies (lanes P) and from individual single colonies (lanes 1 though 5) from child-mother pairs from individual households. The sequences of the four RAPD primers used (primers 1254, 1281, 1283, and 1290) are given in Materials and Methods. (A) Profiles in which predominant strains from the child and mother are closely matched (as was seen in about 30% of the cases) and also the profile of a rare instance in which the child seemed to carry more than one strain in abundance (compare the pooled and the multiple single colonies). The glr gene fragments from the isolates from child 1 and mother 1 (767 bp) were 100% identical. (B) Profiles showing that predominant strains from the child and the mother are unrelated (as was seen in about 70% of the cases) and also that just one strain is predominant in each person. The glr gene fragments (768 bp) from the isolates child 1 and mother 1 were 3.4% divergent (26 base substitution differences), as is typical of unrelated strains (see Fig. 2).

Gene sequencing. Determination of the sequences from at least one H. pylori housekeepinggene (glr [glutamate racemase] and/or cysS [cysteinyl tRNA synthetase])from each participating household member was used as an independenttest of strain relatedness either as a quantitative complementto RAPD profiling or, when pure H. pylori cultures were notavailable, as the sole means of determining relatedness. Segmentscontaining these genes were amplified by PCR from DNAs extractedfrom cultures grown from single colonies or from string testsamples (14). In cases in which PCR products were made fromstring test sample DNA, to obtain unambiguous H. pylori genesequences it was sometimes necessary to reamplify the segmentswith new nested gene-specific primers whose 3' ends were justinterior to those of the primers used in the first amplifications.The PCR products were purified and sequenced at the Universityof Washington (Seattle) Sequencing Center or the WashingtonUniversity (St. Louis, MO) PNACL core facility. Percent sequenceidentities were scored by using ClustalW software.

Determining strain relatedness. When culture isolates were available, the strains were consideredclosely related genetically if the RAPD profiles were similar(no more than 1 or 2 of the $~$ 30 bands did not match, as illustratedin Fig. 1) and if the glr DNA sequences were $≤$ 0.5% divergent(>99.5% identical). This cutoff was based on the frequencydistribution of divergent sequences from paired strains shownin Fig. 2, which reveals well-separated peaks centered on <0.5%and on 3% to 4.5% divergence. The glr sequences of most strainpairs with closely matched RAPD patterns also matched, and viceversa. When H. pylori culture was not successful, the cysS genewas also sequenced to test the validity of inferences from testsof just one gene. Additional housekeeping genes (atpA, glmM,ppa, recA, and/or cysS) (14) were also sequenced when the RAPDanalysis and glr data were discordant (i.e., when the data matchedby one criterion but not another) to better understand the relatednessof strains from pairs of family members.

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FIG. 2. Distribution of percent DNA sequence divergence in glr genes in pairs of H. pylori strains from a child and a parent. The numbers on top of each vertical bar indicate the number of strain pairs from family members that exhibited a particular level of divergence.

To determine the frequencies of alleles that were $≥$ 99.5% identicalin the community at large, a list of every possible pair ofstrains that came from members of different families was assembled.Pairwise alignments of the sequences of glr genes were scoredat each position as matched (score, +1) or mismatched (score,0) for both transitions and transversions by using a pairwisealignment program implemented in the Perl program (http://www.perl.org/).The statistical significance of differences between the frequenciesof matched alleles within households and the community at largewas calculated by using the Stata program (www.stata.com).

Epidemiologic data analysis. H. pylori strains from mother-offspring pairs and also fromfather-offspring, sibling-sibling, and spouse-spouse pairs wereclassified as concordant or discordant, as defined above. Toseek the factors associated with intrafamilial transmission,we analyzed contingency tables and modeled the probability thatthe pairs of each of the four categories shared a concordantstrain by multiple logistic regressions. The covariates (possiblecausal determinants) of interest were relationship classes andrange of age in each pair compared. We adjusted for potentialconfounders such as sociodemographic variables, housing characteristics,place of food consumption by the families, and the presenceor the absence of domestic animals and animal feces inside housesand tested for interactions and second-order effects.

Nucleotide sequence accession numbers. The nucleotide sequences determined in this study were depositedin the NCBI GenBank database under accession numbers EU930438to EU930645.

RESULTS

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RESULTS

Experimental goals and design. We tested if the H. pylori strains predominating in childrenin a Peruvian shantytown were often related genetically to thoseof their parents, in particular, their mothers, as is the casein industrialized societies. Samples were obtained from participantsas young as 1 year of age by using the minimally invasive stringtest (42, 49), with informed consent or assent, as appropriate,in protocols approved by Peruvian and U.S. human studies committees.H. pylori was cultured from 135 of 166 (81%) individuals thatwere found to be H. pylori infected by ureB gene PCR with stringtest sample DNA. There was less success in culturing H. pylorifrom children under age 5 years ( $~$ 10%) than from older individuals( $~$ 48%) (P < 0.01); the reasons for this difference are notknown.

Occasional findings of two unrelated H. pylori strains in thesame person are interesting in epidemiologic and evolutionarycontexts (26, 41) and could be important to the present study.To assess if mixed infections are common in this shantytownpopulation, we fingerprinted by RAPD analysis five single coloniesand a pool of five or more additional colonies from the firstdozen mother-child pairs from whom H. pylori was cultured. Allbut 2 of these 24 people seemed to carry just one strain perperson. The only exceptions are shown in Fig. 1A: an indicationof a second strain was seen by comparing the profiles obtainedfrom the DNAs derived from a pool of colonies and from the fiveseparate single colonies of H. pylori culture from the child;in addition, profiles obtained with two primers (primers 1254and 1283) but not the other two primers indicated that isolate3 from this child's mother may have diverged from the otherisolates. In each of the other 22 cases analyzed in this way,however, each participant seemed to carry just one predominantstrain, as illustrated by the profiles shown in Fig. 1B, whichwere obtained with isolates from a mother-child pair from adifferent household. The generally high uniformity inferredfrom these RAPD data were supported by the homogeneity (theabsence of mixed peaks) of the sequence tracings from H. pylorigene PCR products from total string test sample DNA (data notshown). Also in agreement, we had previously found uniform H.pylori populations (no mixed infections) in each of another20 shantytown residents in similar RAPD tests of single coloniesand pools (49). Accordingly, for efficiency in identifying thepredominant or only strain that a person carried in the greatmajority of cases, we focused further analyses on DNA from onecolony per person or, when culture was not successful, on thesequences of the PCR products generated from DNA extracted fromstrings.

Relatedness of H. pylori strains within and between families. H. pylori strains from 133 pairs of infected family memberswere analyzed: 103 pairs by RAPD fingerprinting and PCR-basedsequencing of the glr gene and 30 pairs by PCR-based sequencingof the glr and cysS genes. For the latter group, the genes werefrom DNAs extracted from strings for cases in which samplesfor culture were not obtained from one or both family members.In the glr sequence comparisons, 99 of 133 pairs (74%) formedone peak of >1% divergence (average, 3% to 4.5%), and theother 34 (26%) pairs formed a second distinct peak of <0.5%divergence (Fig. 2). A threshold of <0.5% DNA sequence divergencewas therefore used to infer whether the alleles of a given genefrom different isolates were closely related or not. Similarly,divergent RAPD patterns ( $≥$ 3 bands different among the $~$ 30 bandsscored, on average) were found for 77 of 103 (75%) pairs, andidentical or nearly identical RAPD patterns ( $≤$ 2 different bands)were found for 26 of 103 (25%) pairs.

Only 12 of 133 pairs were discordant (different indicationsfrom the glr sequence and the RAPD profile or from the glr andcysS sequences). Sequencing of four or five additional locifrom discordant pairs revealed sequence identity at these otherloci. Strain pairs that were discordant at only one of the sixloci sequenced were considered to be closely related derivativesof a recent common ancestor, with one isolate potentially divergedfrom the other by interstrain gene transfer. These pairs werecounted as nearly matched in the tally in Fig. 3.

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FIG. 3. Distribution of relatedness among child-mother H. pylori strain pairs according to the age of the offspring. Taken across all age groups, 24 of 81 (30%) pairs of strains from the child and the mother were perfectly matched or nearly matched (closely related). Similarly, 2 of 11 (18%), 1 of 4 (25%), and 12 of 37 (32%) pairs of strains from the child and father, the mother and the father, and one sibling and another sibling, respectively, were perfectly matched or nearly matched (closely related). All other pairs of strains were not related in their RAPD patterns and gene sequences.

In terms of distributions within families, on the basis of theRAPD profiles and the glr or other housekeeping gene sequences,24 of 81 strain pairs (30%) from children and mothers were closelyrelated (matched or nearly matched) and the other 70% were not(Fig. 3). Similarly, 2 of 11 (18%) strain pairs from childrenand fathers, 12 of 37 (32.4%) strain pairs from siblings, and1 of 4 (25%) strain pairs from spouses were closely related(see the Fig. 3 legend). These results further emphasize thatthere was no obvious relationship between the strains from pairsof family members in most families.

There were 13,512 possible pairs of glr sequences from membersof different households in the broader community in our study.Only 63 (0.4%) of them had glr alleles whose sequences were $≤$ 0.5% divergent. Thus, our results indicate both that the sharingof closely related strains among family members is markedlyless common in this shantytown than in industrialized societysettings and, equally, that strains from members of the samefamily are far more likely to be related than random strainsfrom the community are.

Association analyses. No significant association was seen between related strainsand any of several likely covariates in any family category(between the mother or father and the child, between siblings,or between spouses). Mother-child and sibling pairs were pooledto increase the sample size (118 pairs) and statistical power;and the data were adjusted for sociologic, demographic, andhousing characteristic variables and assessed for interactionsand second-order effects. Again, no covariate tested was significantlyassociated with the probability of the sharing of a relatedstrain.

DISCUSSION

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DISCUSSION

It is only in developing countries that most people are stillchronically infected by H. pylori, that frequent exposure makeseradication therapy ineffective, and that infection-associatedgastric cancer remains one of the most frequently lethal ofmalignancies (8, 13, 33, 44). In contrast, in industrializedcountries, less than half of all people are currently infectedwith H. pylori, and an essentially H. pylori-free future isanticipated (25). Thus, as with many pathogens, the epidemiologyof H. pylori infection and associated diseases varies markedlyamong human populations. In the study described here, we examinedthe relatedness of H. pylori strains from members of familiesin a shantytown in Peru to test the applicability of two typesof transmission models in a representative developing countrysetting: (i) the all-in-the-family model (12, 27), which wasdeveloped with data from industrialized countries, and (ii)a community-at-large/environmental reservoir model, which isencouraged by recent laboratory findings of the survival ofH. pylori for at least a few days in several nonchlorinatedwaters (5, 31, 40), H. pylori biofilm formation (11, 50), andH. pylori proliferation during coculture with amoebae (51).We found that $~$ 70% of strains from children in our study werenot closely related to those from their mothers, the parentimplicated in most transmission in industrialized societies,or to those from other family members. This suggested that manyinfections were community acquired. However, since strains fromchildren were still more frequently closely related to thoseof another family member ( $~$ 30%) than they were to those fromthe community at large (<1%), transmission within the familyor from common sources must also be significant in this society.

A theoretical model of frequent transmission from mother toinfant, with occasional displacement of the strain in the childby superinfecting strains in subsequent years, would have predictedthat closely related mother-child strain pairs would be farmore common in cases of very young children than of older children.Remarkably, however, this was not observed: matched pairs wereabout as common with older children as with younger children.This suggests that when transmission is intrafamilial, eachhousehold member may be equally prone to serve as the sourceof infection for other family members, independent of age, andthat even cases of closely related mother-child strain pairsneed not stem from early parent-to-child transmission and thenthe lifelong persistence of the initial strain.

We do not imagine, however, that most children had never beenexposed to their mothers' strains. Prior studies have indicatedthat infections are often transient in infants (29, 47) andalso in an experimentally infected monkey model (17), that individualhosts can differ in their susceptibilities to particular H.pylori strains, and that one strain can sometimes graduallydisplace another (16). These features may reflect differencesamong hosts and changes over time in traits that can be importantto individual strains, e.g., the types of host glycans usedas receptors for H. pylori adherence, the nature and intensityof inflammatory responses, and gastric pH (4, 6, 18). Such hostfeatures encourage the thinking that resident H. pylori strainsmust continuously adapt to their individual hosts during long-termchronic infection, such that a strain that has adapted to oneperson for many years may often be less well suited for colonizationof the next person who ingests it (16, 45). Since exposure toinfectious doses of H. pylori is relatively frequent in thiscommunity (the rate is estimated to be $~$ 20% per person per yearfor adults and higher for infants [29, 44]), this frameworkpredicts that many of the first strains to infect infants (presumablyoften those of their mothers), if they get established at all,will often be displaced by superinfecting unrelated strainsthat happen to be better suited for the infant's physiology.Such a dynamic might reflect the differences between the juvenilestomach, which may have little gastritis and normal acidity,and the stomach of an adult, which may have severe gastritis,often with hypochlorhydria, due to the years of chronic H. pyloriinfection.

Just one H. pylori strain per person was found to predominatein the great majority of shantytown residents (Fig. 1) (49).This high degree of uniformity suggests either that most superinfectingstrains fail to establish themselves because already establishedstrains are difficult to displace or that displacement by amore fit superinfecting strain tends to be rather rapid andcomplete. Such displacement of one strain by another has beenseen in experimentally infected monkeys (16) and mice (M. Zhangand D. E. Berg, unpublished data). We suggest that only rarelyin this environment would two strains be so well balanced infitness or tissue tropism that they would coexist for long periods,each at a high abundance, as was seen in mice with one exceptionalstrain pair (2).

How easily one strain is completely replaced by another in developing-countrysettings and how effectively a resident strain persists despiterepeated invasion by competitors and further adapts by mutationor interstrain recombination with superinfecting strains isnot known. The rapid emergence of new high-capacity, low-costgenome sequencing, the discovery of single-nucleotide polymorphisms,and transcript profiling technologies (34, 43) and the setsof strains found here should be particularly useful for theexamination of the mechanisms of adaptive evolution in high-riskhuman populations and the loci at which selection for changeoperates. Most immediately, however, this study indicates thatH. pylori is often transmitted from outside the family in adeveloping-country setting. In this setting, only community-basederadication measures will succeed in markedly reducing the prevalenceof H. pylori infection and the burden of gastric cancer, pepticulcer, and other associated diseases.

ACKNOWLEDGMENTS

We thank the members of the community of PSJM for their collaborationand Paula Maguiña for her able assistance with the administrativeaspects of this study.

This research was supported by grants from the U.S. NationalInstitutes of Health (grant RO1 DK63041 to D.E.B. and grantsT35 1007646 and D43TW006581 to R.H.G.). Other studies in D.E.B.'slaboratory, distinct from those reported here, are funded bya contract from Ondek Pty.; Washington University may receiveincome based on a license of technology to Ondek.

The contents of this report are solely the responsibility ofthe authors and do not necessarily represent the official viewsof the U.S. National Institutes of Health.

FOOTNOTES

* Corresponding author. Mailing address for Douglas E. Berg: Department of Molecular Microbiology, Campus Box 8230, Washington University Medical School, 4940 Parkview Place, St. Louis, MO 63110. Phone: (314) 362-2772. Fax: (314) 362-1232. E-mail: berg{at}borcim.wustl.edu. Mailing address for Robert H. Gilman: The Johns Hopkins School of Public Health, Department of International Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 614-3959. Fax: (410) 614-6060. E-mail: rgilman{at}jhsph.edu

Published ahead of print on 8 October 2008.

REFERENCES

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