Helicobacter pylori with East Asian-type cagPAI genes is more virulent than strains with Western-type in some cagPAI genes

(1)

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

Helicobacter

pylori

with

East

Asian-type

cagPAI

genes

is

more

virulent

than

strains

with

Western-type

in

some

cagPAI

genes

Xiao-yan

Yuan,

Jin-Jun

Yan,

Ya-chao

Yang,

Chun-mei

Wu,

Yan

Hu,

Jian-li

Geng

∗

WeihaiMunicipalHospitalafﬁliatedtoDalianMedicalUniversity,DepartmentofClinicalLab,Weihai,Shandong,PRChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14December2015 Accepted7July2016

Availableonline22December2016 AssociateEditor:Elizabethde AndradeMarques Keywords: Helicobacterpylori cagPAI Virulence IL-8

a

b

s

t

r

a

c

t

TheseverityofHelicobacterpylori-relateddiseaseiscorrelatedwiththepresenceandintegrity ofacagpathogenicityisland(cagPAI).cagPAIgenotypemayhaveamodifyingeffectonthe pathogenicpotentialoftheinfectingstrain.AfteranalyzingthesequencesofcagPAIgenes, somestrainswiththeEastAsian-typecagPAIgeneswereselectedforfurtheranalysisto examinetheassociationbetweenthediversityofthecagPAIgenesandthevirulenceofH. pylori.Theresultsshowedthatgastricmucosalinflammatorycellinfiltrationwas signifi-cantlyhigherinpatientswithEastAsian-typecagPAIgenesH.pyloristraincomparedwith mosaicismcagPAIgenesH.pyloristrain(p<0.05).H.pyloristrainswiththeEastAsian-type cagPAIgeneswerecloselyassociatedwithIL-8secretioninvitroandinvivocomparedwith H.pyloristrainswiththemosaicismcagPAIgenes(p<0.01).H.pyloristrainswithEast Asian-typecagPAIgenesareabletostronglytranslocateCagAtohostcells.Theseresultssuggest thatH.pyloristrainswithEastAsian-typecagPAIgenesaremorevirulentthanthestrainsof cagPAIgene/genesthatareWesterntype.

licenses/by-nc-nd/4.0/).

Introduction

Helicobacterpylori(H.pylori)isagram-negativebacteriumand is the main pathogenic factor of gastroduodenal diseases. H.pylori containsmany virulencefactors,including thecag pathogenicityisland(cagPAI),thatareinvolvedinthe patho-genesis of several diseases.1,2 _H. _pylori _cagPAI _consists _of

approximately30genesencodingforproteinsofthetypeIV secretionsystem(T4SS),whichtransfersCagAand peptido-glycanintohostepithelialcells,resultinginincreasedcellular

∗ _{Corresponding}_author.

E-mail:gengjianli1962@outlook.com(J.Geng).

release ofinterleukin 8(IL-8).3,4 _Studies _have_also

demon-stratedthatH.pyloristrainswithafunctionalT4SSaremore frequentlyassociatedwiththeetiologyofH.pylori.5,6

H. pylori is a highly genetically diverse species that is attributed toits high rate of DNA recombination.7–9 _It_has

beenreportedinJapanthatthegeneticdiversitywithinthe cagPAImayhavemodifyingeffectsonthepathogenic poten-tialofinfectingstrains.10 _The_similarity_coefﬁcient_revealed

thatallcagPAIgenescouldbeplacedintotwomajorgroups, EastAsian-typeorWestern-type.MostofcagPAIgenesbelong totheEastAsian-typeandisfoundassociatedwithhighrisk

http://dx.doi.org/10.1016/j.bjm.2016.12.004

(2)

ofdevelopinggastriccancerinsomeregions.Noticeably, in someregions, some strainsof the Western-typeofseveral cagPAIgene/genesareofEastAsian-typeofothercagPAIgenes, potentiallyduetothemosaicismofcagPAIgenesinH.pylori andgeneticrecombination.11

Much work has been reported on the genetic diversity ofcagPAI,particularlyonthegenotypeofcagA.12,13_Minimal

research has been conducted in applying cagPAI genotype analysistoevaluatethevirulenceofH.pylori.InsomeH.pylori strains,thetwopartsofthecagPAIgenesareinterposedbya segmentcalledinsertionsequence605(IS605):theupstream ascagIIregion,andthedownstreamascagIregion.Inthis study,somecagPAIgenesincagII(cagT,cagX)andcagIregion (cagI,cagL,cagM)wereselectedforsequencing.Thevirulence ofthestrainswithEastAsian-typecagPAIgeneswasevaluated bycomparingstrainswithmosaiccagPAIgenes.

Materials

and

methods

Patientsandsampling

Patientswithabdominalsymptomswereclinicallyexamined, and biopsy samples were taken for H. pylori isolation and histologicalanalysisinWeihaiMunicipalHospital(afﬁliated withDalianMedicalUniversity)fromJune2010toJune2014. Noneofthepatientsunderwentantimicrobialtherapy,orhave taken proton pump inhibitors (PPIs) or non-steroidal anti-inﬂammatorydrugs a month before their inclusion in the study.Writtenandinformed consentwasobtainedfromall patients,andthestudywasconducteduponapprovalbythe adhocEthicalCommitteeofWeihaiMunicipalHospital.

H.pyloricultivationandidentiﬁcation

Biopsyspecimenswerecollectedinbrainheartinfusionbroth (Oxoid,UnitedKingdom),anddispersedbyusingthetissue homogenizer.Everyhomogenatewasinoculatedonto Campy-lobacteragar(Oxoid,UnitedKingdom)with8%sheepblood andH.pyloriselectivesupplement(Oxoid,UnitedKingdom) undermicroaerophiliccondition(5%O2,10%CO2and85%N2)

at37◦C for72h. Small dewdrop coloniesofH.pylori were

selectedforfurtherphenotypicanalysisbyPCRofthe16SrRNA genesequenceaspreviouslydescribed.11

SequenceanalysisofcagPAIgenes

ThepresenceofﬁvecagPAIgenes,whichspreadoverthecag I(cagI,cagL,cagM)andcagIIregions(cagT,cagX)inH.pylori strains,wasanalyzedbyPCRusingﬁvesetsofprimerslisted

inTable1.Theampliconswereselectedforsequence

anal-ysis byLife TechnologiesCorporation.After the full-length aminoacidsequencesofeachgeneweretranslatedfromthe nucleotide sequences byPrimer 5.0,the phylogenetic trees ofCagPAIproteinswereconstructedbytheneighbor-joining methodofSaitouandNeibyusingaprogramcalledMEGA. AllcagPAIgenesintheisolatescouldbeplacedintotwomajor groups.MostH.pyloristrainsfromtheregionareeitherEast Asian-typeorWestern-type.Threepreviouslyreported West-ernstrainshavebeenidentiﬁedsofar:26695(UK),J99(USA), andNCTC11637(Australia).

Inthepresentstudy,thestrainswereclassifiedbasedon thegenotypesofcagPAIgenes:EastAsian-typecagPAIgenes group (all five cagPAI genes), and mosaicism cagPAI genes group (lessthan fivecagPAIgenes identifiedasEast Asian-type,andcontainingmorethanoneWestern-typecagPAIgene exceptcagA).

AnalysisofcagAstatusand3variableregionofcagA

PCR analyses were performed to analyze cagA status and cagA3 variableregionusingspeciﬁcprimers(Table1).After PCR, the ampliﬁed PCR products were electrophoresed in 2%agarosegels andexamined underUVillumination.The ampliconsofcagA3variableregionweresequencedbyLife TechnologiesCorporation.

Histologicalanalysis

Stomach biopsy specimens from each patient were exam-inedbyanexperiencedpathologist.Foreachbiopsyspecimen, thegradesofinﬂammatorycellinﬁltration,gastricmucosal atrophy, and H. pylori density were scored on the basisof

Table1–Polymerasechainreaction(PCR)primerpairsusedforcagPAIgenesinHelicobacterpyloriisolates.

Region Primer Sequence Ampliconsize(bp)

cagI f: ATAGAATTCACAGAAGTAGTAATAACGCTTGAAC 1086 r: AGACTCGAGTTTGACAATAACTTTAGAGCTAG cagL f: GATGGATCCGAAGATATAACAAGCGGTTT 654 r: GCCCTCGAGTTTAACAATGATCTTACTTGA cagM f: TAGGGATCCGAGCAGTTTGGTTCATTT 1149 r: CGCCTCGAGCTATTCAAAGGGATTATTC cagT f: TCCGGATCCATGAAAGTGAGAGCAAGTGT 843 r: GCCAAGCTTTCACTTACCACTGAGCAAAC cagX f: GGAATTCATGGGGCAGGCATTTTTTA 1569 r: GGGTCGACTTATTTATCTCTGACAAGAGGGAG

cagAconservativeregion f: GATAGGGATAACAGGCAAGC 297 r: GGGGGTTGTATGATATTTTC

(3)

Table2–HistologicaldifferencesamongthetwocagPAIgroups.

Parameter EastAsian-typecagPAIgenesgroup(n=42) MosaicismcagPAIgenesgroup(n=26)

H.pyloridensity 1.52±0.41 1.63±0.52

Inﬂammatorycellinﬁltration 1.71±0.68a _1.03_±_0.42

Atrophy 0.51±0.17 0.47±0.16

a _p_<_0.05.

theupdatedSydneySystem(0,none;1,mild;2,moderate;3, severe).14

MucosalIL-8levelanalysisinbiopsysamples

IL-8 levels in the biopsy specimens were measured after homogenization using an enzyme-linked immunosorbent assay (ELISA) (Invitrogen). Brieﬂy, the supernatants from homogenized specimens were obtained by centrifugation (10,000×g for15min),and the total proteinsofthe super-natantsweremeasuredbyusingtheBradfordassay(Bio-Rad, Richmond,CA).IL-8levelsinsampleswereexpressedaspg/mg ofprotein.

DeterminationofIL-8secretionfromGES-1cells co-culturedwithH.pyloristrains

Thestrainswereselectedandco-culturedwithGES-1cellsfor analysisofIL-8secretion.Brieﬂy,GES-1cellswereseededata densityof8×104_cells/well_in_96-well_plates._H._pylori_was

har-vestedfromagar dishesand washedtwicewithPBS before beingadded toculturewellsataMOI of100.Thecell cul-turemediawerecollectedat24h.IL-8levelsweredetectedby ELISA(Invitrogen)accordingtothemanufacturer’sprotocol. Theexperimentswereperformedtwiceindependently.

CagATranslocationAssay

GES-1cellsinfectedwithH.pylori(1:100)asdescribedabove werewashedwithPBSuntilnoH.pyloriwerefoundadheredto cellsasobservedunderamicroscope.Cellswerethenscraped andcentrifugedat4000rpmfor30min.Cellsweresuspended inRIPALysisBufferon icefor30min.Aftercentrifugingat 13,500rpmfor30min,thesupernatantwascollectedto ana-lyzeCagA byWestern blotting.Colony-forming units (CFU) assaywasperformedtoconﬁrmthatnoH.pyloriweredetected onscrapedcells.

Statisticalanalysis

Differences between the Asian-type strains group and the mosaic-typestrainsgroupwereanalyzedusingStudent’sttest andChi-squaretest.pvalueslessthan 0.05wasconsidered signiﬁcant.

Results

AftersequenceanalysisofcagPAIgenes(Fig.1),76strainswith EastAsian-typecagPAIgeneswereisolatedfrom gastroduo-denaldiseases,includingchronicgastritis(CG,n=29),gastric

ulcer (GU, n=21), duodenal ulcer (DU, n=19), and gastric cancer(GC,n=7).Twenty-sixstrainswithmosaicismcagPAI geneswereisolatedfromchronicgastritis(CG,n=14),gastric ulcer (GU,n=6),duodenalulcer(DU,n=4),andgastric can-cer(GC,n=2).IsolateswithEastAsian-typecagPAIgeneswere foundatlowerfrequencyinCG(38.2%,29/76)thanthosewith mosaicismofcagPAIgenesinCG(53.8%,14/26,p<0.01).

DetectionofcagAstatusandsequencingofthecagA3 variableregion

PCRproductswereobtainedfromallisolatesofH.pylori.After nucleotideanalysis,cagA3variableregionweregroupedas EastAsian-typeorWestern-type.TheEastAsian-type(521bp) possessedthreeEPIYAmotifs,whiletheWestern-type(502bp) possessed twoEPIYA motifs and oneEPIYT (Fig. 2). All 76 strainswithEastAsian-typecagPAIgenesharboredEast Asian-type,andonly2strainspossessedWestern-typein26strains withmosaicismcagPAIgenes.

HistologicalanalysisofH.pyloridensity,inﬂammatory cellinﬁltration,andatrophyinthebiopsyspecimens

Furtherhistopathologicevaluationsofbiopsyspecimenswere performedon42patientswithEastAsian-typecagPAIgenes H.pyloristrainsand26patientswithmosaicismcagPAIgenes H. pylori strains. Thebiopsy specimens of East Asian-type cagPAI genesgroupwereselected randomlyforhistological analysis.

Gastricmucosalinflammatorycellinfiltrationwas signif-icantly higher in East Asian-type cagPAI genes group than mosaicismcagPAIgenesgroup(p<0.05)(Fig.3).However,there werenosignificantdifferencesinthegradeofH.pylori den-sityandgastricmucosalatrophyaccordingtothediversityof cagPAIgenes(Table2).ThissuggestedthatthestatusofcagPAI genotype isassociatedwithprogressionofgastric mucosal inflammatorycellinfiltration.

IL-8productioningastricmucosa(invivo)andfrom GES-1cells(invitro)

Werandomlyselected36patientswithEastAsian-typecagPAI genesH.pyloristrainsand26patientswithmosaicismcagPAI genesH.pyloristrainstomeasuregastricmucosalIL-8levels. Thegastric mucosal IL-8 levelinpatients withEast Asian-type cagPAI genesH. pylori strainswas signiﬁcantly higher (112.7±33.1pg/mg)thanthosecontainingmosaicismcagPAI genesH.pyloristrains(75.8±24.7pg/mg,p<0.01).

IL-8 production in GES-1 cells that were co-cultured with 20 H. pylori strains was also examined. These

(4)

WH1-DU WH15-CG WH20-CG WH29-CG WH4-GU WH9-CG WH15-CG WH10-CG WH36-GU WH32-CG WH23-DU WH35-GU WH13-DU WH26-GU WH5-CG WH14-DU WH34-CG WH31-DU WH19-CG WH24-GU WH28-DU WH8-GC WH30-CG J99 WH2-CG WH33-CG WH12-CG 11 637 26 695 WH22-CG WH30-CG WH35-GU WH19-CG WH14-DU WH6-CG WH8-GC WH34-CG WH21-GU WH5-CG WH29-CG WH31-DU WH32-CG WH20-CG WH13-DU WH7-CG WH12-CG 11 637 26 695 WH2-CG WH33-CG J99 WH23-DU WH8-GC WH24-GC WH22-CG WH6-CG WH5-CG East Asian-type cagPAI genes group East Asian-type cagPAI genes group Mosaicism cagPAI genes group East Asian-type cagPAI genes group Mosaicism cagPAI genes group East Asian-type cagPAI genes group Mosaicism cagPAI genes group Mosaicism cagPAI genes group East Asian-type cagPAI genes group Mosaicism cagPAI genes group WH26-GU WH4-GU WH9-CG WH29-CG WH32-CG WH30-CG WH33-CG WH12-CG WH2-CG WH34-CG WH20-CG WH5-CG WH6-CG WH24-GU WH33-CG WH19-CG WH30-CG WH4-GU WH8-GC WH29-CG WH31-DU 26 695 11 637 WH2-CG J99 WH35-GU WH8-GC WH29-CG WH36-GU WH5-CG WH1-DU WH14-DU WH28-DU WH30-CG WH7-CG WH32-CG WH4-GU WH19-CG WH6-CG WH33-CG WH2-CG WH12-CG 11 637 26 695 J99 0.005 0.005 0.002 0.002 0.002 J99 11 637 26 695

Fig.1–PhylogenetictreeofthecompleteaminoacidsequencesofcagI,cagL,cagT,cagMandcagXinsomestrains.Scalebars

representcalculateddistances.Allthreeproteinsweredividedintotwomajorgroups:EastAsian-grouporWestern-group.

included 10 strainswith East Asian-type cagPAIgenes and 10 strains with mosaicism cagPAI genes. IL-8 production (1423.9±195.3pg/mL)wassigniﬁcantlyhigherinstrainswith EastAsian-typecagPAIgenesthaninthosewithmosaicism cagPAIgenes (1113.9±103.6pg/mL,p<0.01) (Table 3).These results suggested that the cagPAI genotype of H. pylori strainisimportantforinducingIL-8 productioninvivoand invitro.

CagAdeliverytohostcells

TenH.pyloristrainswithEastAsian-typecagPAIgenesand10 strainswithmosaicismcagPAIgeneswererandomlyselected forfurtheranalysisofCagAtranslocation.Afterinfectionof GES-1 cells byH. pylori, cells with translocated CagA were foundmoreincreasedinEastAsian-typecagPAIgenesgroup thanmosaicismcagPAIgenesgroup.Theseresultsindicated

(5)

Fig.2–DeducedaminoacidsequenceofcagA3variableregionfromsomestrains.

Fig.3–HistopathologicevolutionsofbiopsyspecimensintwogroupsaccordingtoH&Estain(100×).(A)EastAsian-type

cagPAIgenesgroup;(B)mosaicismcagPAIgenes.Inﬁltrationofinﬂammatorycells(arrows).

Table3–IL-8levelsingastricmucosa(invivo)andGES-1cells(invitro).

EastAsian-typecagPAIgenesgroup MosaicismcagPAIgenesgroup Gastricmucosa(pg/mg) 112.7±33.1(n=36)a _75.8_±_24.7_(n₌₂₆₎

GES-1cells(pg/mL) 1423.9±195.3(n=10)a _1113.9_±_103.6_(n₌₁₀₎

a _p_<_0.01.

thatthetranslocationofCagAtohostcellswaslesseffective instrainswithmosaicismcagPAIgenes(Fig.4).

Discussion

H.pyloricagPAIisamajorvirulencedeterminantinH. pylori-relateddiseases.IncomparisonwithcagPAI-negativestrains, infection with cagPAI-positive H. pylori strains signiﬁcantly increasestheriskofdevelopingseveregastricmucosal inﬂam-mation,duodenalulcers,andgastric cancers.15,16 _The_cagA

Fig.4–CagAtranslocationbetweentwogroups.(A)East

Asian-typecagPAIgenesgroup;(B)mosaicismcagPAIgenes

(6)

gene is part of cagPAI, and CagA is the primary virulence factorofH.pylori thatsufﬁcientlyinducestumorigenesisin cellandanimal models.CagAhasalsobeenreportedtobe epidemiologicallyassociatedwithhigher riskofdeveloping gastriccancer.17,18 _Based_on_the _sequence _of_the ₃ _region,

cagAwasclassiﬁedinto2types:EastAsian-typeand Western-type.Intriguingly,bothinvitroandinvivostudieshaveclearly showedthatEastAsian-typeCagA proteinismore carcino-genicthanWestern-typeCagAprotein.19_In_this_region,_most

strains possess East Asian-type of cagA with three EPIYA

motifs.

InalittoralregionofNortheastChina,nearly100%ofthe strainspossesscagA,andmostisolatesofcagAwereincluded inEastAsian-type.20_Noticeably,_a_high_incidence_of_atrophic

gastritis and gastric cancer hasalso been reported in this region.21 _Early_studies_showed_that_the_distinct_distribution

ofcagPAIdiversityinthisregionmaybeinvolvedinthe devel-opmentofatrophic gastritis,potentiallyincreasingtherisk ofworsenedoutcomeindifferentdiseasesbasedon phyloge-neticanalysisofcagPAIgenes.11_We_hypothesize_that_the_T4SS

encodedbyEastern-typecagPAIgenesintheregionresultsin theabilityofH.pyloritoincreasetheriskofdevelopinggastric cancer.

Inthe study,isolateswith EastAsian-type cagPAIgenes were foundatlower frequency inCG, comparedtostrains withmosaicismcagPAI genes inCG.Two cagPAIgenotypes of H. pylori strains were selected based on the phyloge-neticanalysisoffull-lengthCagPAI proteins,includingEast Asian-typecagPAIandmosaicismcagPAIgenesstrains.Some strainswereselectedtostudytheirvirulence,andto exam-ine whether there is an association between the diversity ofthecagPAIgenesandthevirulenceofH.pylori.Wefound thatgastricmucosalinflammatorycellinfiltrationwas signif-icantlyhigherinpatientswithEastAsian-typecagPAIgenes H. pylori than others with mosaicismcagPAI genes strains. TheseobservationssuggestthatcagPAIgenotypeisassociated withprogressionofgastricmucosalinflammatorycell infiltra-tion.Meanwhile,wealsofoundthatH.pyloristrainswiththe EastAsian-typecagPAIgenesarecloselyassociatedwithIL-8 secretioncomparedwithH.pyloristrainswiththemosaicism cagPAI genes. Intriguingly, H. pylori strains with the East Asian-typecagPAIgenescaneasilytranslocateCagAtohost cells.

H. pylori exhibits extensive genetic diversity and rapid allelicdiversiﬁcationattributedtoitshighmutationrateand frequentrecombinationindifferentdiseases.22–24_We_believe

thatthemosaicismincagPAIgenesisbestexplainedbygenetic recombination. Thus, the genetic recombination in cagPAI betweenthewest strainand Eaststraincould increasethe diversiﬁcationandvirulenceofH.pylori.

In conclusion, H. pylori with the East Asian-type cagPAI genesisassociatedwithgastricmucosal inﬂammatorycell inﬁltrationandIL-8secretion,whichseemstobemorevirulent comparedwithotherstrains.

Conﬂicts

of

interest

Theauthors declarethattheyhave noconﬂictsofinterest concerningthisarticle.

Acknowledgments

ThisworkwassupportedbyShandongProvincialNatural Sci-enceFoundation,China(No.ZR2015HM072)andJiangsuKey LaboratoryofMedicalScienceandLaboratoryMedicine(No. JSKLM-2014-014).

r

e

f

e

r

e

n

c

e

s

1.Sánchez-ZaucoNA,TorresJ,Pérez-FigueroaGE,etal.Impact

ofcagPAIandT4SSontheinﬂammatoryresponseofhuman

neutrophilstoHelicobacterpyloriinfection.PLOSONE.

2013;8(6):e64623.

2.NguyenLT,UchidaT,TsukamotoY,etal.Clinicalrelevance

ofcagPAIintactnessinHelicobacterpyloriisolatesfrom

Vietnam.EurJClinMicrobiolInfectDis.2010;29:651–660.

3.OdenbreitS,PülsJ,SedlmaierB,GerlandE,FischerW,HaasR.

TranslocationofHelicobacterpyloriCagAintogastricepithelial

cellsbytypeIVsecretion.Science.2000;287:1497–1500.

4.VialaJ,ChaputC,BonecaIG,etal.Nod1respondsto

peptidoglycandeliveredbytheHelicobacterpyloricag

pathogenicityisland.NatImmunol.2004;5:1166–1174.

5.TegtmeyerN,WesslerS,BackertS.Roleofthe

cag-pathogenicityislandencodedtypeIVsecretionsystem inHelicobacterpyloripathogenesis.FEBSJ.2011;278: 1190–1202.

6.VanniniA,RoncaratiD,SpinsantiM,ScarlatoV,DanielliA.In

depthanalysisoftheHelicobacterpyloricagpathogenicity

islandtranscriptionalresponses.PLOSONE.2014;9(6):e98416.

7.CensiniS,LangeC,XiangZ,etal.cag,apathogenicityisland

ofHelicobacterpylori,encodestypeI-speciﬁcand

disease-associatedvirulencefactors.ProcNatlAcadSciUSA.

1996;93:14648–14653.

8.CovacciA,TelfordJL,DelGiudiceG,ParsonnetJ,RappuoliR.

Helicobacterpylorivirulenceandgeneticgeography.Science.

1999;284:1328–1333.

9.AlmRA,LingLS,MoirDT,etal.Genomic-sequence

comparisonoftwounrelatedisolatesofthehumangastric

pathogenHelicobacterpylori.Nature.1999;397:176–180.

10.AzumaT,YamakawaA,YamazakiS,etal.Distinctdiversity

ofthecagpathogenicityislandamongHelicobacterpylori

strainsinJapan.JClinMicrobiol.2004;42:2508–2517.

11.WangMY,LinJ,SunSB,YuanXY.Identiﬁcationof

Granulicatellaelegansfromacaseofinfectiveendocarditis,

basedonthephenotypiccharacteristicsand16SrRNAgene

sequence.JCardiovascMed(Hagerstown).2015;16(suppl

2):S138–S140.

12.Bustamante-RengifoJA,MattaAJ,PazosA,BravoLE.Invitro

effectofamoxicillinandclarithromycinonthe3regionof

cagAgeneinHelicobacterpyloriisolates.WorldJGastroenterol.

2013;19(36):6044–6054.

13.MatosJI,deSousaHA,Marcos-PintoR,Dinis-RibeiroM.

HelicobacterpyloriCagAandVacAgenotypesandgastric

phenotype:ameta-analysis.EurJGastroenterolHepatol.

2013;25(12):1431–1441.

14.DixonMF,GentaRM,YardleyJH,CorreaP.Classiﬁcationand

gradingofgastritis.TheupdatedSydneySystem.

InternationalWorkshopontheHistopathologyofGastritis,

Houston1994.AmJSurgPathol.1996;20:1161–1181.

15.HatakeyamaM.Helicobacterpyloriandgastriccarcinogenesis.

JGastroenterol.2009;44:239–248.

16.KumarS,KumarA,DixitVK.Evidencesshowingassociation

ofinterleukin-1Bpolymorphismswithincreasedriskof

gastriccancerinanIndianpopulation.BiochemBiophysRes

(7)

17.OhnishiN,YuasaH,TanakaS,etal.Transgenicexpressionof

HelicobacterpyloriCagAinducesgastrointestinaland

hematopoieticneoplasmsinmouse.ProcNatlAcadSciUSA.

2008;105(3):1003–1008.

18.HatakeyamaM.OncogenicmechanismsoftheHelicobacter

pyloriCagAprotein.NatRevCancer.2004;4(9): 688–694.

19.MiuraM,OhnishiN,TanakaS,YanagiyaK,HatakeyamaM.

Differentialoncogenicpotentialofgeographicallydistinct

HelicobacterpyloriCagAisoformsinmice.IntJCancer.

2009;125(11):2497–2504.

20.WangMY,ChenC,GaoXZ,etal.DistributionofHelicobacter

pylorivirulencemarkersinpatientswithgastroduodenal

diseasesinaregionathighriskofgastriccancer.Microb

Pathog.2013;59–60:13–18.

21.GaoXZ,ChuYL,QiaoXL,etal.Narrowbandimaging

endoscopyfordiagnosisofmalignantandpremalignant

gastriclesions.ChinJDig.2009;29(5):289–292.

22.XiangZ,CensiniS,BayeliPF,etal.Analysisofexpressionof

CagAandVacAvirulencefactorsin43strainsofHelicobacter

pylorirevealsthatclinicalisolatescanbedividedintotwo

majortypesandthatCagAisnotnecessaryforexpressionof

thevacuolatingcytotoxin.InfectImmun.1995;63(1):94–98.

23.KrebesJ,DidelotX,KennemannL,SuerbaumS.Bidirectional

genomicexchangebetweenHelicobacterpyloristrainsfroma

familyinCoventry,UnitedKingdom.IntJMedMicrobiol.

2014;304(8):1135–1146.

24.FalushD,WirthT,LinzB,etal.Tracesofhumanmigrations

inHelicobacterpyloripopulations.Science.