Primary antibiotic resistance and its relationship with cagA and vacA genes in Helicobacter pylori isolates from Algerian patients

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

Clinical

Microbiology

Primary

antibiotic

resistance

and

its

relationship

with

cagA

and

vacA

genes

in

Helicobacter

pylori

isolates

from

Algerian

patients

Meryem

Bachir

a,∗

_,

_Rachida

_Allem

a

_,

_Abedelkarim

_Tifrit

a

_,

_Meriem

_Medjekane

a

_,

Amine

El-Mokhtar

Drici

b

_,

_Mustafa

_Diaf

b

_,

_Kara

_Turki

_Douidi

c

a_Bioresources_Laboratory,_Department_of_Biology,_Faculty_of_Natural_and_Life_Sciences,_Hassiba_Ben_Bouali_University_of_Chlef_(UHBC),

Chlef,Algeria

b_Laboratory_of_Molecular_{Microbiology,}_Proteomics_and_Health,_Department_of_Biology,_Faculty_of_Natural_and_Life_Sciences,_University_of

DjillaliLiabes(UDL),Sidi-Bel-Abbes,Algeria

c_Department_of_{Gastroenterology,}_University_Hospital_Hassani_Abedelkader,_{Sidi-Bel-Abbes,}_{Sidi-Bel-Abbes,}_Algeria

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22December2016 Accepted27November2017 Availableonline13February2018 AssociateEditor:JorgeSampaio

Keywords: Helicobacterpylori Antibioticresistance vaca caga Algeria

a

b

s

t

r

a

c

t

TheepidemiologyofHelicobacterpyloriresistancetoantibioticsispoorlydocumentedin AfricaandespeciallyinAlgeria.Theaimofourstudywastodeterminetheantibiotic resis-tancerates,aswellasitspossiblerelationshipwithVacAandCagAvirulencemarkersof isolatesfromAlgerianpatients.OnehundredandfiftyoneH.pyloriisolatewereobtained between2012and2015from200patientswithupperabdominalpain.Antimicrobial suscep-tibilitytestingwasperformedforamoxicillin,clarithromycin,metronidazole,ciprofloxacin, rifampicinandtetracycline.MolecularidentificationofH.pyloriandthedetectionofvacA

andcagAgeneswereperformedusingspecificprimers.WefoundthatH.pyloriwaspresent in83.5%ofcollectedbiopsies,54.9%ofthesampleswerecagApositive,49.67%werevacA s1m1,18.30%werevacAs1m2and25.49%werevacAs2m2.Isolateswerecharacterizedby noresistancetoamoxicillin(0%),tetracycline(0%),rifampicin(0%),ahighrateof resis-tancetometronidazole(61.1%)andalowerrateofresistancetoclarithromycin(22.8%)and ciprofloxacin(16.8%).NostaticallysignificantrelationshipwasfoundbetweenvagAandcagA

genotypesandantibioticresistanceresults(p>0.5)exceptforthemetronidazole,whichhad relationwiththepresenceofcagAgenotype(p=0.001).

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

Introduction

Helicobacterpylori(H.pylori)isagramnegativespiralchapped bacterium, described in 1982 by Marshal and Warren. It

∗ _{Corresponding}_author_at:_Department_of_Biology,_Faculty_of_Natural_and_Life_Sciences,_Hassiba_Ben_Bouali_University_of_Chlef,_Laboratoire

debioressources,02000Chlef,Algeria. E-mail:[email protected](M.Bachir).

speciﬁcally infects the gastric mucosa. This discovery had changed all dataabout gastro-duodenal disorders.H. pylori

infects approximately ﬁfty per cent ofthe world’s popula-tion,itisanimportantriskfactorinchronicgastritis,peptic ulcerdisease,gastriccarcinomaandmucosa-associated lym-phoid tissue(MALT) lymphoma.It chronicallyinfectsmost

https://doi.org/10.1016/j.bjm.2017.11.003

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peopleinthe developingcountries, andalsoremainsa sig-niﬁcantpathogeninindustrializedcountries.1_In_Algeria,_its

prevalenceisaverage80%inpepticulcer.2_Fortunately,_many

H.pyloriassociatedpathologiescanbepreventedorcuredby theeradicationofthebacterium.

Until now, in Algeria eradication of H. pylori is recom-mended by a triple therapy which includes amoxicillin, clarithromycinormetronidazolecombinedwithprotonpump inhibitors (PPI) for7–10 days.3 _It_is _effective _for_79–85% _of

patientsbutafterthepublicationoftheMaastrichtV,Toronto andSpanishConsensusaboutmanagementofH.pylori infec-tion and its treatment in adults,4,5 recommendations for eradication,therapyin Algeriashould bechanged,atleast inlength inorder togain an eradication >=90%oftreated patients.

Eradication efforts fail for a signiﬁcant proportion of patients in Algeria for several reasons, including bacterial resistance to clarithromycin. Several studies have demon-stratedthatprimaryresistancetoclarithromycinisamajor factorfortherapeuticfailure; therate ofresistanceto clar-ithromycinissigniﬁcantlyincreasingandthehighrateofthe resistancetometronidazoleleadstoavoidthecombinationof thesetwoantibioticsinthetreatmentregimens.3_In_another

way,resultsofsomestudieshavesuggestedthatthe eradi-cationrate inpatients withgastritisislower thaninthose withpepticulcerdiseases.6_Since_the_worldwide_increase_of

thedrugresistanceratesrepresentsaproblemofrelevance, someresearcheshavebeenconductedonantibioticresistance relationshipwithbacterialgeneticfactorssuchasthe cyto-toxinassociatedgeneA(cagA)andvaculatingcytotoxingene A(vacA).7,8

BecauseofthelimiteddataabouttheresistanceofH.pylori

toantibioticsinAlgeria,theaimofthisstudywasto inves-tigate the resistance rates of amoxicillin, clarithromycin, metronidazole,tetracycline,ciproﬂoxacinandrifampicin,and tostudytherelationbetweenH.pylorigenotypesandthe resis-tancetoantibiotics.

Materials

and

methods

patients

From2012to2015,fourHospitalswereinvolvedinthepresent study:MustaphaPachaUniversityHospital(Algiers),Halouche clinicofGastroenterology(Chlef),FirstNovember1954 Uni-versityHospitalofOranandSidiBelabessUniversityHospital. Twohundredpatientswithupperabdominalpainfromfour different regions in Algeria (Algiers, Chlef, Oran and Sidi Belabess),wereenrolledinthepresentstudy(91malesand 109females).ThepatientswereAlgeriancitizensagingfrom 18to86yearsandprovidedwritteninformedconsentbefore endoscopy.Patientseitherreceivingprotonpumpinhibitory drugsorhavebeentreatedwithantimicrobialswereexcluded fromthestudy.Allsubjectsunderwentendoscopytoobtain twoantrumbiopsiesfromeachpatient.Theﬁrstwasusedfor screeningofH.pyloripositivespecimensbyarapidureasetest (RUT).Whilethesecondpiecewasplacedin1mLofsterile phosphatebuffersalinesolutionandwastransported imme-diatelyfortheisolationofH.pylori.Thisstudyprotocolwas approvedbytheAlgeriannationalethicscommittee.

Bacterialculture

H. pylori was isolated from grinded gastric biopsy samples onbrainheartinfusion(BHI)agar(bioMérieux)supplemented with10%deﬁbrinatedhorseblood,0.4%IsoVitaleX,5mg/Lof trimethoprim, 5mg/Lofcefsulodin, 10mg/Lofvancomycin, and 8mg/L of amphotericin B. The plates were incubated at37◦Cunder microaerobicconditionsfor7days ormore.

H.pyloriwasidentiﬁedbycolonyaspect,microscopic morphol-ogy,positiveurease,catalaseandoxidasetests.Strainswere storedat−80◦_C_in_BHI_broth_with_20%_glycerol.9,10

Antimicrobialsusceptibilitytesting

Antibiotic susceptibility was determined using the E-test and agar dilution methods as recommended by EUCAST. Thetestedantibioticswereamoxicillin(AMX),clarithromycin (CLA), ciproﬂoxacin (CIP), metronidazole (MTZ), rifampicin (RIF) and tetracycline(TET). The breakpoints usedto clas-sifyisolatesassusceptibleorresistantaccordingtotheMIC valueaccording toEUCAST: theisolateis resistantto AMX if MIC >0.12mg/L, resistant to CLAif MIC >0.5mg/L, resis-tanttoCIPifMIC>1mg/L;resistant toMTZifMIC>8mg/L; resistant toRIF if MIC >1mg/Land resistantto TETif MIC >1mg/L.11_{Mueller–Hinton}_agar_supplemented_with_7%

deﬁb-rinated horse blood (bioMérieux) was used as the culture medium. An H. pylori culture suspension with a turbidity equivalent ofa 3.0McFarlandstandard wasusedto inocu-latetheplatescontainingserialdilutionsofantibiotics:CLA, 0.016–256mg/L; AMX, 0.016–32mg/L; MTZ, 0.016–256mg/L; TET,CIPandRIF,0.016–32mg/L(bioMérieux),orplates with-outantibioticsontoE-teststripsofeachantibiotictestedwere applied(bioMérieux).Theplateswereincubatedat37◦Cfor 3daysundermicroaerophilicconditions.11

DNAextractionandpuriﬁcation

TotalgenomicDNAwasextractedusingcommercialkits: Mag-azorbDNAmini-prepkit(Promega)forbiopsiesandWizard GenomicDNAPuriﬁcationKit(Promega)forstrainsaccording tothemanufacturer’sguidelines.GenomicDNAfrombiopsies andstrainswasstoredat−20◦_C.

MoleculardetectionofH.pylori

MoleculardetectionofH.pyloriinbiopsyDNAandstrain iden-tiﬁcation wereperformedusingtheprimersinTable1.The 25␮Lreaction mixture consisted of1× PCR buffer, 1.5mM magnesium chloride,200␮Mofeach dNTP,20pmolofeach primer and 1U hot start Taq DNA polymerase (Promega). AmpliﬁcationwascarriedoutinanEppendorfMastercycler gradientusingthecyclingparametersinTable1.ThePCR prod-uctswereseparatedona2%agarosegeland100bpladderwas usedasDNAmolecularweightstandard.12,13

ThecagAgenedetection

Thepresenceofthe genotypecagAwasdetermined byPCR usingprimersinTable1.DNAsamplefromH.pyloriJ99strain wasusedaspositivecontroland steriledistilledwaterwas

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Table1–Primersusedforpolymerasechainreactionanalysis.

Primer Sequence(5–3) PCRconditions SizeofPCRproduct Reference HEL-F AACGATGAAGCTTCTAGCTTGCTA Initialdenaturationat94◦Cfor

10min;35cyclesof94◦Cfor 30s,56◦Cfor1minand72◦C for1min;ﬁnalextensionof 72◦Cfor10min

399bp 12

HEL-R GTGCTTATTCSTNAGATACCGTCAT

glmM-F GGATAAGCTTTTAGGGGTGTTAGGGG 294pb 13

glmM-R GCTTACTTTCTAACACTAACGCGC .

cagA-F1 GATAACAGGCAAGCTTTTGAGG Initialdenaturationat94◦Cfor 10min;40cyclesof94◦Cfor 30s,58◦Cfor30sand72◦Cfor 1min;ﬁnalextensionof72◦C for10min

349pb 14,15

cagA-B1 CTGCAAAAGATTGTTTGGCAGA

VA1-F ATGGAAATACAACAAACACAC 259pb 14

VA1-R CTGCTTGAATGCGCCAAAC

VA1-F ATGGAAATACAACAAACACAC Initialdenaturationat94◦Cfor 10min;35cyclesof94◦Cfor 30s,60◦Cfor30sand72◦Cfor 30s;ﬁnalextensionof72◦Cfor 10min.

286pb 14

VA1-R CTGCTTGAATGCGCCAAAC

VAG-F CAATCTGTCCAATCAAGCGAG 570pb 14

VAG-R GCGTCAAATAATTCCAAGG

VAG-F CAATCTGTCCAATCAAGCGAG 645pb 14

VAG-R GCGTCAAATAATTCCAAGG 14

usedasanegativecontrol.The10␮Lreactionmixture con-sistedof1×PCRbuffer,1.5mMmagnesiumchloride,200␮M dNTP,0.5pmolofeachprimer,0.5Uhotstarttaqpolymerase (Promega)and2␮LoftemplateDNA.Ampliﬁcationwas car-ried out in an Eppendorf Mastercycler gradient using the cyclingparametersinTable1.ThePCRproductswere sepa-ratedon a2%agarosegeland a250bpladder wasusedas DNAmolecularweightstandard.14,15

ThevacAgenedetection

ThepresenceofthegenotypesofvacAalleles(s1,s2,m1and

m2)wasdeterminedbymultiplexPCRusingtheprimersin

Table1. DNAsamplesfrom J99H.pyloristrainwas usedas

positivecontrolsandsteriledistilledwaterwasusedasa neg-ativecontrol.The20␮Lreactionmixtureconsistedof1×PCR buffer,1.5mMmagnesiumchloride,400␮MdNTP,0.5pmolof eachprimer,1Uhotstarttaqpolymerase(Promega)and4␮L oftemplateDNA.Ampliﬁcationwascarriedoutinan Eppen-dorf Mastercyclergradientusing the cycling parameters in

Table1.ThePCRproductswere separated ona2%agarose

gelanda100bpladderwasusedasDNAmolecularweight standard.14

Statisticalanalysis

ThedatawereanalyzedusingSPSSsoftware(Version17.SPSS Inc, United States) and p values were calculated using

Chi-squaretesttofindanysignificantrelationship.p<0.05was consideredstatisticallysignificant.

Results

Isolatesandantibioticsusceptibilitytesting

Oftwohundredpatients,H.pyloriinfectionwasfoundin163 individuals(81.5%)onthebasisofpositiverapidureasetest. Atotalof151(76%)H.pyloriwereisolatedandtheantibiotic susceptibilitywasdetermined.AccordingtoEUCAST break-points,only26isolatesweresusceptibletoallantibioticsand allisolatestestedweresusceptibletotetracyclin(MICrange, 0.016–0.064mg/L), rifampicin (MIC range, 0.016–0.032mg/L) and amoxicillin (MIC range, 0.016–0.064mg/L. The resis-tance rate to clarithromycin (MIC range, 0.5–32mg/L) was (25.2%,n=38)andforciproﬂoxacin(MICrange,1–32mg/L),it was (18.5%,n=28). Theisolateswerecharacterizedbyhigh ratesofresistancetometronidazole(MICrange,8–256mg/L) (67.5%, n=102) (Table 2). Twenty six isolates were resis-tant to both metronidazole and clarithromycin. Twelve of the 28 ciproﬂoxacin-resistant isolates were also resistant to metronidazole and one was resistant to clarithromycin. Metronidazole-resistantisolateswerefoundmorefrequently inwomencomparedwithmen(43.05%versus24.4%,p<0.05) but there were norelationbetween thegenderor age and resistancefortheotherantibiotics(p>0.5).

ByPCRH.pyloriwasdetectedin167outof200(83.5%)where both16SrRNAandglmMgenesweredetected.Generally,of167 positivegastricbiopsysamples,97samples(58%)werecagA

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Table2–ResistancetoantibioticsofH.pyloriisolatesin malesandfemalesfrom2012to2015,Algeria.

Antimicrobial Resistance(%) (n/total) Resistancein females(%) (n/total) Resistancein males(%) (n/total) AMX 0(0/151) 0(0/151) 0(0/151) CIP 18.5(28/151) 9.93(15/151) 8.61(13/151) CLA 25.2(38/151) 15.89(24/151) 9.27(14/151) MTZ 67.5(102/151) 43.05(65/151) 24.50(37/151) RIF 0(0/151) 0(0/151) 0(0/151) TET 0(0/151) 0(0/151) 0(0/151) AMX,amoxicillin; CLA,clarithromycin; CIP, ciproﬂoxacin;MTZ, metronidazole;RIF,rifampicin;TET,tetracycline.

positiveandallsampleshadampliﬁedbandsforbothvacAs

andmregions.Overall,100(59.88%)sampleshadvacAs1m1,

30(17.96%)hadvacAs1m2,37(22.15%)hadvacAs2m2.No stat-icallysigniﬁcantrelationshipwasfoundbetweenvacAorcagA

genotypesandantibioticresistanceresults(p>0.5)exceptfor themetronidazole,whereithadrelationwiththepresenceof

cagAgenotype(p=0.001)(Figs.1and2).Amongtheresistant isolates,thevacAs1m1/cagA+genotypewasthemost preva-lentfollowedbythevacAs1m1/cagA−genotype(Table3).

Discussion

H.pyloriinfectionrepresentsthemostprevalentchronic bac-terialdiseaseintheworldwitharangethataffectsmorethan halfofthepopulation.16_The_prevalence_of_H._pylori_differs

sig-niﬁcantlybetweencountries,withhighratesofinfectionbeing associatedwithlowsocioeconomicstatusandhighdensities ofliving.Forinstance,inJapan,SouthAmerica,Turkeyand Pakistan,theprevalenceismorethan80%,whileinEnglandit islower(20%).17_H._pylori_was_detected_in₁₆₇_(83.5%)_of_a_total

of200biopsyspecimensbythePCRmethod.Ourﬁndingsare similartootherreportsinIran.18_This_rate_is_higher_than_the

ratefoundwhenusingculture(76%).Thisisduetothe sen-sitivityandthespeciﬁcityofthePCRandtheexigencyofthe bacteriumincultureconditionswhichmakesitsisolationvery

0 5 10 15 20 25 30 35 40 45 50

MTZ-R MTZ-S CLA-R CLA-S CIP-R CIP-S cagA positive cagA negative % F re q u en cy Antibiotic

Fig.2–Prevalenceofantibiotic-resistantincagApositive andcagAnegativeH.pyloriisolates.MTZ-R,metronidazol resistant;MTZ-S,metronizaolsusceptible;CLA-R,

clarithromycinresistant;CLA-S,clarithromycinsusceptible; CIP-R,ciproﬂoxacinresistant;CIP-S,ciproﬂoxacin

susceptible.

difﬁcultwithoutforgettingthatculturehasthegreat advan-tageofallowingperformanceofantimicrobialsusceptibility.

TheglmMgeneishighlyconservedandhasbeenusedto identifyH.pyloriingastricbiopsies.Ithasbettersensitivity thantheureAgene.19_One_of_the_advantages_of_using_the_glmM

genetoidentifyH.pyloridirectlyingastricbiopsiesisitshigh degreeofsensitivityandspeciﬁcity,becauseithasadetection rateof10–100H.pyloricells,whichisbetterthan histopathol-ogy and culture.20 _Our _ﬁndings _revealed _a _rate _of _positive

H.pyloriinthetestedbiopsiesof83.5%basedondirect molec-ular detectionbyPCRusingtheglmMgenes. Althoughitis referredthatglmMgenedetectionismoresensitivethanother genes,inthisstudy,detectionof16SrRNAgenehadthesame sensitivityasglmMgene.Hoshinaetal.alsoused16SrRNA

sequencesforthespeciﬁcdetectionofH.pylori.Thespeciﬁcity ofH.pyloridetectionreportedbyHoshinaandcolleaguesis similartoourresults.21

InAlgeria,theprevalenceofH.pyloriisveryhighsoitisvery importanttoknow theratesofits resistancetoantibiotics, especially when antibiotic resistance represents a serious

0 10 20 30 40 50 60 70 80 90

MTZ-R MTZ-S CLA-R CLA-S CIP-R CIP-S

vacA positive vacA negative % F re q u en cy

Antibiotic

Fig.1–Prevalenceofantibiotic-resistantinvacApositiveandvacAnegativeH.pyloriisolates.MTZ-R,metronidazol resistant;MTZ-S,metronizaolsusceptible;CLA-R,clarithromycinresistant;CLA-S,clarithromycinsusceptible;CIP-R, ciproﬂoxacinresistant;CIP-S,ciproﬂoxacinsusceptible.

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Table3–ResistancetoantibioticsofH.pylorigenotypes.

Genotypes MTZ-R(%)(n/total) CLA-R(%)(n/total) CIP-R(%)(n/total)

vacAs1m1/cagA+ 43.04(65/151) 11.9(18/151) 7.28(11/151)

vacAs1m1/cagA- 0.66(1/151) 0(0/151) 1.3(2/151)

vacAs1m2/cagA+ 1.3(2/151) 0.66(1/151) 0(0/151)

vacAs1m2/cagA- 7.28(11/151) 3.31(5/151) 2.64(4/151)

vacAs2m2/cagA- 12.5(19/151) 5.96(9/151) 1.98(3/151)

publichealth probleminAlgeria andaround the world.In

adults,theratesofprimaryresistancetoclarithromycinand

metronidazolewere documentedas15% forclarithromycin

and37%formetronidazolein2013.3_There_is_an_increase_in

therateofclarithromycinresistancewith25.2%andthesame formetronidazol(67.5%)foundinourpresentstudy. Concern-ingclarithromycinprimaryresistance,ourprevalencewould seem higher than that foundinCongo (1.7%),22 _in_Iceland

(9%)23 _and _Tunisia_(14.6).24 _This_rate _was_almost _similar _to

thosefoundinChina(28.9%),25 _and_in_Spain_(17.1%).26 _This

differenceinclarithromycinresistanceratebetweenseveral countriesmightbeduetotheprescriptionofthisantibiotic. Since clarithromycin is widely used as antimicrobial drug toheal infections inother organ systems suchas respira-torysystemandpreviousconsumptionofmacrolides(cross resistance),theprevalenceofclarithromycin-resistantis con-tinuouslyincreasing.27

Metronidazoleresistanceishigherinthedeveloping coun-tries(50–80%).Ourresultwasmuchhigherthanthatfound inEurope(33.1%)28,29_and_in_Iceland_(1%),23_but_was_similar_to

theratefoundinChina(63.8%)25_and_lower_than_that_found_in

Columbia(82%).30_We_found_a_higher_primary_{metronidazole}

resistanceinfemalepatientscomparedwithmaleones,most likelyduetothewideuseofthisantibioticforgynecological infections.Inaccordancewithdatapresentedby Glupczyn-skietal.andBatnavalaetal.,metronidazoleresistantisolates were found altogether morefrequently infemales than in malesandinnonEuropeannatives.31,32 _Koletzko_et_al._also

foundthatmetronidazolewaswidelyprescribedforinfections suchasparasiticorfemalegenitalinfectionsinAfricaandin Asiawhichexplainsourresults.33

Fluoroquinolone resistance was found in 28 out of 151 (18.5%). In Asia, different values among countries were detected,theresistanceratebeing14.9%inJapan,34_and_28%

inChina.25_In_a_single_Italian_study,35_{levoﬂoxacin}_resistance

washigherwithold(>45years)thanyoungpatients(28.4%vs. 14.4%).InKoreatheresistancerateofH.pyloritociproﬂoxacin wasreportedtobe(33.8%),36_this_data_agree_with_our_results.

ButinSwedenalowrateofresistance(3.3%)wasreported withciproﬂoxacin.37_This_increasing_resistance_rate_could_be

explainedbytheuseofﬂuoroquinolonesforurinaryinfections andacrossresistancebetweenthedifferentmoleculesofthis antibioticgroup.

Noamoxicillin,rifampicinandtetracyclinresistancewas observed.Foramoxicillinresistanceﬁnding,itwassimilarto thosedeterminedbyWolleetal.38_in_their_study_in_Japan._In

Europe,availabledatafromtwostudiesenrolling599patients foundaprevalencerate<1%.28_A_high_prevalence_was_reported

InAsia andSouthAmerica(38%).39,40 _Amoxicillin_is_widely

usedinAlgeriabut theabsenceofresistancecould bedue tothecomplexityofamoxicillinresistancemechanisms(pbp

gene mutations, membranepermeability alterations, efﬂux pumps,etc.).Rifampicinresistancestainsare rare;thiswas conﬁrmedintwostudies,showingthepresenceofaresistant strainin22outof1585patientsinGermany(1.4%),41_and_in

17out of255patientsinEngland(6.6%;95%).42 _This

antibi-oticisrarelyusedforH.pylori eradicationtreatmentwhich explainstheabsenceofresistantstrainsinAlgeria.In accor-dance withourstudy,tetracyclineresistancewasabsentin patients from Iceland,23 _and _very _low _in_Congo _(2.5%) _and

inChina(3.9%).22,25_In_contrast,_increased_values_were_found

inChilly(27%).43_{Unfortunately,}_{tetracycline-based}_regimens

requiretheuseofbismuthsaltswhicharenolongeravailable inseveralcountriesduetopossibleside-effects.

ItisevidentnowthatthepresenceofH.pylorivirulence genesandtheirdifferentgenotypiccombinationsinthestrain colonizingapatient,affects thedevelopmentofthe gastric disease.Some characteristicsofH. pylori strainshave been associatedwiththe progressionofinfection tomoresevere disease.Some genotypes,suchasalleles s1 and m1 ofthe

vacA gene and presence ofthe cagA gene, are considered pathogenicitymarkerssincetheyare associatedwith cyto-toxinproductionandtheinductionofmoreintenseepithelial lesionsandinﬂammatoryreactions.ThecagAwasidentiﬁed in58%patients,thisresultishighertothoseobtainedin Pak-istan (24.2%),44_but _lower _than_those_obtained_in_Tunisia_of

61.6%,45_Iran_(76%)_and_Iraq_(71%).46_The_cagA_positive_strains

inaMexicanstudywere86%.47_In_Japan,_the_rate_of_cagA_is_very

high(90%),48_which_is_correlated_with_the_high_prevalence_of

gastriccancerinthatcountry.

ThemajorityofH. pyloriisolatesinourstudy werevacA

positive.Thevirulenceoftheisolatehasstrongrelationwith themosaiccombinationofsandmregionallelictypeswhich is responsible of the production ofthe cytotoxin; the pre-dominantcombinationofvacAallelesinourstudywass1/m1

(59.88%).Theseresultsareinaccordancewithseveralother studies.InBrazil,49_and_in_Mixico,50_vacA_s1/m1_was_the

pre-dominantgenotypeinthesecountriestoo.Numerousstudies haveprovidedevidenceforastrongerassociationofdisease outcomesinindividualsinfectedwithH.pyloristrains possess-ingactivevacAgenotypessuchass1andm1thanthatwithless ornon-activevacAtypes.51,52

ConcerningcagAandvacAallelescombination,wefound thatthepredominantwascagA+/s1m1.Thisﬁndingisin accor-dancewithotherstudiesperformedinLatinAmericasuchus Faundez etal.whofoundthatcagA+/s1m1strainswere the mostcommoninBrazilianpatients53 _and_{Morales-Espinosa}

etal.alsofoundthatcagA+/s1m1strainshadahigher preva-lence in Mexicanpatients.54 _{Additionally,} _this _genotype _is

the mostprevalentinAsianpopulation too.51 _cagA_is_most

commonly associated with the vacA s1 genotype, particu-larlyinpatientswithpepticulcerorgastriccancer.Therefore,

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thecagA+vacAs1genotypeispresentinpatientswithhigh expressionoftumornecrosisfactorreceptor-associated fac-tor1,tumornecrosisfactorreceptorsuperfamilymember9,or B-celllymphoma-extra-large.55_In_our_study,_we_have_selected

patientswithupperabdominalpain,thisexplainourlowrate of58%cagAand59.88%s1/m1whichislowcomparablywith othercountries,theprevalenceofthesegenotypesinpatients withpepticulcer and gastriccancer issigniﬁcantlygreater thanamongthosewithgastritisalone.53

Therelationofvirulencefactorsandtheresistanceresults describesnodirectrole ofvacAor cagAgenes inantibiotic resistancewhich agrees with Ghotaslou et al. results. The

vacA s1m1/cagA+ genotype is associated with more severe gastricdiseasesduetothefactthatstrainswiththis geno-typecausesevereinﬂammationwithincreasedproductionof IL-1␤andTNF-␣,inducinginhibitorsofsecretionof hydrochlo-ricacid andincreasegastric pH;conditions thatmay favor theactionofantibiotics.56_The_{metronidazole}_resistance_had

relation withthe presence ofcagA genotype in our study, thisdatadisagreewiththosereportedbyKhanetal.57_where

theyfoundthattheabsenceofcagAgenecontributesinthe acquisitionofresistance.Apparently,infectedpatientswith

vacA s1m1/cagA+ resistant strains are at increased risk of progressiontomoresevereconditionsbyfailureinH.pylori

eradication.Thus,inthispopulation,infectedpatientswith

vacA s1m1/cagA+ resistant strains are at increased risk of severediseasethaninfectedpatientswithvacAs1m1/cagA− orvacAs2m2/cagA−,however,allmustbecloselymonitored.

InpatientsfromAlgeriawithupperabdominalpain,the prevalenceofH.pyloriis83.5%,67.5%oftheisolateswere resis-tanttometronidazole,25.2%wereresistanttoclarithromycin and18.5%wereresistanttociprofloxacin.Theseratesof resis-tancetometronidazole andclarithromycinare higherthan internationallyacceptedrangesandallowstoppingtriple ther-apyforH.pylorieradication andnewtherapeuticstrategies areneededinAlgeria.Metronidazole,mightwellberefrained fromusinginordertopreventtheemergenceofmoreresistant strainsespeciallybecauseofthehighrateofmetronidazole resistance in Algeria. About clarithromycin, this antibiotic should notbe used, or aclarithromycin susceptibility test shouldbeperformed,beforeprescription.Therefore,itis nec-essarytomonitortheevolutionoffluroquinoloneresistance inthis area. ThevacA s1m1/cagA+ genotype was the most prevalentamongresistantisolates.Therewasnosignificant difference found in resistance with H. pylori genotypes. It is likely that infected patients with resistant strains vacA s1m1/cagA+areatincreasedriskofprogressiontomoresevere conditionsbyfailureinH.pylorieradication.Thesedataareof agreatimportancefortheregionalhealthsystemsofAlgeria.

Ethics

Thiswork isregistered inanational researchproject enti-tled:Universityresearch project(CNEPRU),Financedbythe AlgerianResearchMinistryandhasthisfollowingregistration code:D01N01UN020120150001.Thisstudy wasapprovedby theethicscommitteesofallhospitalswheretheendoscopy wasperformed and all patients providedwritten informed consentbeforeendoscopy.

Funding

This work was ﬁnanced by “Laboratoirede bioressources”, DepartmentofBiology,FacultyofSciences,HassibaBenBouali University,02000Chlef,Algeria.

InAlgeria,allresearchprojectsareﬁnancedbythe Alge-rianResearchMinistrythroughresearchlaboratoriesineach University.ProjectgrantnumberD01N01UN020120150001.

Authors’

contributions

Conceived and designed the experiments: MB, RA, AT and MM. Performed the experiments: MB, AT, AED and MM. Analyzed the data: MB, AT and MD. Contributed reagents/materials/analysistools: MB,MM and KTD. Wrote the paper:MB,RA.All authorsreadand approvedthe ﬁnal manuscript.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

WethankDrSamirHalouche(CliniquedeGastroenterology, 02000Chlef,Algeia)andPrBousria(FirstNovember1954 Uni-versityHospitol,Oran,Algeria)fortheirtechnicalassistance, and alsothecliniciansinvolvedinthis study.WethankDr HaceneMahmoudiandDrAhmadAichounifortheirmoral support.

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