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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
caBioresourcesLaboratory,DepartmentofBiology,FacultyofNaturalandLifeSciences,HassibaBenBoualiUniversityofChlef(UHBC),
Chlef,Algeria
bLaboratoryofMolecularMicrobiology,ProteomicsandHealth,DepartmentofBiology,FacultyofNaturalandLifeSciences,Universityof
DjillaliLiabes(UDL),Sidi-Bel-Abbes,Algeria
cDepartmentofGastroenterology,UniversityHospitalHassaniAbedelkader,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).
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Helicobacterpylori(H.pylori)isagramnegativespiralchapped bacterium, described in 1982 by Marshal and Warren. It
∗ Correspondingauthorat:DepartmentofBiology,FacultyofNaturalandLifeSciences,HassibaBenBoualiUniversityofChlef,Laboratoire
debioressources,02000Chlef,Algeria. E-mail:m.bachir@univ-chlef.dz(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
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
peopleinthe developingcountries, andalsoremainsa sig-nificantpathogeninindustrializedcountries.1InAlgeria,its
prevalenceisaverage80%inpepticulcer.2Fortunately,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 Itis effective for79–85% 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.3Inanother
way,resultsofsomestudieshavesuggestedthatthe eradi-cationrate inpatients withgastritisislower thaninthose withpepticulcerdiseases.6Sincetheworldwideincreaseof
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◦CinBHIbrothwith20%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.11Mueller–Hintonagarsupplementedwith7%
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 25Lreaction mixture consisted of1× PCR buffer, 1.5mM magnesium chloride,200Mofeach 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
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.The10Lreactionmixture con-sistedof1×PCRbuffer,1.5mMmagnesiumchloride,200M dNTP,0.5pmolofeachprimer,0.5Uhotstarttaqpolymerase (Promega)and2LoftemplateDNA.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.The20Lreactionmixtureconsistedof1×PCR buffer,1.5mMmagnesiumchloride,400MdNTP,0.5pmolof eachprimer,1Uhotstarttaqpolymerase(Promega)and4L 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
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.16TheprevalenceofH.pyloridiffers
sig-nificantlybetweencountries,withhighratesofinfectionbeing associatedwithlowsocioeconomicstatusandhighdensities ofliving.Forinstance,inJapan,SouthAmerica,Turkeyand Pakistan,theprevalenceismorethan80%,whileinEnglandit islower(20%).17H.pyloriwasdetectedin167(83.5%)ofatotal
of200biopsyspecimensbythePCRmethod.Ourfindingsare similartootherreportsinIran.18Thisrateishigherthanthe
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.19OneoftheadvantagesofusingtheglmM
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.
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%formetronidazolein2013.3Thereisanincreasein
therateofclarithromycinresistancewith25.2%andthesame formetronidazol(67.5%)foundinourpresentstudy. Concern-ingclarithromycinprimaryresistance,ourprevalencewould seem higher than that foundinCongo (1.7%),22 inIceland
(9%)23 and Tunisia(14.6).24 Thisrate wasalmost similar to
thosefoundinChina(28.9%),25 andinSpain(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%)28,29andinIceland(1%),23butwassimilarto
theratefoundinChina(63.8%)25andlowerthanthatfoundin
Columbia(82%).30Wefoundahigherprimarymetronidazole
resistanceinfemalepatientscomparedwithmaleones,most likelyduetothewideuseofthisantibioticforgynecological infections.Inaccordancewithdatapresentedby Glupczyn-skietal.andBatnavalaetal.,metronidazoleresistantisolates were found altogether morefrequently infemales than in malesandinnonEuropeannatives.31,32 Koletzkoetal.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,34and28%
inChina.25InasingleItalianstudy,35levofloxacinresistance
washigherwithold(>45years)thanyoungpatients(28.4%vs. 14.4%).InKoreatheresistancerateofH.pyloritociprofloxacin wasreportedtobe(33.8%),36thisdataagreewithourresults.
ButinSwedenalowrateofresistance(3.3%)wasreported withciprofloxacin.37Thisincreasingresistanceratecouldbe
explainedbytheuseoffluoroquinolonesforurinaryinfections andacrossresistancebetweenthedifferentmoleculesofthis antibioticgroup.
Noamoxicillin,rifampicinandtetracyclinresistancewas observed.Foramoxicillinresistancefinding,itwassimilarto thosedeterminedbyWolleetal.38intheirstudyinJapan.In
Europe,availabledatafromtwostudiesenrolling599patients foundaprevalencerate<1%.28Ahighprevalencewasreported
InAsia andSouthAmerica(38%).39,40 Amoxicilliniswidely
usedinAlgeriabut theabsenceofresistancecould bedue tothecomplexityofamoxicillinresistancemechanisms(pbp
gene mutations, membranepermeability alterations, efflux pumps,etc.).Rifampicinresistancestainsare rare;thiswas confirmedintwostudies,showingthepresenceofaresistant strainin22outof1585patientsinGermany(1.4%),41andin
17out of255patientsinEngland(6.6%;95%).42 This
antibi-oticisrarelyusedforH.pylori eradicationtreatmentwhich explainstheabsenceofresistantstrainsinAlgeria.In accor-dance withourstudy,tetracyclineresistancewasabsentin patients from Iceland,23 and very low inCongo (2.5%) and
inChina(3.9%).22,25Incontrast,increasedvalueswerefound
inChilly(27%).43Unfortunately,tetracycline-basedregimens
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%),44but lower thanthoseobtainedinTunisiaof
61.6%,45Iran(76%)andIraq(71%).46ThecagApositivestrains
inaMexicanstudywere86%.47InJapan,therateofcagAisvery
high(90%),48whichiscorrelatedwiththehighprevalenceof
gastriccancerinthatcountry.
ThemajorityofH. pyloriisolatesinourstudy werevacA
positive.Thevirulenceoftheisolatehasstrongrelationwith themosaiccombinationofsandmregionallelictypeswhich is responsible of the production ofthe cytotoxin; the pre-dominantcombinationofvacAallelesinourstudywass1/m1
(59.88%).Theseresultsareinaccordancewithseveralother studies.InBrazil,49andinMixico,50vacAs1/m1wasthe
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 andMorales-Espinosa
etal.alsofoundthatcagA+/s1m1strainshadahigher preva-lence in Mexicanpatients.54 Additionally, this genotype is
the mostprevalentinAsianpopulation too.51 cagAismost
commonly associated with the vacA s1 genotype, particu-larlyinpatientswithpepticulcerorgastriccancer.Therefore,
thecagA+vacAs1genotypeispresentinpatientswithhigh expressionoftumornecrosisfactorreceptor-associated fac-tor1,tumornecrosisfactorreceptorsuperfamilymember9,or B-celllymphoma-extra-large.55Inourstudy,wehaveselected
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-1andTNF-␣,inducinginhibitorsofsecretionof hydrochlo-ricacid andincreasegastric pH;conditions thatmay favor theactionofantibiotics.56Themetronidazoleresistancehad
relation withthe presence ofcagA genotype in our study, thisdatadisagreewiththosereportedbyKhanetal.57where
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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