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Vol.
3,No.
8,529-533(2011)Healthdoi:10.
4236/health.
2011.
38088Copyright2011SciRes.
Openlyaccessibleathttp://www.
scirp.
org/journal/HEALTH/ITSandpB2.
5geneexpressionofNaegleriafowleriindrugresistanceJundeeRabablert1,SupathraTiewcharoen2*,VirachJunnu21DepartmentofBiology,FacultyofScience,SilpakornUniversity,NakhonPathom,Thailand;2DepartmentofParasitology,FacultyofMedicineSirirajHospital,MahidolUniversity,Bangkok,Thailand;*CorrespondingAuthor:sistc@mahidol.
ac.
thReceived8May2011;revised22June2011;accepted13July2011.
ABSTRACTNaegleriafowleriwascausativeagentofpri-maryamoebicmeningoencephalitis(PAM).
Ac-crodingtothefailureoftreatment,severalre-searchesreportedtheactivityofchemothera-peuticdrugsagainstN.
fowleributwedidnotknowthedrugresistanceoftheamoebae.
Thepurposeofthisstudywastoexaminetheef-fectsofdrugs(amphotericinB,artesunate,azi-thromycin,voriconazole,chlorpromazine,fluco-nazoleandgentamicinsulphate)onITSandpB2.
3genesofNaegleriafowleritrophozoites.
OurstudydemonstratedgeneexpressionoftreatedN.
fowleribyRT-PCR.
Theresultsre-viewedthatITSgeneofN.
fowlerishowedupregulatetoamphotericinB,azithromycinandgentamicinsulphate,whilepB2.
3geneshowedupregulatetoartesunate.
Theseresultscom-paredwithbetaactin(housekeepinggene)ex-pressionattimeintervals15-120min.
Thecha-ngeofgeneexpressionoftreatedN.
fowleriwaspossiblytocauseofdrugresistance.
Theme-chanismofdrugresistancegenesITSandpB2.
3ofN.
fowlerishouldbeclarifiedinfurtherstudy.
Keywords:NaegleriaFowleri;ITS;pB2.
3;DrugResistance1.
INTRODUCTIONNaegleriafowlericausesseveremeningoencephalitismainlyinchildrenandyoungadults.
Duetothetreat-mentshavenotbeensucceeded,mostofpatientsdiefromN.
fowleriinfection[1].
TheeffectofdrugagainstN.
fowlerihasbeencarriedoutbothinvitroandinvivostudieswhichprovidedforclinicaltrendsfortreatment[2].
Apreludeofdrugresistanthasbeenfrequentlydocumentedinrecentyears[3].
SeveralresearchesonantifungalresistanthavebeenfocusedonelucidatingthemolecularbasisandtranscriptionalregulationofazoleinCandidaspp.
[4].
TheresistancetoazoleuptakeofC.
albicanscanbeachievedwiththeintroductionofkeypointmutationinand/orupregulationofgeneexpres-sionwhichencodesoneffluxpump;EGR11,MDRin-cludingATP-bindingcassettetransportermoleculesCDR2[5].
Inaddition,antifungalresistantwasinvolvedsteroluptakewhichwascontroledbyUPC2gene[6].
OwingtofailuretreatmentofPAM,oneofthemajorproblemswasdrugresistantfromgenealteration.
Uptodate,astudyhasbeenaddressedtheintrinsicresistantonnfa1andMp2Cl5geneswhichregulatedpathogenesisoftheamoebae.
Theresultsdemonstratedthateithernfa1re-sistanttofluconazoleorMp2Cl5resistanttoamphote-ricinB,azithromycinandartesunateofN.
fowleriwerefound[7].
OwingtothedominantITS,locatedinthe5.
8SrRNAgeneandspecies-specificchromosomalDNApB2.
3geneswereusedforidentifypathogenicN.
fowleri[8]anddiversityofN.
fowleriatmolecularlevel[9],weinvestigatedtheactivityofdrugsonITSandpB2.
3genesofN.
fowleri.
ThisreportrevealedtheabilityofNaegle-riagenesagainstdrugsofchoice.
2.
MATERIALSANDMETHODS2.
1.
NaegleriaFowleriCultivationFreelivingN.
fowleritrophozoites(Khon-Kaenstrain)wereculturedinNelson'smediumsupplementedwith5%heat-inactivatedfetalcalfserum(FCS)withoutanti-bioticsat37C.
Trophozoitesweretestedwiththecon-centrationofamphortericinB,voriconazole,fluconazole,chlorpromazine,artesunate,azithromycinandgentami-cinatIC50[10]during15-120min,triplicate.
Untreatedtrophozoitewasusedfornegativecontrol.
Atindicatedtimes,trophozoitesweretwicewashedwithnormalsa-lineandfrozenat–80Cuntilrequired.
2.
2.
RNAExtractionTotalRNAwasextractedfromuntreatedortreatedJ.
Rabablertetal.
/Health3(2011)529-533Copyright2011SciRes.
Openlyaccessibleathttp://www.
scirp.
org/journal/HEALTH/530amoebaetrophozoitesusingTriReagent(Sigma-Aldrich,USA).
Forpositivecontrol,nfactingene(housekeepinggene)ofN.
fowleriwasconfirmedbyprimer5′-ACTCTGGTGATGGTGTCTCTCACAC-3′and5′-CTCTGACAATTTCTCTCTCAGTGG-3′.
TheampliconsofamoebaewerepreparedfromprimersofITS(ITS1;5′-GAACCTGCGTAGGGATCATTT-3′andITS2;5′-TTTCTTTTCCTCCCCTTATTA-3′)andpB2.
3(p3f;5′-GTGAAAACCTTTTTTCCATTTACA-3′)andp3r;5′-AAATAAAAATTACCATTTGAAA-3′)byone–stepSuper-scriptPCR(Invitrogen,Gransland).
MWofeachampliconwasdetectedby1.
5%AgaroseGelElectro-phoresisat100Vfor30min.
3.
RESULTSANDDISCUSSIONInourstudieswedemonstratedtheresponsibilityofN.
fowleritotheeffectsofdrugs(amphotericinB,artesu-nate,azithromycin,voriconazole,chlorpromazine,flu-conazoleandgentamicinsulphate)onITSandofpB2.
3genebyRT-PCR.
ThenumberofN.
fowleritreatedoruntreatedN.
fowleriwasnotsignificantdifferentbe-tweentwogroupsattimeintervals(theresultswasnotshown)(t<0.
005).
TotalRNAwasextractedfromthistwogroupsusingTriReagent(Sigma-Aldrich,USA)atindicatedtime.
Asapositivecontrol,weusednfactintoamplifyunderthesameRT-PCRconditionandregula-tionofexpressionofnfactingenewasdetectedat170bpat15-120min.
Theupregulateofnfactinwascom-paredwithtreatedoruntreatedamoebaeateverypointoftime.
WefoundthatuntreatedN.
fowlerishowedupregulateofITSgeneat450whileITSgeneexpressionfromtreatedtrophozoiteswithvoriconazole,fluconazoleorchlorpromazinewasnotobservedduring120min.
Incontrast,trophozoitestreatedwithamphotericinBwasfoundatleast30minwhereastrophozoitestreatedwithazithromycinorgentamicinwasshownatleast45min(Figure1).
Similarly,wetestedthedrugsactivityagainstpB2.
3geneexpressionofamoebaetrophozoites.
Theuntreatedtrophozoitesshowedbrightbandfragmentat310bpasshowninFigure2.
Interesting,wedidnotobservepB2.
3geneexpressionfromtreatedtrophozoiteswithapanelofdrugsat120min,exceptartesunate.
ItispossiblysuggestedthatITSgeneofN.
fowleritropho-zoitewasresistedtoamphotericinB,azithromycinorgentamicinincludingthepB2.
3genewasresistedtoar-tesunate.
Figure1.
EffectofdrugsonITSgeneofN.
fowleribyRT-PCRat15,30,45,60and120min(a1-e1)comparedwiththepositiveexpressioncontrol,nfactingeneatthesametime(a2-e2).
UntreatedN.
fowlerishowedbrightbandfragmentat450bp(lane1).
TreatedN.
fowleriwithamphotericinB(lane2),voriconazole(lane3),fluconazole(lane4),chlorpromazine(lane5),artesunate(lane6),azithro-mycin(lane7),andgentamicinsulphate(lane8),respectively.
J.
Rabablertetal.
/Health3(2011)529-533Copyright2011SciRes.
Openlyaccessibleathttp://www.
scirp.
org/journal/HEALTH/531Figure2.
EffectofdrugsonpB2.
3geneofN.
fowleribyRT-PCRat15,30,45,60and120min(f1-j1)com-paredwiththepositiveexpressioncontrol,nfactingene(170)atthesametime(f2-j2).
UntreatedN.
fowlerishowedbrightbandfragmentat310bp(lane1).
TreatedN.
fowleriwithamphotericinB(lane2),voriconazole(lane3),fluconazole(lane4),chlorpromazine(lane5),artesunate(lane6),azithromycin(lane7),andgen-tamicinsulphate(lane8),respectively.
AmphotericinBhasbeengenerallyrecognizedforfungalandprotozoatreatment,manypublicationsre-portedamphotericinB-resistantgenesinCandidalusita-niae,Saccharomycescerevisiae[11]andCryptococcusneoformans[12].
However,ithasbeenreportedinpath-ogenicprotozoa;Entamoebahistolytica,Giardialam-blia,Trichomonasvaginalisandalsooccurredanaerobicprotozoa;Blastocystishominis,Cryptosporidiumparvum,Isosporaspp.
,Cyclosporssporidiaspp.
[13].
Inaddition,amphotericinBresistantgenewasalsofoundinvectorbornprotozoa,Leishmaniatarentolae[14].
ThemechanismofamphotericinB,polyeneresistantofC.
albicansandS.
cerevisiaecausedbymutationofEGRgeneswhichcontroltheproductionofergosterolandsensitivitytopolyenes.
AstheresultofEGR6mu-tantstrainofC.
lusitaniaetranscription,thereducedergosterolcontentwasappeared[15].
AstudyofEGR6mutantcausedunabletoformamphotericinB-generatedporesinthecellmembraneonCandidaspp[16].
OurstudyrevealedamphotericinBresistantITSgeneofN.
fowleriwasfirstlyappeared.
Moreover,drugresistant;azithromycin,gentamicinsulphatetoITSandartesunatetopB2.
3ofN.
fowleriwerealsodemonstrated.
AfewpublicationsreportedtheazithromycinresistantinPseu-domonasaeruginosa[17]andgeneticmutationat23SrRNAregionofUreaplasmaurealyticumandNeisseriagonorrhoeae[18].
Generesistant,ermG,toazithromy-cinwasestablishedinBacteroides.
Mechanismofazith-romycinresistantestablishedatMexCD-OprJpumpofPseudomonasaeruginosabiofims[19].
Gentamicinhasbeengeneralusedincultivation;En-terococcusfaecali[20],Pseudomonasaeruginosa[21].
Theoccurrenceofgentamicin-resistantgenesofgramnegativebacteria;Enterobacteriaceae,Pseudomonas,Acinetobacterwereisoloatedfromdifferentenvironmentmainlyoriginatingfromsewage,faces,coastalwaterJ.
Rabablertetal.
/Health3(2011)529-533Copyright2011SciRes.
Openlyaccessibleathttp://www.
scirp.
org/journal/HEALTH/532pollutedwithwastewaterandthedistributionoftheseresistantgenescouldbroadlytransfertohosts[22].
Gentamicin-resistanttoN.
fowlerihasbeenfoundinvitrosince2009[10].
Thisstudyshowedthatgentamicinresistantconcernedbothgeneexpressionandaminoglycosideactivity[20].
Artesunatewasusedtotreatmentforfalciparummalariaduetoartesunateaccumulatedlipidsbodiesandinducedoxidativemembranedamage[23].
Furthermore,itblockedproteinsynthesisofyeastcells[24]andinhibitedrepli-cationofcytomegalovirus[25].
Accordingtogeneraluseofartesunate,theartemisininresistancetomalariawasfoundinclinicaltrial[26].
Theresistancegenemdr1,cg10,tctp,andatp6toartemisininofPlasmodiumcha-baudichabaudiweredevelopedandtransmittedtoitsderivatives[27].
ThepB2.
3resistantgeneofN.
fowleriwasappeared,therebyitlocatedinmitochondriaandchromosomeoftheamoebae.
Inconclusion,oneofthedrugtreatmentfailurefocusedonITSandpB2.
3genesofN.
fowleri.
4.
ACKNOWLEDGEMENTSThisworkwaspartiallysuppotedby:grantNumberRGP2552/02fromDepartmentofBiology,FacultyofScience,SilpakornUniversityatSanamchanPalace,NakhonPathom,Thailand.
WethanktheheadofDepartmentParasitology,FacultyofMedicineSirirajhospital,MahidolUniversityforprovidedtheresearchfacility.
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