Allovahlkampfia Spelaeais a Potential Environmental Host for Pathogenic Bacteria

Mohamed et al., J Bacteriol Parasitol 2016, 7:1

Allovahlkampfia spelaeais a Potential Environmental Host for Pathogenic Bacteria

Mona EmbarekMohamed1*,Enas AbdelhameedMahmoudHuseein2, HaiamMohamedFarrag2, FatmaAbdelAziz Mostafa3and AlaaThabetHassan4

1Department of Microbiology andImmunology, Faculty of Medicine,Assiut University, Assiut, Egypt

2Department of Parasitology, Facultyof Medicine, AssiutUniversity, Assiut, Egypt3Department of Botany andMicrobiology, Faculty of Science,Cairo University, Giza, Egypt4Department of ChestDiseases, Assiut UniversityHospitals, Assiut, Egypt

*Correspondingauthor:MonaEmbarekMohamed,DepartmentofMicrobiologyandImmunology,FacultyofMedicine,AssiutUniversity,71515Assiut,Egypt,Tel:

+20-882413500/2411899;Fax:+20882333327,E-mail:

Received date: November 19, 2015; Accepted date: December 28, 2015; Published date:January 04, 2016

Copyright:©2016MohamedME,etal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords:Allovahlkampiaspelaea;Amoeba-bacteriainteractions;Co-cultureexperiments;Gramnegativebacteria;Intracellularsurvival

Introduction

Allovahlkampiaspelaea(A.spelaea)belongstothegenusAllovahlkampiain thefamily Vahlkampiidae,classHeterolobosea,andPhylumPercolozoa[1]thatwasidentiiedfortheirsttimein2009[2].A.spelaeaisafreelivingamoeba(FLA)andhasbeenfoundtobeassociatedwithkeratitisasevidentinaresearchconductedbyTolbaetal.FLAisubiquitousinnature[3]andsomeofthemproduceserioushumaninfections[4].Amoebainnaturemayhavecontactwithothermicroorganismsincludingbacteria.Amoebaearethedominantbacterialconsumers,contributingtorecyclingofnutrientsandmaintainingthestructureofthemicrobialcommunity[5].MostFLAgeneraarecharacterizedbyabiphasiclifecycleconsistingofavegetativetrophozoitestageandaphysiologicallystaticcyststage[2].Cystsarehighlyresistantandremainviable(andinfective)forseveralyearswhichfacilitatesspreadingandcolonizationof newecologicalniches[6].Ontheotherhand,bacteriahavedevelopedseveralantipredatorstrategiesincludingcellsizereduction,modiiedcellmorphology,modiicationofcellwallcharacteristics,high-speedmotility,bioilmormicrocolonyformation,andproductionofexopolymersortoxins[7].Inthiscase,amoebaemayactasaprotectivehostforsomebacterialpathogensagainstharshenvironmentalconditionsthatnormallykill.heroleofFLAinsurvival and protection of pathogenic bacteria is increasingly

recognized[8].heamoebaeaidinbacterialtransmissiontosusceptiblehoststhusconstitutesaproblemtotheecosystemhealth[4].Additionally,bacteriabecomemoreresistanttodisinfectants[5,8,9].ManymicroorganismsareknowntobehostedbyFLAincluding;Acinetobacterspp.,Aeromonasspp.,Enterobacterspp.,Escherichiacoli(E.coli),Klebsiellapneumonia(Kl.pneumonia),Pseudomonasaeruginosa(Ps.aeruginosa),Salmonellaspp.,andStaphylococcusaureus(Staph.aureus)[10].hehypothesisinourstudywasthatA.spelaeamayplayaroleforsurvivalandmultiplicationofbacterialpathogens.

MaterialsandMethods

MolecularcharacterizationofA.spelaeathatwasconductedattheDepartmentofMedicalGenomics,GraduateSchoolofFrontierSciences,heUniversityofTokyo,Japan.

CultureandmolecularcharacterizationofA.spelaea

A.spelaeausedinthisstudywasobtainedfromapatientwithkeratitis.A.spelaeawasisolatedon1.5%non-nutrientagarmadewithPage’ssaline(PAS)andseededwithE.colikeptat30°Cfor7days.CultureswereexaminedusinginvertedmicroscopeforpresenceofFLA andsubculturedevery 10to 14days byinvertinga sliceon a newagarplateasdescribedpreviously[11].hemorphologyofthetrophozoiteandcysts(non-stainedandGiemsa’sStained)wereidentiiedusinglightmicroscopeandinvertedmicroscopeaccordingto Smirnov and Goodkov [12]. Molecular characterization of A.

spelaeabypolymerasechainreaction(PCR)ofthe18SribosomalRNAandsequencingwasperformed usingprimersdescribedpreviously[11,13-15].

Bacterialculturesandantibioticsusceptibilitytests

Bacterialstrainsusedinthisstudywereisolatedfromcaseswithlowerrespiratorytractandurinarytractinfections.hetestedstrainswereMethicillin-resistantStaph.aureus(MRSA),Enterobacteriaceae[E.coli1,Kl.pneumoniae,Enterobacteraerogenes(E.aerogenes),Citrobactercloaca(C. cloacae),Proteus mirabilis(Prmirabilis),Raoultellaterrigena(R.terrigena),Raoultellaornitholytica(R.ornitholytica)],andotherGramnegativebacteria;Aeromonashydrophila(A.hydrophila)andPs.aeruginosa.GramnegativebacteriawereidentiieduptothespecieslevelbyAPI20Esystem(BioMérieux,France)whiledetectionofMRSAbasedoncolonial morphology,Gramstaining,andstandardbiochemicalreactionsaccordingtotheBergey'sManualofSystematicBacteriology[16].OurGramnegativebacteriawereallsensitivetoimipenemandmeropenem(Oxoid,England)whileMRSAweresensitivetolinezolid.SusceptibilitytestswereperformedusingthediskdifusionmethodasrecommendedbytheClinicalandLaboratoryStandardsInstitute(CLSI)guidelines[17].Bacteriawereculturedinbrainheartinfusionbrothovernightat37°Cwithoutshakingpriortoexperimentationandwereusedatthestationarygrowthphase.

Co-culture experiments

A.spelaeawasgrownwithoutshakingin15mlPYGmedium(0.75

%,w/v,proteosepeptone;0.75%,w/v,yeastextract;1.5%,w/v,glucose)intissueculturelasksat30°C,asdescribedpreviously[18].hemediumbeingrefreshed17-20hpriortoallexperimentation.hisresultedinmorethan95%oftheamoebaeinthetrophozoiteform.

SupernatantsfromA.spelaeacultureswerecentrifugedat2800×gfor30min.NewpelletswereresuspendedinPYGbeforebeingprocessedtorecovercultivablebacteria.Co-cultureexperimentswereperformedwithaslightmodiicationtoapreviousmethod[19].A.spelaeawasincubatedinaconcentrationof1×106amoebae/mLPYGmedium/wellin24wellplatesuntilconluent.hecellswerewashedoncewithPAS.Next,Diferentbacterialstrainswereaddedinaconcentrationof1×107colonyformingunits(c.f.u)/well/mLPYGgivingamultiplicity

ofinfection(MOI)of 10. PHwasadjustedto 7.2andtheplates wereincubatedfor1hat30°Ctopermitbacterialuptake.Tokillresidualextracellularbacteria,mediumwasreplacedwithPYGsupplementedwith16mg/Limipenem,aconcentrationgreaterthanthehighestMICobservedforallGram-negativestrainsandlinezolidinconcentrationof1.5µg/mlwasusedforMRSA.Plateswereincubatedfor1hat30°C.Antibioticswereremovedbywashingthree-timeswithPAS.Attheinalwash,thediscardedsupernatantswerealsoplatedontonutrientagarplatestodeterminebacterialpresenceand100μlfreshPASwereaddedtowells.hemicrotiterplateswereincubatedagainat30°C(designatedtime0h).hewellswereprocessedat0h,8h,24h,48h,and72h.Tocounttheextracellularbacteria,PASwascarefullyaspiratedandsampled.Todeterminethenumberofintracellularbacteria,100μloffreshPASwereaddedtothewellsandthesurfaceofeachwellbottomwasscrapedtoremoveadherentcells.Finally,amoebaewerelysedbyaddingsodiumdodecylsulphate(SDS)in0.5%inalconcentrationtoeachwellfor20min,andthenumberofbacteriawasenumeratedbyplatingonnutrientagarplates[20].A.spelaeaviabilitywasmonitoredusingtheeosindyeexclusionassayusinglightandinvertedmicroscopeaccordingtoWangandAhearn[21].

Statisticalanalysis

heSPSSprogramversion20.0wasusedforthestatisticalanalysisofdata.Datawerepresentedasnumberandpercentage,ormean±SDasappropriate.ANOVAtestwasusedbeforedataweretransformed(Log10),p value<0.05wasconsidered statisticallysigniicant.

ResultsandDiscussion

Asnewlydiscoveredin2009inthekarstcavesofSlovenia,dataon

A.spelaea(Figure1)andtheirinteractionswithbacteriaarelacking.ForidentiicationandcharacterizationofA. spelaea, we dependedupon18SrRNAgenesequencingthatrevealedourstraintobeA.spelaeastrainSK1.BeingamemberofFLA,theroleofA.spelaeainsurvivalandmultiplicationofpathogenicbacteriashouldbeconsidered.So,weaimedinthisstudytoinvestigatesurvivaland/ormultiplicationofthetestedbacteriainsideamoebacells.Ourbacterialstrainswereisolatedfromcasesofurinaryandrespiratorytractinfectionsandthemajorityofthemareknowntobenaturalcontaminants of thewater and foodsystems.

Co-cultureofA.spelaeaandbacteria

Inourco-culturesystem,wefollowedthesurvivalofbacteriainside

A.spelaeaat30°C.BacteriaalonewereincubatedwithvariousconcentrationsofSDS,anditwasfoundthat0.5%SDShadnoefectonbacterialviability.

BacterialsurvivalandmultiplicationinsideA.spelaea

Figure2showstheintracellularbacterialcountsinthepresenceof

A.spelaea.WiththeexceptionofPr.mirabilisthatshowedlowcountsthroughouttheexperiment,ourresultsshowedthatpathogenicbacteriasurvivedandmultipliedwithintheamoebahost.Inparticular,Ps.aeruginosaandA.hydrophilawherethecellcountsexceeded5logcyclesattime0hwithhighlysigniicantdiferencesversusotherbacterialstrains(P<0.001forboth)andincreasedsigniicantlyinnumbertoreach>8and>7logcycleattime24hofco-incubation,respectivelyincomparisontootherisolates(p<0.001forboth).hehighmultiplicationrateofA.hydrophilainsideFLAinourstudyhasbeendemonstratedbefore[22,23].Ps.aeruginosaisanenvironmentalGram-negativebacillusthatcolonizeshospitalwatersystemsandcausesnosocomialinfections[24].Additionally,Ps.aeruginosa-amoebaco-infectionshavebeendescribedinkeratitispatients[25].OurA.spelaeawasisolatedfromapatientwithkeratitis,sotheinteractionsbetweenA.spelaeaandPs.aeruginosaareofspecialconcerninthosepatients.heisolationofFLAnaturallyinfectedwithPs.aeruginosa[5,26-29]demonstratedtheroleofamoebaeandtheircystsasvectorsfortheseintracellularbacteria[30].OurresultsshowedtheintracellularmultiplicationofPs.aeruginosaassupportedpreviously[20,31,32].Incontrasttoourindings,anotherreport[33]supportedtheextracellularmultiplicationmodeofPs.aeruginosawithbettergrowthoutsidethaninsideeukaryoticcells.Inourwork,MRSAintracellularcountswere>4logcycleattime0hthatsigniicantlydiferedfromotherbacteria(p<0.001)andincreasedby>1logcycleattime24hofco-incubationthatdiferedfromPs.aeruginosaandA.hydrophila(p<0.001)butshowednosigniicantdiferences(p0.05)againsttheEnterobacteriaceaegroup.Huwsetal.[34]demonstratedtheproliferationofepidemicstrainsofMRSAinsideFLA.OurEnterobacteriaceaegroupstartedwith~1logcyclegrowthattime0hthatincreasedupto4logcycleforC.cloacaeandE.coli1,and>2logcycleforKl.pneumoniae,E.aerogenes,R.terrigena,andR.ornitholytica. Previous data demonstratedthatEnterobacteriaceaecansurviveandmultiplywithinamoebahost[23,35-39]whichisconsistentwithourindings.At48hco-incubation,theintracellularcountsforallbacteriainourstudydecreasedonwards.hedecreaseater2-3daysofincubation,hasbeenreportedpreviously[19,40]whichmaybeattributedtothelimitedintracellularlifeofbacteria,orthepresenceofviablenotcultivablecells[41].Inourwork,theintracellularviablecountofPr.mirabilisshowedsigniicantlythehighestlevelsattime0h(p=0.045,0.021,0.01,and0.005versustime8h,24h,48h,and72h,respectively).AsthecomparativecountsofPr.mirabilisinthepresenceofamoebaewerelower,then,thiswasevidenceofpredationbyA.spelaea.

Extracellularbacterialcounts

heextracellularbacterialcountsinourwork,asshowninigure2,occurredasaresultofintracellularmultiplicationandsubsequentreleaseofvesiclescontaininglivebacteriaasreported[8,42].heywerecharacterizedbygradualincreasefromtime0hto8hco-incubation(p≤0.001)untilsigniicantlymaximumextracellularcountsattime24h co-incubation (p0.001). henbacterial counts decreased

signiicantlytoveryloworundetectedlevelsat72hco-incubation(p>0.05versustime0h).Signiicantmaximumextracellularviablecountsat24hco-incubationweredetectedforbacteriathatshowedthehighestgrowthintracellulary;Ps.aeruginosaandA.hydrophila(p<0.001forbothversusotherbacteriatested).Pr.mirabilisextracellularcountsreachedverylowlevelswith insigniicantdiferencesbetweentime8hco-incubationuntiltheendofexperiment(p>0.05).here arevaryingaccountsintheliteratureonthetypesofinteractionbetweenbacteriaandFLAwithdiferenttypesofendocytosisandintracellularbehaviorsincludingintracellularlysisofbacteria,followedbyitsdigestionbyamoebaeorintracellularsurvivalandmultiplicationofbacterialeadingtoamoeballysiswhichmaybedependentonvirulenceofbacteria[43].Asshowninourwork,thenon-invasivebacteriaaretakenupbyamoebaasafoodsource(inourcasePr.mirabilis),whiletheinvasivebacteriaareabletoresideandmultiplyinsideamoebaewithoutbeingkilled(inourcasetheotherbacteria)[35],wheretheyuseamoebaeasatransmissionvehicleanddevelopresistanceagainstotherphagocyticcellsinhosttissues[4,44].Nevertheless,theprecisemechanismsofintracellularsurvivalofourtestedbacteriaremainunclearandhavetobedetermined.AlthoughapreviousreporthasdemonstratedtheabilityofbacteriatoinhibitthefusionoflysosomeswithphagosomesasacriticalstepintheintracellularsurvivalinsideAcanthamoebacastellanii(A.castellanii)[45],so,ourbacterialstrainsmayusesimilarmechanismstoevadetheamoeba-celldefenses.Otherreports[34,40,46]thatdemonstratedthemultiplicationofStaph.aureuswithinFLA,suggestedthatStaph.aureuspossessnospeciicmechanismforevadingdigestionbuthavepost-ingestiondefensessuchasathickercellwall,oranantioxidantyellowcarotenoid. Pickup etal.[46] stated that Kl.pneumonaehastheability to resistphagocytosis and digestionasa resultofpolysaccharidecapsule.heabilityofmanybacterialpathogenstosurviveintracellularly inA.spelaeamaybeakeystep intheevolutionofthosebacteriatoproducehumanandanimalinfections.

hesurvivalrateofA.spelaeainthepresenceofbacteria

NumberofviableA.spelaeainabsenceofbacteriaincreasedfrom1×106cell/mL(100%)ontime0hto~0.96×107cells/mL(110%)and1.88×107(119%)cells/mLon24hand48h,respectively,andthensurvivedto~0.8×106cell/mL(82%)at72h(Figure3).GrowthofcocultivatedA.spelaea,exceptwithPr.mirabilis,wasinhibitedwithvaryingdegrees.hestatisticalanalysisshowedhighlysigniicantdiferencesinsurvivalratesofalone-cultivatedcomparedtothecocultivatedA.spelaea(p<0.001for24hand48h,andp<0.002for72hexceptforR. ornitholyticaandE.aerogenes where p=0.097forboth).

A.spelaeagrowthat24hco-incubationwashighlysigniicantlyafectedby Ps.aeruginosaandA.hydrophilawherep were<0.008and

<0.01versusotherbacterialstrains,respectively.Inthiscontext,itiswellknownthattheinvasivepropertyofPs.aeruginosatargettheamoebawiththeirtoxinsthatcausecelllysis[33,47].A.spelaeagrowthwas signiicantly enhanced by Pr. mirabilis (p<0.002, <0.003, and

<0.003for24h,48h,and72hco-incubationversusotherbacterialstrains,respectively).hedecreaseinsurvivalratesofA.spelaeawhenco-incubatedwithourbacterialstrainssuggeststhatamoeballysisoccurredasaresultofbacterialmultiplication.Nevertheless,bacterialgrowthdidnotresultintotalkillingoftheamoebalcellsthatsurviveduntilendoftheexperimentat72h,whichsuggeststheadaptationofourbacteriatotheintracellularenvironmentwithoutcausingtotalprotozoallysisasreportedpreviously[19].Ourresultsareinaccordancewithpreviouslypublisheddata[5,38,48-50]thatdetecteddecreasedamoebalsurvivalinpresenceofpathogenicbacteria.

hepredation/survival/intracellularreplicationdataforourbacteriadiferedinsomeinstancesfromsomeworkonbacteriaandFLAinteractionsalreadypublished[6,51]thatrevealedadose-dependentproliferativeresponseofFLAwhenco-incubatedwithbacterialikeE.coli,Staph.aureus,Ccloacae,andPs.aeruginosa.WeattributethisdiscrepancybetweenourresultsandpreviousdatatotheuseofA.spelaeaisolatethathasnotbeeninvestigatedbefore,diferentbacterial

strainsused,diferentMOI, ordiferentco-cultureconditions asyetreported[19].hepredatoryactivityofFLAisknowntobeinluencedbyseveralfactorsincludingthetypeandamountofsurroundingbacteria[52,53].Asobviousinourindings,A.spelaeamayactasabacterialpredator,orasareservoirforbacteria,withenvironmentalandclinicalimplications.Ourresultsalthoughmaynotrelectallpossiblemodalityofinteractionsasasingleamoebalhosthavebeen

employed,demonstratedthatmanypathogenicbacteriaareabletointeract with A.spelaea, even specieswhichwerenotexpected tohaveanintracellular life cycle. A.spelaeaefecton health ecosystemhas twoproblems.First,A.spelaeaserveasreservoirsforpathogenicbacteria.Second,A. spelaeaspeciescanthemselvescausediseaseinhumansoranimals. In the environment, the interactions of bacteria and A.

spelaeaareexpectedtobemuchmorecomplexthanreportedhere,asdiferentbacterialpreyarepresentindiferentnichesthatcanbecolonizedbycompetingbacterialandprotozoan predators.Decipheringthemechanismsofbacteria-protozoainteractionwillassistinabetterunderstandingofA.spelaeaandbacteriallifestyle.

ConlictofInterest

heauthorsdeclarethattheyhavenoconlictofinterest.

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