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ARTICLEOPENDegradationintermediatesofpolyhydroxybutyrateinhibitsphenotypicexpressionofvirulencefactorsandbiolmformationinluminescentVibriosp.
PUGSK8GeorgeSeghalKiran1,SethuPriyadharshini1,AlanDWDobson2,3,ElumalaiGnanamani4andJosephSelvin5Luminescentvibriosareubiquitousinthemarineenvironmentandarethecausativeagentsofvibriosisandmassmortalityinmanyaquaticanimals.
Inaquaticenvironments,treatmentscannotbelimitedtothediseasedpopulationalone,thereforetreatmentoftheentireaquaticsystemistheonlypossibleapproach.
Thus,theuseofantibioticstotreatpartoftheinfectedanimalsrequiresadosebasedontheentirebiomass,whichresultsinthetreatmentofuninfectedanimalsaswellasnon-targetnormalmicrobialora.
Atreatmentmethodbasedonanti-virulenceorquorumquenchinghasrecentlybeenproposedasaneffectivetreatmentstrategyforaquaticanimals.
Polyhydroxybutyrates(PHB)arebacterialstoragemolecules,whichaccumulateincellsundernutritionalstress.
ThedegradationofPHBreleasesshort-chainβ-hydroxybutyricacid,whichmayactasanti-infectivemolecule.
Todate,thereisverylimitedinformationonthepotentialanti-infectiveandanti-virulencemechanismsinvolvingPHB.
Inthisstudy,weaimtoexaminetheeffectofPHBoninhibitionofthevirulencecascadeofVibriosuchasbiolmformation,luminescence,motilitybehaviour,haemolysinandquorumsensing.
AluminescentVibrioPUGSK8,tentativelyidentiedasVibriocampbelliiPUGSK8wastestedinvitroforproductionofextracellularvirulencefactorsandthenestablishedasapotentialshrimppathogenbasedoninvivochallengeexperiments.
TheabilityofVibrioPUGSK8toformbiolmsandtheeffectofPHBonbiolmformationwastestedina96-wellmicrotitre-plateassaysystem.
ThemotilitybehaviourofVibrioPUGSK8wasevaluatedusingtwitching,swimmingandswarmingplateassays.
ReporterstrainssuchasChromobacteriumviolaceumCV026andAgrobacteriumtumefacienswereusedtodetectquorum-sensingmolecules.
Gaschromatography–massspectrometryspectralanalysiswasperformedtoelucidatethefragmentationpatternandstructureofN-hexanoylhomoserinelactone.
PHBdepolymeraseactivityinVibrioPUGSK8wasquantiedastheamountoftheenzymesolutiontohydrolyse1μgofPHBpermin.
AninvivochallengeexperimentwasperformedusingagnotobioticArtemiaassay.
Ofthe27isolatestested,theVibrioPUGSK8strainwasselectedfortarget-specicassaysbasedonthehighintensityofluminescenceandproductionofvirulencefactors.
ThevirulencecascadedetectedinVibrioPUGSK8includeluminescence,motilitybehaviour,biolmformation,quorumsensingandhaemolysinproduction.
Thusinhibition/degradationofthevirulencecascadewouldbeaneffectiveapproachtocontainVibrioinfectionsinaquaticanimals.
Inthisreport,wedemonstratethatthedegradationintermediateofPHBeffectivelyinhibitsbiolmformation,luminescence,motilitybehaviour,haemolysinproductionandtheN-acyl-homoserinelactone(AHL)-mediatedquorum-sensingpathwayinPUGSK8.
Interestingly,thegrowthofVibrioPUGSK8remainsunaffectedinthepresenceofPHB,withPHBdegradationbeingdetectedinthemedia.
PHBdepolymeraseactivityinVibrioPUGSK8resultsinthereleaseofdegradationintermediatesincludeashort-chainβ-hydroxybutyricacid,whichinhibitsthevirulencecascadeinVibrioPUGSK8.
Thus,amoleculethattargetsquorumsensingandthevirulencecascadeandwhichisspecies/strain-speciccouldprovetobeaneffectivealternativetoantimicrobialagentstocontrolthepathogenesisofVibrio,andtherebyhelptocontainVibriooutbreaksinaquaticsystems.
npjBiolmsandMicrobiomes(2016)2,16002;doi:10.
1038/npjbiolms.
2016.
2;publishedonline15June2016INTRODUCTIONMemberoftheVibriogenusarecommoninhabitantsofvariousaquaticenvironments.
Theytypicallyexisteitherasfree-livingorganismsorassociatedwithhostssuchaszooplankton,whichareknowntoprotecttheVibriofromavarietyofdifferentenvironmentalstresses.
Vibriohavealsobeenreportedtobeassociatedwithmanyhigherorganismsinmarineenvironmentsincludingcorals,crabs,molluscsandshamongothers.
Althoughthemajorityoftheseassociationsarenotharmfultothehost,thereareexampleswhereVibriospp.
arepathogenicwithVibriocoralliilyticusandVibriotubiashiibeingthecausativeagentsofdiseaseincommerciallyimportantorganismssuchasoystersandcorals,respectively.
OthersincludetheluminescentstrainVibrioharveyi,whichisthepotentialcausativeagentofmassmortalityinshrimpaquacultureworldwide,1togetherwithVibrioharveyiandVibrioalginolyticuswhicharethemostcommonpathogensofgiantblacktigershrimpPenaeusmonodoninAsiaandposetheprincipalthreatfacedbyshrimphatcheriesallovertheworld.
1DepartmentofFoodScienceandTechnology,PondicherryUniversity,Puducherry,India;2SchoolofMicrobiology,UniversityCollegeCork,Cork,Ireland;3EnvironmentalResearchInstitute,UniversityCollegeCork,Cork,Ireland;4DepartmentofChemistry,StanfordUniversity,Stanford,CA,USAand5DepartmentofMicrobiology,SchoolofLifeSciences,PondicherryUniversity,Puducherry,India.
Correspondence:JSelvin(jselvin.
mib@pondiuni.
edu.
in)Received9October2015;revised28January2016;accepted8March2016www.
nature.
com/npjbiolmsPublishedinpartnershipwiththeNanyangTechnologicalUniversityVibrioparahaemolyticusisbelievedtobetheprimarycausativeagentoftherecentmassmortalityinshrimpduetoearly-mortalitysyndrome.
2Thebiolm-formingcapacityofV.
choleraeiswelldocumented,bothinnaturalhabitatsandunderlaboratoryconditions.
3–5AmongtheshrimpVibriopathogens,thebiolm-formingcapacityofV.
harveyihasbeenestablishedoncementslab,plasticandsteelcouponsurfaces.
6Adhesionandprolifera-tionwithinthebiolmisanestablishedmechanismofpathogen-esisandinfectionofV.
harveyiinP.
monodon.
7Inadditionseveralstudieshavesuggestedthatbiolmsareimportantforsurvival,virulenceandstressresistanceinVibriospp.
,4,8–12withbiolmformationbeingcommonlyassociatedwithcolonisationandsubsequentpathogenesisinhostsbyvibriosinmarineenvironments.
13Todate,onlyafewstudieshavebeencarriedoutonbiolminhibitioninVibriospp.
14–16Bacteriainbiolmsaresurroundedbyanextracellularmatrixthatcanrestrictdiffusionofantimicrobialagents.
17Inadditionchangesinthemembranesterolcompositioninbacteriaduringbiolmdevelopmentcanalsoincreasethemicrobialcell'sresistanttoantibiotics.
18,19Quorumsensingisacell-to-cellcommunicationprocessinbacteriathatinvolvestheproduction,release,detectionandcollectiveresponsetoextracellularsignalmoleculescalledautoinducers,whichcontrolthephenotypicexpressionofbioluminescence,biomassdevelopment,ecologicalsuccession,competence,biolmformation,motilityandtheproductionofvirulencefactors.
20,21Gram-negativequorum-sensingcircuitsrelyonLuxIdependentacylhomoserinelactones(AHL)andaLuxR-typeautoinducerbindingtranscriptionalregulatorprotein.
Thequorum-sensingcircuitofV.
harveyiisknowntoconsistofathree-channelmodelsignaltransductionpathway.
Therstchannelismediatedbyacylatedhomoserinelactone-autoinducer1(HAI-1),thesecondchannelbyafuranosylboratediester—autoinducer-2andthethirdbyacholeraautoinducer-1(CAI-1).
22–24UpdatesonthetaxonomicrevisionsofVibriocladesarestillinconclusive,andrequirecomprehensiverevisionsparticularlywithrespecttoluminescentVibrioclades.
ForinstanceluminescentV.
campbelliibelongingtotheV.
harveyicladeareknowntousethequorum-sensingmoleculeN-hexanoylhomo-serinelactone.
25,26Thequorum-sensingmoleculesidentiedinluminescentvibriosincludeC6homoserinelactoneinV.
scheriandC4homoserinelactoneinV.
harveyi.
Butthereislittleconclusiveinformationonquorum-sensingmoleculesproducedinvariousotherspeciesintheV.
harveyiclade.
Giventhatquorumsensingisamolecularmechanisminvolvedintheexpressionofvirulencefactorsinmanypathogenicbacteria,theninterferencewithquorumsensingandalterationofquorumsensingcircuitscaneffectivelyregulatevirulenceexpressionandpathogenicity.
Polyhydroxybutyrate(PHB)isacommonbacterialintracellularbiopolymerthatappearasgranulesandareproducedinbacterialcellsundernutritionallimitationorwhentheyareinenviron-ments,whichareunsuitableforcellgrowth.
ThePHBpolymercanbehydrolysedtoshort-chainβ-hydroxybutyricacid,whichhasbeenshowntobeaneffectiveanti-infectivemoleculeinthegastrointestinaltractoftheshrimpproviding73%protectiontotreatedanimal.
27,28PHBcanalsoactasaninducerofHSP70,whichprovideprotectionagainstV.
campbelliinfectioninArtemia.
29However,themechanismofactionofPHBandanypotentialeffectthatitmayexertonquorumsensing/quenchinghastodatenotbeenfullyestablished.
Onthebasisofcurrentreportsintheliterature,potentialmechanismsfortheanti-infectiveeffectofPHBmayoccurasaresultofeither(i)PHBbeinghydrolysedto3-hydroxybutyricacidbythedigestiveenzymespresentinthegastrointestinaltractofthetreatedanimalor(ii)owingtothePHBdepolymeraseactivityofresidentbacteriainthegut.
Inthispaper,wereportthatthedegradationintermediatesofPHBincludeβ-hydroxybutyricacidwhichexhibitsantibacterialactivityagainstVibrioPUGSK8inbothinvitroplateassayandinvivochallengeexperimentsinagnotobioticArtemiamodel.
TheshrimppathogenVibrioPUGSK8showedPHBdepolymeraseactivity,resultingintheproductionofaPHBdegradationintermediate,whicheffectivelyshutsdownthephenotypicexpressionofvirulencefactorsandbiolmformation.
Interest-ingly,thePHBdegradationintermediatedoesnotappeartohaveadetrimentaleffectonthegrowthofVibrioPUGSK8resultinginthemicrobialbiomassremainingunaffected.
Inthisstudy,wereportontheanti-infectiveeffectofPHB,whichinhibitsthevirulencecascadeinVibrioPUGSK8.
ThedegradationintermediateofPHBreducesmotilitymediatedbyagellarandpiliadhesionfactorsresultinginthedisruptionofbiolmformation,andinhibitedphenotypicexpressionofbioluminescence,haemolysinandquorum-sensingmediatedthroughAHL.
RESULTSIdenticationandcharacterisationofpathogenicVibrioPUGSK8BacterialisolateswereobtainedfromdiseasedshrimpsamplescollectedfromashrimphatcherylocatedonthesoutheastcoastofIndia.
Amongthe68colonies,theisolatesweregroupedintoluminescentandnon-luminescentbacteria.
All27luminescentcoloniesshowedinvitroexpressionofvirulencefactorssuchasphospholipaseandhaemolysin(SupplementaryTableS1).
Amongthese,thestrainPUGSK8waschosenasitdisplayedveryhighlevelsofluminescenceanindirectfactor,whichisproducedaspartofthevirulencecascade.
StrainPUGSK8wastentativelyidentiedasVibriocampbellibasedonbiochemicalandphyloge-neticanalysis.
ThestrainPUGSK8wassensitivetoO/129andtestedpositiveforanumberofextracellularvirulencefactorsincludingphospholipase,haemolysin,elastase,chitinaseandcellsurfacehydrophobicity.
PhylogeneticanalysisofthePUGSK816SribosomalRNA(rRNA)sequenceshowedclosetmatchesof99%withVibriocampbellibelongingtotheVibrioharveyiclade(Figure1a).
ThesequencedataweresubmittedtoGenbankwiththeaccessionnumberKR024645.
V.
campbelliiPUGSK8formedbiolmgrowthonvarioussurfacessuchasglass,polystyreneandplastic(SupplementaryFigureS1).
Thestrainwasfurthertestedinvivotoestablishitspathogenicitytocauseshelldiseaseinhealthychallengedshrimps.
StrainPUGSK8wasestablishedasapotentialshrimppathogenbasedonchallengeexperiments(SupplementaryFigureS2).
BacterialgrowthandluminescenceGiventhatbioluminescenceinVibriospeciesisoneofthephenotypeswhichiscontrolledbyquorumsensing,weexaminedthepossibilitythatPHBmayaffectbioluminescenceinVibrioPUGSK8.
InitialexperimentswereconductedtodeterminethattheadditionofPHB(50μg/ml)toVibrioPUGSK8culturesdidnothaveadetrimentaleffectoncellgrowth(Figure1b).
ThegrowthprolesforPUGSK8growninthepresenceandabsenceofPHBweresimilar,withculturesenteringlogarithmicphaseafter9h,withdecreasedgrowthbeingobservedafter17h.
Thus,nodetrimentaleffectonthegrowthofVibrioPUGSK8wasapparent.
However,whenbioluminescencewasmeasuredbetween13and15hfollowingincubationhighlevelsofbioluminescencewereobservedinthecontrol,whereasinthePHB-treated(50μg/ml)asksreducedlevelsofbioluminescencewereobserved,inbothPHB-treatedculturessuchasPHBMSI04andPHBstandard(Sigma-AldrichCorporation,Bangalore,India)(Figure2).
At16h,bioluminescenceinthecontrolVibrioPUGSK8cultureshaddecreasedmarkedly,whilenobioluminescencewasobservedinthePHBcultures.
Thus,giventhatPHBappearstoinhibitbioluminescenceinVibrioPUGSK8cultures,wethenexaminedthepossibilitythatthisinhibitionmayaffecttheproductionofvariousvirulencefactors,whichlikebioluminescenceareknowntoberegulatedbyquorumsensinginVibrio.
DegradationintermediatesofPHBGSeghalKiranetal2npjBiolmsandMicrobiomes(2016)16002PublishedinpartnershipwiththeNanyangTechnologicalUniversityInhibitionofmotilityandbiolmformationMotilityisanimportantvirulencefactorinthevirulencecascadeofV.
campbelli,asitcontributestobiolmformation.
VibrioPUGSK8displayedpronouncedagellarandpilimediatedmotilityasevidentbythetwitching,swimmingandswarmingassays(Figure3A).
TheadditionofPHB(50μg/ml),however,inhibitedthemotilityofPUGSK8whichinturnreducedthecolonisationcapacityofthestrainonvarioustestedsurfaces.
PHBcompletelyinhibitedtheswimming,swarmingandtwitchingmotilityofVibrioPUGSK8,whereasacleareffectonbiolmformationwasalsoevidentfromthemicrotitre-plateassayandconfocallaserscanningmicroscopy(CLSM)imageanalysis(Figure3B),withaconcentrationof150μgPHBbeingthemosteffectiveinreducingthebiolmformation.
GiventhatPUGSK8caneffectivelycolonisevarioussurfacessuchasglass,polystyreneandplastic(Figure1c),itappearslikelythattheobservedinhibitionofmotilitymayresultinreducingtheadhesion,andcolonisationcapacityofVibrioPUGSK08.
HaemolyticandPHBdepolymeraseactivityofVibrioPUGSK8ProductionofthevirulencefactorhaemolysinwasinhibitedaroundthewellsofPHB-treatedcell-freesupernatant(CFS)ofVibrioPUGSK8.
ThehaemolysinactivitywasreducedintheplatestowhichCFScollectedfrom24-hculturesofVibrioPUGSK8treatedwithPHBhadbeenadded(Figure3B).
CompleteinhibitionofhaemolysinproductionwasobservedinplatestowhichCFScollectedfrom48-hculturesofVibrioPUGSK8treatedwithPHBhadbeenadded(Figure3C).
Thisinhibitionofhaemolyticactivity(Figure3C),providesdirectevidentthatPHBcontrolsthephenotypicexpressionofthisvirulencefactorinthestrain.
PHBdepolymeraseactivityinVibrioPUGSK8couldbeclearvisualisedonPHBagarplates(Figure4).
DegradationofPHBoccurredinbothminimalagarandZobellmarineagar(ZMA,Himedia,Mumbai,India)indicatingthatPHBdepolymeraseactivityinVibrioPUGSK8doesnotappeartobedependentonthenutritionalavailabilityinthemedium.
TheturbidometricassayshowedthatVibrioPUGSK8producedamaximumactivityof25.
72U/mlofPHBdepolymeraseat48h,withproductionofdepolymeraseincreas-ingattheonsetofstationaryphase.
ESI-HRMSanalysisofenzymehydrolysedPHBproducesapeakatm/z105.
05,correspondingtothemassofbutyricacids(SupplementaryFigureS3).
N-acyl-homoserinelactonedegradationVibrioPUGSK8appearstopossesstheabilitytodegradeAHLasevidencedbyalossinpurplecolourinthereporterstrainChromobacteriumviolaceumCV026inthepresenceofextractsfromPUGSK8growninthepresenceof50μg/mlPHB(Figure5,b);withtheeffectbeingobservedevenafter72h(Figure5,b3).
PurplecolourwasobservedinCV026inthepresenceofextractsfromPUGSK8,whichwasnotgrowninthepresencePHB(Figure5,a1–3).
TofurtherstudythisapparentAHLdegradation,followingextractionoftheAHLthefragmentationpatternanalysisfollowingTLCplatesidentiedauniquefractionwithanRfvalueof0.
67,whichwhencomparedwithanAHLstandard(sigma)indicatedthatthefractionmaycontainaN-hexanoylhomoserinelactonesignallingmolecule.
ToconrmthechemicalidentityoftheTLCfraction,massspectralanalysiswasperformed.
Themassspectrum(MS)oftheVibrioPUGSK8fractioncloselymatchedthespectrumfromastandardC6-AHL(Figure6a),conrmingthepresenceofaC6HSL(N-hexanoylhomoserinelactone)compound(Figure6b).
FurtheranalysisofsomeoftheseselectedfragmentsindicatethatFigure2.
TheintensityofluminescenceduringthegrowthcycleofVibrioPUGSK8.
(a)TheintensityofluminescencedecreasedinthemediasupplementedwithPHB(MSI04)andPHB(sigma).
Theluminescencereacheddetectablelevelsatthe13thhofincubationandproductionbecameundetectableafter16thofincubation.
(b)LuminescenceinVibrioPUGSK8following13hincubation.
Figure1.
PhylogeneticandgrowthcharacteristicsofVibrioPUGSK8.
(a)PhylogenetictreeofVibriosp.
PUGSK8.
MaximumparsimonyconsensusphylogenetictreeconstructedusingMEGA6.
0basedon16SrRNAgenesequenceofVibriosp.
PUGSK8showingrepresentativesofotherrelatedtaxa.
Thephylogeneticanalysisshowed99%toVibrioharveyiandVibriocampbellii.
OnthebasisoftheAHLmoleculeproducedbythestrainPUSK8,itwastentativelyidentiedasVibriocampbelliistrainPUGSK8.
(b)TheeffectofPHBongrowthofVibrioPUGSK8.
ThegrowthwasrecordedatOD600nm.
Theisolate(control)enteredlogarithmicphaseafter9hofgrowthandstartedtodeclineafter17h.
ThetrendofgrowthpatternwasnotaffectedinthemediasupplementedwithPHB(50μg/ml).
DegradationintermediatesofPHBGSeghalKiranetal3PublishedinpartnershipwiththeNanyangTechnologicalUniversitynpjBiolmsandMicrobiomes(2016)16002thefragmentionatm/z143maybeduetoaMcLaffertyrearrangement,whichisatypicalcarbonylgrouphavingahydrogenatomintheγ-position(Figure6d).
Thisrearrangementwouldgiverisetoanenolicfragmentandanolenlossofwaterfromtheiongivingrisetoanm/z143.
ThelossofthecharacteristicAHLpeakintheGC-MSspectrainPHB-treatedVibrioPUGSK8culturesisindicativeofAHLdegradation,whichaccompaniedthelossinquorum-sensingsignallingasevidencedbythelossinpurplecolourinthereporterstrainC.
violaceumCV026(Figure6c).
PHBinhibitionofVibriovirulenceinArtemiaPHBappearstoexhibitanti-virulenceeffectsinvivoandmayregulatethephenotypicexpressionofvirulencefactorsinvolvedintheinvasionofArtemiaduringVibrioinfections.
PHBitselfisnottoxictoArtemiawiththesurvivalratebeingunaffectedatPHBconcentrationsbetween50and200μg/ml.
IninvivochallengeexperimentsthetreatmentofArtemiawithPHBatconcentrations50μg/mlappearssufcienttoelicitcompleteprotectionagainstinfectionscausedbypathogenicVibrio,withprotectionbeingmaintainedupto48hpostchallenge.
LowersurvivalrateswereFigure3.
TheeffectofdegradationintermediatesofPHBonthephenotypicexpressionofvirulencefactors.
(A)TheeffectofPHBonthemotilitybehaviourofVibrioPUGSK8.
ThePHBtreatmenteffectedthelossoftwitching,swarmingandswimmingbehaviours.
TheeffectofPHBontwitching(a–control,d—treated),swarming(b—control,e—treated)andswimming(c—control,f—treated).
(B)CLSMimagesshowstheeffectPHBonbiolmformationbyVibrioPUGSK8.
Theaiscontrolandb,canddisshowingtheeffectofPHBonbiolmformationatconcentrationsof50,100and150μg/ml,respectively.
(C)HaemolyticactivityofVibrioPUGSK8onbloodagarplate.
(a)Thecell-freesupernatant(CFS)ofuntreatedVibrioPUGSK8showinghaemolyticactivity(b)HaemolyticactivityofCFScollectedfromPHB-treatedVibrio(24h)(c)CompleteinactivationofhaemolyticactivityofCFScollectedfromPHB-treatedVibrio(48h).
DegradationintermediatesofPHBGSeghalKiranetal4npjBiolmsandMicrobiomes(2016)16002PublishedinpartnershipwiththeNanyangTechnologicalUniversityobservedatlowerPHBconcentrations(10and25μg/ml),with60%and80%survival,respectively,indicatingthatPHBataconcentrationof50μg/mlwasmosteffectiveintheArtemiamodelexperiments(Figure7).
ConverselyintheabsenceofPHB,survivalratesinchallengedArtemiastartedtodeclineafter6hwithmortalityratesreaching95%at24h,postchallengewithVibrio.
DISCUSSIONInthisstudy,27luminescentVibrioswereisolatedfromthehepatopancreasofinfectedshrimpsamplescollectedfromshrimpfarmslocatedinsoutheastcoastofIndia.
Fromthese27strains,thestrainPUGSK8wasselectedfortarget-specicassaysbasedonitsabilitytoproducehigh-intensityluminescence,whichunlikesomeoftheotherisolatescontinuedtobeluminescentevenafteranumberofsub-culturingsteps.
Luminescenceisapartofthequorum-sensingsystemofV.
harveyiandV.
campbellithatisinvolvedintheestablishmentofthepathogeninthehost.
30,31ThoughbacteriafromtheV.
harveyicladeandrelatedbacteriaareoftenreferredtoasluminescentVibrios,alargedifferencebetweendifferentstrainswithrespecttoluminescencehasbeenreported.
32,33Thenon-luminescenceweobservedintheotherstrainsmaybeduetodefectsintheautoregulationofthegenesinvolvedintheluxoperon.
24BiochemicalandmorphologicalanalysisofPUGSK8indicatedthatitwasfromtheVibriogenus,whilephylogeneticanalysisshoweda99%matchwithVibriocampbelliistrains.
VibrioPUGSK8exhibitedagellarandpilimediatedmotility,haemolyticactivity,luminescence,togetherwiththeabilitytoformbiolmsonglass,polystyreneandplastic.
Pathogenicvibriosarethecausativeagentofvibriosisandaresuspectedtobeinvolvedinearly-mortalitysyndromeoracutehepatopancreaticnecrosissyndrome(EMS/AHPNS),whichisamajorthreadtotheshrimpaquacultureindustry;causing100%mortality,leadingtothelossof41billionUSdollarsinrecentoutbreaksinmanycountries.
CurrenttreatmentforVibrioinfectionsinshrimpinvolvereactivetreatmentwithantibioticsandaswiththeuseofmanyantibioticsposesthethreatoftheemergenceofdrug-resistantinvibrios.
Indeed,antibioticsarebecomingincreasinglyineffectiveinthecontrolofpathogenicvibriosandthiscoupledwiththefactthattheuseofantibioticsinanimalhusbandryisbannedinmanycountries;hasresultedinanincreasedinterestintheuseofalternatetreatmentmethodstotreatVibriooutbreaksinshrimpaquaculture.
Withthisinmindwetargetedtheidenticationofanti-infectiveagents,whichwouldinterferewiththequorum-sensingsysteminluminescentVibrioPUGSK8,whichhadbeenisolatedfromhepatopancreasoftheinfectedshrimpP.
monodon.
Thenon-specicsurfaceadhesionbehaviourofVibrioPUGSK8wasevidentanditexhibitedastrongbiolm-formingpotential.
Inthisstudy,weusedPHBproducedbyBrevibacteriumcaseiMSI04asapotentialanti-infectivemoleculeagainstluminescentVibrioPUGSK8,aswehadpreviouslyshownthatPHBmoleculesareeffectiveinthecontrolofbiolmformationbyvibrios.
17Themicrotitre-plateassaysystemcoupledwiththeconfocallaserscanningmicroscopyimagesrevealedthatPHBwasveryeffectiveinthecontrol/disruptionofbothbiolmformationandpreformedbiolms.
AsinotherVibriospeciesthevirulencecascadeinVibrioPUGSK8includesmotilitybehaviour,biolmformation,quorum-sensingsystemsandhaemolysinproduction.
Thus,interferingwiththisvirulencecascadeinanywaycouldproveaneffectiveapproachtohelpcontainVibrioinfectionsinaquaticsystems.
Thequorum-sensingsystemofVibrioPUGSK8ishighlyactiveandislikelytoinvolveAHLmolecules,giventhattheMSdatashowedcharacteristicpeaks34inextractsfromthestrain,whicharesimilartomassfragmentsofN-hexanoylhomoserinelactoneinNitrosomonaseuropaea.
35PHBeffectivelycontrolsbiolmforma-tioninVibrioPUGSK8,theexpressionofbioluminescence,Figure4.
PHBdepolymeraseproduedbyVibrioPUGSK8atvarioustimeintervals.
(a)isshowingPHBdepolymeraseactivityinU/ml.
ThePHBdepolymeraseactivityonminimalmediasupplementedwith100mg/lPHB(b),200mg/lPHB(c)and250mg/lPHB(d).
Figure5.
EffectofPHBonthedegradationofAHLproducedbyVibrioPUGSK8.
TheassaywasperformedonCV026withAHLextractofVibrioPUGSK8.
a1,a2anda3showpurplecolourasanindicatorofAHLexpressionaroundthewellslledwithAHLextractofVibrioPUGSK8.
b1,b2andb3areshowinglossofpurplecolourindicatingAHLdegradationbyPHB(50μg/ml)treatedVibrioPUGSK8extract.
DegradationintermediatesofPHBGSeghalKiranetal5PublishedinpartnershipwiththeNanyangTechnologicalUniversitynpjBiolmsandMicrobiomes(2016)16002colonisationcapacityandvirulencecascadeincludingmotilityandhaemolysin,therebyreducingpathogenicity,andindoingsodisruptsAHL-mediatedquorum-sensingpathway.
GiventhatthegrowthofVibrioPUGSK8remainunaffectedbyPHB,itappearslikelythattheobservedeffectsmaybeasaresultofPHBmetabolism.
AsimilargrowthindependentinhibitionofquorumsensinghasrecentlybeenreportedforcoumarininPseudomonasaeruginosa.
36Figure6.
GC-MSanalysisofAHLsignallingmoleculesproducedbyVibrioPUGSK8.
(a)isshowingGC-MScharacteristicpeaksofstandardAHL.
(b)CharacteristicpeaksofAHLproducedbyVibrioPUGSK8evidencesthecellsignallingmoleculeasN-hexanoylhomoserinelactone.
(c)AbsenceofcharacteristicGC-MSpeaksofAHLindicatingthePHBshutsoffPHBmediatedcellsignallinginVibrio.
(d).
ThefragmentationpatternelucidatedbasedontheMSspectraofAHLproducedbyVibrioPUGSK8showedconformitywithCataldietal.
(2008).
DegradationintermediatesofPHBGSeghalKiranetal6npjBiolmsandMicrobiomes(2016)16002PublishedinpartnershipwiththeNanyangTechnologicalUniversityItiswellestablishedthatswarmingmotilityandquorumsensingisnecessarytodevelopcolonialbacterialpopulationbothinside/outsidethehost.
37Motilitybehaviourandquorum-sensingmoleculesareimportantforcelldifferentiation,proliferationandsensinginbacterialpopulations.
ButinourndingsitappearsthatinthePHB-treatedVibrioPUGSK8themotilitybehaviourwascompletelyreducedindicatingthequorum-quenchingnatureofPHB.
Recently,ithasbeenreportedthatmotilitybehaviourinV.
harveyiisregulatedthroughquorumsensing.
38MotilityisalsowellestablishedasafactorinthevirulencecascadeofpathogenicV.
harveyi.
Aswehavealsodemonstratedhereinthisstudy,inhibitionofmotilitycansignicantlyreducevirulenceofV.
harveyi.
38Bacteriawithinbiolmsarehighlyresistanttowardsantibiotics.
39Whenthebiolm-formingcapacityofbacteriaisreduced,theresistancetowardsantibioticsandpotentialofpathogenesisisalsoreducedinthefree-livingVibriopopulation.
TheexperimentaldatageneratedhereindicatethatPHBmayaffectthepathogenicityofVibriobyinterferingwiththesignallingmolecules.
Thequorum-quenchingactivitywasrevealedinthegaschromatographydata,whichshowednocharacteristicpeaks/masswithrespecttotheAHLmolecule.
ThereporterstrainplateassayshowedlossofcharacteristiccolourformationinthePHB-treatedplatesofAgrobacteriumtumefaciensandCV026.
Thequorum-quenchingeffectofPHBmaybeduetothehydrolysisofthePHBbyPUGSK8bysecretingPHBdepolymerase,whichdegradesthePHBintoβ-hydroxybutyricacid.
ThisistherstreportonluminescentVibriosecretingPHBdepolymerase,whichwasevidentasaclearzoneformationonthePHBplates.
IthasrecentlybeenreportedthatPHBataconcentrationof100mg/lprovidescompleteprotectiontotheArtemiaagainstV.
campbelliiinfection.
29However,wereportherethatPHBataconcentrationof50mg/lwaseffectiveinprovidingcompleteprotectiontoArtemiaagainstinfectionbyVibrioPUGSK8inchallengeexperiments.
Thebacteriostaticeffectofshort-chainfattyacidsonEnterobacteria40haspreviouslybeenreportedandmayhavepromisingeffectsonthecontrolofbacterialdiseasesinaquaculture.
41PHBhaspreviouslybeensuccessfullyusedasananti-infectiveingnotobioticstudieswithArtemiafranciscana.
25,42ThepolymerPHBisnotwatersoluble,andneedstobedegradedintoβ-hydroxybutyratemonomersandoligomersinthegastrointestinaltractofthetreatedanimals.
PreviousreportsrevealedthatpretreatmentofPHBwithNaOHfollowedbydigestionwithgutenzymesincreasesthedegradationofPHB.
43,44DegradationofPHBcanalsobeachievedbyPHAdepolymeraseproducingbacteriaandfungi.
45ThemechanismofPHBdegradationintreatedanimals,however,stillremainsunclear.
IthasbeensuggestedthatPHBmayprovideadditionalenergytoactasanimmunostimulantinthetreatedanimals,28orthatPHBanditsdegradationproductsmaylowerthepHintheArtemiagutthroughnon-ionicdiffusioncausingcellularacidica-tiontherebyinhibitingthevirulenceorpathogenesisofVibrio.
Incontrasttothis,thedatapresentedhereappeartoindicatethatthedegradationintermediateofPHBregulatesthephenotypicexpressionofthevirulencecascadeinluminescentVibrioPUGSK8.
DegradationofPHBbyVibrioPUGSK8wasobservedonthemediaindicatingthatPHBdepolymeraseactivitycaninhibitthevirulencecascadeofVibrio.
ThedegradationintermediateofPHBwaseffectiveininhibitingpathogencolonisationthroughbiolmformationandphenotypicexpressionofvirulencefactors.
PHBexhibitedaprotectiveeffectonArtemiaagainstVibrioinfections.
Disruptionofthequorum-sensingsystemsinVibriowouldbeaneffectiveandenvironmentallyfriendlieralternatetoantimicrobialagentstocontrolthepathogen.
Thus,PHBtreatmentscouldbeaneco-friendlynon-invasiveeffectivelystrategytocontainVibrioinfectioninshrimpaquaculture.
Itiswellestablishedthatthetreatmentofindividualinfectedaquaticanimalsisneitherfeasiblenorpossible,withentiresystemtreatmentapproachesinvolvingantibioticsculminatinginresistantandresidualimpacts.
ThePHB-basedtreatmentwouldbeaneffectiveanti-infectivestrategytoachieveaquaticsystemtreatmentapproachestocontainVibriooutbreaksinshrimpaquaculture.
Althoughhalogenatedfura-noneshavebeenshowntobeeffectiveindisruptingsignallingmoleculesinGram-negativebacteria,46,47theadministrationofhigherdosesoffuranonestoshrimplarvaeisbothhighlytoxicandnoteffectiveinallthespeciesandstrainsofVibrioisolatedfrombrineshrimp;thus,alternatemoleculesfortreatmentsarerequired.
48Anaddedadvantageisthatanti-infectivestargetingquorum-sensingsystemscanbegenerated,whicharetarget-specicandwhicharespeciesorstrain-specic,thereforeminimisingthepossibledevelopmentofresistanceoraandthekillingofuntargetedmicrobialora.
MATERIALSANDMETHODSIsolationandselectionofpathogenicVibriocampbelliThepathogenicbacteriausedinthisstudywereisolatedfromthehepatopancreasofaninfectedshrimpcollectedfromashrimphatcheryinKollam(8°54′N76°38′E),whichislocatedonthesouthwestcoastofIndia.
ZMAandnutrientagarwith2%NaClwasusedforbacterialisolation,withpureculturesbeingobtainedfollowingincubationat28°Cfor24h.
SelectiveisolationofVibriowasperformedonthiosulfate-citrate-bilesalts-sucroseagar(Himedia).
Vibriocultureswereexaminedforcolonyluminescenceandintensityusingaspectrourometer(Fluorolog-FL3–11,Kyoto,Japan).
DNAwasisolatedfromPUGSK8andPCRamplicationofthe16SrRNAwasperformedusingtheprimers27F5′-AGAGTTTGATCMTGGCTCAG-3′and1492R5′-CGGTTACCTTGTTACGACTT-3′,generatinga1,500-bpsizefragment.
TheampliedDNAwasclonedusingtheTOPOTAcloningkit(Invitrogen,Carlsbad,CA,USA)forsequencing(Macrogen,Seoul,Korea).
TheforwardandreversesequenceswereobtainedandcomparedfortheirpairwisesimilarityusingtheNCBIBLAST.
MultiplealignmentsofthesesequenceswerecarriedoutbyClustalW1.
83versionofEBI(www.
ebi.
ac.
uk/cgi-bin/clustalw/)withatransitionweightof0.
5.
PhylogenetictreeswereconstructedusingMEGA5.
0version(www.
megasoftware.
net)bymeansoftheneighbourjoining(NJ)andtheunweightedpairgroupmethodalongwiththearithmeticmean(UPGMA)algorithms.
ThenucleotidesequencesweredepositedinGenbankwiththeaccessionnumberKR024645.
SourcesofPHBThestrainB.
caseiMSI04(GenbankaccessionnumberKU510053)usedforPHBproductioninthisstudywasisolatedfromthemarinespongeDendrillanigra,withitsproduction,structuralcharacterisation,anti-adhesiveactivityagainstVibriohavingpreviouslybeingreported.
17StandardPHB(Sigma)wasusedforefcacycomparisoninthepreliminaryexperiments.
Figure7.
EffectofPHBandVibrioonsurvivalofArtemia.
PositivecontrolincludesArtemiachallengedwithVibrio.
NegativecontrolwasArtemiawithoutPHBtreatment/Vibriochallenge.
DegradationintermediatesofPHBGSeghalKiranetal7PublishedinpartnershipwiththeNanyangTechnologicalUniversitynpjBiolmsandMicrobiomes(2016)16002Biolmformation/disruptionassayTheeffectofPHBonbiolmformationinPUGSK8wasassessedina96-wellmicrotitre-plateassay.
Briey,anovernightcultureofVibrioPUGSK8wasinoculatedin200μlofZobellmarinebrothinpolystyrenemicrotitreplates.
ThetestwellscontainedPHBatconcentrationsrangingfrom50–150μg/mlwithwellswithoutPHBandinoculumactingaspositiveandnegativecontrolsrespectively.
After48-hincubationat28°C,thewellswerewashedtwicewithphosphate-bufferedsaline(pH7.
0)andadheredcellsstainedwith0.
1%crystalvioletandtheamountofbiolmformationwasquantiedinamicroplatereader(Labnics,Mumbai,India)at590nm.
17Thebiolm-formingabilityofVibrioPUGSK8wasassessedwithbiolmsbeingallowedtodeveloponvarioussurfacessuchasglass,stainlesssteel,aluminiumandpolystyrene.
Biolmswerestainedwithcrystalvioletandexaminedunderalightmicroscopeat*40magnication(Optika,Ponteranica,Italy).
TovisualisetheeffectofPHBonthebiolm-formingabilityofVibrioPUGSK8,biolmwasallowedtoformoncoverslipsimmersedinZobellmarinebrothcontainingvaryingconcentrationsofPHB(50–150μg/ml)andincubatedat28°Cfor24h,withbrothnotcontainingPHBactingasacontrol.
ThecoverslipswerethenwashedwithPBS,stainedwith0.
1%acridineorangeandobservedunderconfocallaserscanningmicroscopy(LSM710,CarlZeiss,Oberkochen,Germany).
MotilityassayThemotilitybehaviourofVibrioPUGSK8wasevaluatedusingtwitching,swimmingandswarmingplateassays.
49,50Swarmplateswerepreparedbyadding0.
7%bactoagartonutrientbroth(Himedia),supplementedwith0.
5%glucoseand100μgPHB.
Swimmingassayswereperformedusingtryptoneswimagarplatescontaining1%tryptone,0.
5%NaCland0.
3%bactoagarsupplementedwith100μgPHB.
Twitchagarplateswerepreparedbyadding1%tryptonebroth,5%yeastextractand1%NaCltoLBbrothsupplementedwith1%bactoagarand100μgPHB.
PlateswithoutPHBadditionservedascontrolsandallplateswereallowedtodryovernightatroomtemperature.
Twitchingplateswerestabinoculatedusingasteriletoothpickatadepthof3mmtothebottomofthepetridish.
Swarmplateswerespot-inoculatedontheagarsurface.
Swimmingplateswereseededbelowtheagarsurfaceusingasterileinoculationneedle.
Theplateswereincubatedat37°Cforbetween18and48h.
Thediametersofswimming,swarmingandtwitchingzonesweremeasuredandimageswerecapturedonadigitalcamera.
(Nikon,Tokyo,Japan)Screeningofquorum-sensingmoleculesecretedbyVibrioPUGSK8Preparationofcell-freelysateforTLC.
ReporterstrainswereculturedinLBbrothcontainingkanamycin20μg/mlforC.
violaceumCV026,andtetracycline20μg/mlforA.
tumefaciens.
VibrioPUGSK8wasinoculatedinZMB,andallstrainswereincubatedat28°Cfor24h.
Brothcultureswerecentrifugedat10,640gfor5min.
Thesupernatantswerethenpassedthrough0.
1-μmMilliporelters(Merck)andtheltratewasextractedtwicewithanequalvolumeofacidiedethylacetate.
Pooledextractsweredriedoveranhydrousmagnesiumsulphate,evaporatedtodrynessandre-suspendedin50–100μlofHPLCgradeethylacetate.
ThepresenceofAHLsintheextractswasevaluatedbyC18reverse-phaseTLCplates(Merck)51followedbyuorescenceemissionusingaultraviolettransillu-minator.
AHLswereidentiedbycomparingtheretentionofsyntheticstandardAHLs(Sigma)andrespectivetestAHLspots.
DetectionofAHLbyGC-MS.
ExtractsfromVibrioPUGSK8andAHLstandards(sigma)wereanalysedbygaschromatography–massspectro-metry(PerkinElmer-Clarus680model,Shelton,CT,USA)usingtheElite5MScolumn(PerkinElmer)(30m*0.
25mmIDand250-μmlmthickness)operatinginanelectronimpactmodeat70eVwithheliumasthecarriergasataowrateof1ml/min.
ThemassspectralanalysiswasperformedbycomparingthemassvaluesofAHLstandardswiththeAHLofV.
campbellii.
AssessmentofcellregulationandactivityassaysBacterialgrowthandluminescence.
AnovernightcultureofVibrioPUGSK8wasdilutedtoanOD600of0.
1nmandinoculatedintofreshZMBcontaining5mg/100mlPHBandincubatedat28°Cfor24h.
FlaskswithouttheadditionofPHBwereusedascontrols.
ThegrowthandluminescenceofVibrioPUGSK8wasrecordedafter6h(OD600nm)at1-htimeintervalsfor20husingaspectrophotometerandspectrourometer(Fluorolog-FL3–11).
GrowthandluminescenceofVibrioPUGSK8inthepresenceofPHBproducedbyB.
caseiMSI04wascomparedwiththatobservedwiththestandardPHB(sigma).
HaemolyticactivityFifty-microlitreovernightVibrioculturesgrowninthepresenceof0.
5μgPHBfor24and48h,respectively,wascentrifugedat10,621gfor10minandtheCFSwasltredusing0.
45-micronltre.
TheassaywasperformedasdescribedbyBeecherandWong52withsuitablemodications.
Briey,theCFS(75μl)wasaddedtowellinabloodagarcontaining5%(v/v)humanbloodandincubatedat37°Cfor24h.
Afterincubation,theplateswereobservedfortheappearanceofzonesofhaemolysisaroundthewell.
TheCFSofculturesgrownintheabsenceofPHBwasusedasacontrol.
Detectionofquorum-quenchingactivityusingreporterstrainsandGCTheAHLinactivationassaywasperformedasdescribedbyBaruahetal.
53withnecessarymodications.
PUGSK8wasgrownovernightin100mlZMBsupplementedwith50μg/mlPHBandincubatedat28°Cfor48h,54withaskswithoutPHBadditionservingasacontrol.
Followingcentrifugationat10,640gfor5minthecell-freesupernatantwasseparatedandAHLwasextracted,acidiedwithethylacetateandsolventevaporatedtodryness.
ReporterstrainC.
violaceumCV026wasgrownovernightonLBbrothand100μlofCV026wasswabbedontothesurfaceofLBagarplates.
Wellsweremadeusingasterilecorkborer(0.
6mmdiameter)onagarplates,and10mg/μlofextractedandair-driedN-hexanoylhomoserinelactonefromPUGSK8brothculturewithPHBwasaddedintriplicatetowellsintheplatesandincubatedfor36–48h.
AHLextractedwithoutPHBadditionactedascontrols.
PlatesweremonitoredforpurplecolourformationinthereporterstrainCV026.
DegradationofC6-HSLduetoalackofpurplecolourformation,wasfurtherveriedusingGC-MS;withextractsfromVibrioPUGSK8growninthepresenceof50μg/mlPHBbeinganalysed.
PHBdegradationbyVibrioPUGSK8TodeterminePHBdegradationandutilisationbyVibrioPUGSK8,thedegradationassaywasperformedaspreviouslydescribedwithamodicationtothegrowthmedia.
55VibrioPUGSK8wasinoculatedinnutrientrich(ZMA)mediumaswellasintheminimalmediasupplementedwithvariousconcentrationofPHBrangingfrom100mg/l,200mg/l,250mg/lPHB.
Theplateswereincubatedat28°Cfor48handmonitoredfortheformationofzonesofclearance.
TodeterminetheamountofPHBdepolymeraseproducedbyVibrioPUGSK8,aturbidometricassaywasperformedaspreviouslydescribed56,57Briey,VibrioPUGSK8wasculturedinminimalmediasupplementedwith0.
1%PHBat28°Cwithagitationat220r.
p.
m.
fordifferenttimeintervalsrangingfrom12–48h.
Theproductionmediawascentrifugedat10,640gfor10min.
Thecrudeenzymeextractwasthenvacuumlteredthrough0.
22μmMilliporeltres.
PHBdepolymerasewasthenprecipitatedwithequalvolumesofcoldacetoneandtheprecipitatewasdissolvedin10mMacetatebufferpH6.
0.
TheprecipitatedenzymewasfurtherpuriedbysephadexG-200columnchromatography.
Theactiveenzymefractionswerepooledandtheenzymeassaywascarriedout.
Theassaymixwaspreparedwith0.
7mgofPHBin1mlof10mMacetatebufferwithaliquotsbeingmixedthoroughlyusingsonicationfor10min.
Theassaymixwasaddedintriplicateto0.
5mloftheenzymesolutionandincubatedat40°Cfor20min.
Theenzymeactivitywascalculatedasadecreaseintheturbidityat660nmwithPHBwithoutenzymesolutionactingasacontrolblank.
OneunitofPHBdepolymeraseactivitywasdenedastheamountoftheenzymesolution(μl)tohydrolyse1μgofPHBpermin.
Thedegradationproductsformedresultingfromdepolymeraseactivityafter12hofincubationwasanalysedElectrosprayIonisation-HighResolutionMassSpectralanalysis(ESI-HRMS,OrbitrapElite).
ArtemiaassayTheinvivochallengeexperimentwasperformedusingagnotobioticmodelassaywithA.
franciscanaaspreviouslydescribed.
53Theexperi-mentalsetupwasperformedinasterileairlaminarowhood.
Sterilecystsandlarvaewereobtainedbydecapsulationusing3.
3mlNaOH(32%)and50mlNaOCl(50%).
Cystswereaeratedina1-lcapacitysterilisedglasscylinder(jar)containingautoclavedseawater.
Toprovidecompletehydrationofthecysts,anairstonewasplacedinthebottomofthejarseawaterandoxygenatedcontinuouslyusingaeratorpumps.
After24hincubationat28°Cthenewlyhatchedfree-swimmingpink-colouredDegradationintermediatesofPHBGSeghalKiranetal8npjBiolmsandMicrobiomes(2016)16002PublishedinpartnershipwiththeNanyangTechnologicalUniversitynaupliiwerecollectedfromthebottomofthejar.
Theaxenicityofthenaupliiwasconrmedbyplating50μlofthehatchedwaterontoZMAplatesandincubatingat28°Cfor7days.
PreliminarydoseselectingexperimentswereperformedtondtheeffectiveconcentrationofPHBandtodetermineanytoxiceffectofPHBonArtemia.
NewlyhatchedArtemiawereexposedtovariousconcentrationofPHBrangingfrom25to200μg/ml.
Afterexposure,theexperimentalsetupwasincubatedat28°Cunderconstantaerationandillumination.
ThesurvivalofArtemiawasrecordedafter24and48h.
Allexperimentswereperformedintriplicate.
ChallengeexperimentwithgnotobioticArtemiaGroupsof20freshlyhatchednaupliiweretransferredintosterile50-mlbeakerscontaining25mlsterilisedseawater.
Theexperimentswereperformedintriplicatewithparallelnegativecontrol(Artemiawithdriedyeast6mg/ml),positivecontrol(ArtemiachallengedwithluminescentVibrioPUGSK8(105CFU/ml))andtest(ArtemiatreatedwithluminescentVibrioPUGSK8of105CFU/mlalongwith50μg/mlofPHB).
Allexperimentswereperformedinsterileconditionsat28°Cunderlightwithcontinuousaeration.
Thesurvivalratewasdeterminedafter24and48hbytransferringtheliveArtemiatoawatchglassandcountingmanually.
ACKNOWLEDGEMENTSG.
S.
K.
acknowledgestheDepartmentofBiotechnology,MinistryofScienceandTechnologyandIndianCouncilofMedicalResearchforafellowshiptoworkinUCC,Ireland.
J.
S.
thankfullyacknowledgedA.
P.
Lipton,CentralMarineFisheriesResearchInstituteforprovidingArtemiacyst.
ThisworkisfundedbyDepartmentofBiotechnology,MinistryofScienceandTechnology,NewDelhi.
CONTRIBUTIONSJ.
S.
designedthework,G.
S.
K.
guidedtheexperimentsandwritingofthemanuscript,S.
P.
andG.
S.
K.
performedtheexperiments,A.
D.
guidedthedataprocessing,interpretationandmanuscripteditingandE.
G.
didanalysisandchemistryofQSmoleculesandESI-HRMSspectradata.
COMPETINGINTERESTSTheauthorsdeclarenoconictofinterest.
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