a Départementdebiochimie,microbiologieetbio-informatique,Facultédessciencesetdegénie,UniversitéLaval,Canada
b Centrederecherchedel'Institutuniversitairedecardiologieetdepneumologie,UniversitéLaval,Canada
HIGHLIGHTS
• Thispaperpresentsareviewofthechallengesofapplyingmolecularmethods tobioaerosolsstudies
• Comparisonofstudiesusingculturedependentandculture-independent approachestodeterminethemicrobial diversityoverthepastsevenyearsona scaleof50randomlyselectedstudies
• Particularattentionshouldbegivento mouldsandviruseswhenrecovered fromairsamplespriortomolecular analysis
• Thebiasesintroducedbynucleicacid extractionshouldbegivenaparticular attentionwhenapplyingmolecularapproaches,especiallynext-generationsequencingapproaches
• Whenexaminingbioaerosols,optimizationofenvironmentalmicrobiology analysesapproachesaccordingtothe needsofeachprojectismandatoryand ignoringthesecriticalstepswillgreatly decreasetheaccuracyoftheresult
http://dx.doi.org/10.1016/j.scitotenv.2017.05.076 0048-9697/©2017ElsevierB.V.Allrightsreserved.
Bioaerosolsareamongthelessstudiedparticlesintheenvironment.Thelackofstandardizationinsamplingprocedures,difficultiesrelatedtotheeffectofsamplingprocessesontheintegrityofmicroorganisms,andchallenges associatedwiththeapplicationofenvironmentalmicrobiologyanalysesandmolecularandculturemethods frightenmanyyoungscientists.Everymicroorganismhasitsownparticularitiesandactsdifferentlywhenaerosolizedinvariousconditions.Becausetheairisanextremelybiologicallydilutedenvironment,itisnecessaryto concentrateitscontentbeforeanyanalysisisperformed.Challengesfacedwhenapplyingmolecularmethodsto airsamplesrevealtheneedforabetterstandardizationofapproachesforcellandnucleicacidrecovery,the choiceofgeneticmarkers,andinterpretationofdata.Thispaperpresentsafewofthelimitsanddifficultiestackledwhenmolecularmethodsareappliedtobioaerosols,suggestssomeimprovementsbyspecifyingthecritical stagesthatshouldbeconsideredwhenstudyingthemicrobialecologyofbioaerosols,andprovidesthoughtfulinsightsonhowtoovercomethechallengesencountered.
©2017ElsevierB.V.Allrightsreserved. (H.Mbareche), (C.Duchaine).
1.Introduction:challengesinusingmolecularmethodsfor bioaerosolstudies
Theuseofmolecularmethodsinmicrobialstudiesisbasedonthe detectionofgeneticmaterialoforganismspresentinagivensample. Byapplyingthesemethodstodifferentenvironments,itispossibleto identifythemicroorganismspresentandtobetterunderstandtheenvironmentalimpactsandecologicalrolesofthemicrobialcommunity. Molecularmethodsarecriticizedbecausetheydonotrelatetoorganisms'viabilityorintegrity.Propidiummonoazidestainingwasapplied tobioaerosolstudiesofviruses(Bonifaitetal.,2015)andbacteria (Bonifaitetal.,2014),therebyallowingthespecificPCRamplification oftargetedgenesextractedfromintactcellsonly.Othermajorchallengesassociatedwiththedetectionofnucleicacidtoidentifyand quantifymicroorganismscanbesummarizedasfollows:(Bonifait etal.,2015)concentratingandretrievingthebiologicalmaterialfrom aerosolsamplespriortoanalysesand(Bonifaitetal.,2014)choosing therightgenomicregionstoamplifyand/ortosequencetoobtainthe mostaccurateinformationaboutthemicrobialcontentoftheenvironmentsamB19pled.Bacteriacanbequantifiedandidentifiedusingthe conserved16SrRNAprokaryoticgene,aftercentrifugationofliquidor filteredandre-suspendedairsamples.Formolds,twomajorchallenges areconfronted:theycannotbeefficientlyrecoveredfromliquidairsamplesbyusingcentrifugationprotocolssimilartothoseusedforbacteria andarchaea(datanotshown,manuscriptinpreparationandsee Fig.1) anditisdifficulttochooseagenomicregionthatwillleadtoarepresentativeimageofbioaerosolcontent.Theeukaryoticconservedregions, 18Sgeneandinternaltranscribedspacer(ITS)regions,canbeuseddependingonthecontextofthestudy.Eventhoughtheseregionshave highlyconservedfunctionsintheirrespectiveorganisms,thereare somedistinctdifferencesbetweentheirvariabilityineukaryotesand thoseinprokaryotes.Forexample,whiletheV6regionin16Shas beenconsideredasavariableandwellsuitedforassessingbacterialdiversity(Huseetal.,2008),itsequivalentinthe18Sregionismoreconservedineukaryotes,whichisoftenavoidedforthisreason(Sogin, 1991;Hadziavdicetal.,2014).Inaddition,oneofthedisadvantagesin usingmolecularmethodsisthatthemajorityofprotocolsarespecific toeachprojectanddifferfromonestudytoanother.Speci ficgenes fromdifferentorganismscanalsobedetectedusingmolecularapproaches.Virulencefactorgenes,metabolicoperons,andantibioticresistancegenescanbequantifiedfromenvironmentalsamples (Chizhikovetal.,2001;Martínez,2008;Richardsonetal.,2004).David Stahldevelopedtheconceptofmolecularmicrobialecologyonthe basisofthesequencingofthe16SrRNAgeneregionandcomparedit withdifferentdatabases(Amannetal.,1990).Thisconceptwasused inthelastdecadetostudythemicrobialdiversityinenvironmental samples.However,weneedtobeawareoftheparticularitiesandpitfallsofthesemolecularmethods,especiallywhenusingthemwithair samples.Virusesdonotpossessuniversalandconservedmarkers, therebymakingitimpossibletouseablindedmolecularapproach,unlessviralmetagenomeisretrievedbynext-generationsequencing (NGS)methods(Posada-Cespedesetal.,2016).Filtrationstepsforconcentratingthebiologicalmaterialinairsamplesshowedlittleefficacy whenrecoveringvirusesfromairsamples.Thesizeofparticlescarrying airbornevirusesisnotknownasitmayvarydependingonconditions linkedtotheaerosolization.Inaddition,thevirusescontainedinthe dropletnuclei,thedriedresidueformedbyevaporationofaerosolized droplets,maynotberesuspendedduringliquidsamplingandcouldremainasacomplexvirus-containingparticle.Ingeneral,itisnotpossible toconcentratevirusesbecausetheycanbeshelteredinaparticlewhose sizeisnotpredictable,notknown,ormayvaryfromsampletosample orevenwithinagivensampleorthesizeofthevirusesmayvaryfrom thatoftheparticletheyaretrappedin.Forexample,Lindsleyandcollaboratorsshowedthatalthoughinfluenzavirusesare80 120nminsize, theycouldbefoundin1-to4-μmparticlesinhospitalsettings (Lindsleyetal.,2010;Verreaultetal.,2008).
Althoughmolecularapproachesmightnotprovidedirectinformationondisseminationandtransmissionofdiseases,crucialinformation canbeobtainedwhenusedtodetectDNAsequencesofmicroorganisms inanenvironment.Indeed,theseapproachesenableustodirectlylink aerosolcontenttothesourceandtobetterevaluateaircontamination. Forexample,detectionofhighconcentrationsof Aspergillusfumigatus inacompostingenvironmentbyusingqPCRisagoodindicatorthat workersmightbeathigherriskofdevelopinghealthproblems,regardlessoftheviabilityof A.fumigatus inoursamples.Thus,usingtheNGS approach,themostabundantmicroorganismsinanenvironmentcan befound.Subsequently,qPCRcanbeappliedtoobtainaspecificconcentrationpercubicmeterofagivengenus.Finally,theroleofthisgenusin thetransmissionanddisseminationofdiseasescanbeinvestigated.
Fig.1 showstheproposedstandardizedexperimentalprotocolappliedforstudyingthemicrobialecologyofanaerialenvironmentby usingmolecularapproaches,particularlytheNGSapproach.Depending onthetypesofmicroorganismsassessedandtheenvironmentsanalyzed,everystepiscritical.Thesamplingstrategyincludingthenumber andtypesofreplicates,volumeofairsampled,microorganismconcentrationmethod,nucleicacidextractionprotocol,genomictargetstobe amplified,sequencingtechnology,andbioinformaticswork flowshould becarefullychosenandspecificallyadaptedtomeettherequirements oftheproject.
2.Molecularapproaches vs. culture-basedmethods
Exposuretobioaerosols,includingairborneinfectiousagents,indoor allergens,fungalagentsorviruses,isusuallydeterminedbycollecting suchparticlesinsolid,liquid,oragarmedia.Thisisfollowedbythequalitativeand/orquantitativeanalysesofthesamplesbyusingmicroscopic,microbiological,biochemical,andimmunochemicaltechniques, whichofferdifferentdegreesofsensitivityandspecificity.Unfortunately,notallmicroorganismscanbecultured.Therefore,examiningonly thecultivablefractionleadstoanunderestimationofthetotalairborne microorganismconcentrationinasample.Inadditiontothevariations inculturingabilityofmicroorganismsinanenvironment,aerosolization andsamplingprocessescreateadditionalstressesthataffectthecapacityofmicroorganismstogrowinaculture(Heidelbergetal.,1997). Molecular-basedmethodscanleadtoabacterialconcentrationofup to3ordersofmagnitudehigherthanculture-basedmethods(Nehme etal.,2008).Conversely,animportantfactorthatshouldbeconsidered whenusingmoleculardetectionisthatnotallorganismspossessa knownuniqueregioncataloguedindatabases;therefore,notallorganismscanbeidentifiedbythesameway.Virusesrepresenttheprincipal exampleofsuchcases.Moreover,contrarytothecurrentdogma,Shade andhiscollaborators(Shadeetal.,2012)demonstratedthatculturebasedmethodscouldrevealrarebacteriapresentinacommunity. Theirresultsshowedthatbacteriadetectedbyculture-basedmethods wereeitherlessabundantorabsentwhenstudiedbymolecularapproaches.Theseobservationssuggestthat,tohaveatrueportrayalof microbialrichness,culture-basedmethodsshouldbeperformedin combinationwithmolecularmethods.
Becauseofthelesssequencingcosts,multiplestudieshavetriedto establishtherelationshipbetweentheculture-basedandmolecularapproacheswhenstudyingthemicrobialdiversity.Thisdemocratization canbevisualizedin Fig.2 thatshowsthedistributionofthenumberof studiesusingculture-dependentandculture-independentapproaches todeterminethemicrobialdiversityoverthepast7yearsonascaleof 50randomlyselectedstudies.Theselectioncriteriawerebasedonstudiesthatusedeithercultureorsequencingapproachestostudyabroad rangeofmicroorganismsinanenvironment.Studiesthatfocusedona particularphylumwereeliminated.Asshowninthegraph,theuseof culture-independentmethodshascontinuouslyincreasedsincethe NGSmethodshaveinvadedthemarket.However,somestudiesused boththeapproachesonthesamesamplestodeterminetheircommon potentialandrelevance.Althoughtheculture-dependenttechniques
providecomplementaryinformationsometimes,moststudiesshoweda deeperdiversityprofilewhenusingculture-independenttechniques. Table1 presentsareviewofsomestudiesinthelast5yearsthatdemonstratedthelinkbetweenculture-basedandmolecularapproacheswith theiroutcomewhendeterminingthemicrobialdiversityinsamples fromvariousenvironments.
3.Bioaerosolsampling
Variousaerosolcollectorshavebeendevelopedtocaptureandanalyzeairborneparticulatematterforairbornepathogendetectionand quantificationandforbiodiversitydetermination.Airsamplersoperate ineitherofthetwoways: filtrationorgravitational/inertialforces.There areseveralavailableairsamplersthatcanbeusedforsubsequentmolecularbiologicalanalyses.In filtration-basedairsamplers,aerosolsare collectedindifferenttypesof filtersandthenremovedfromthe filters byusinganelutionbuffer(Crook,1995 ).Itismandatorytoremove theparticlestobeanalyzedfromthe filterstoavoidanyinhibition fromthe filtersinthefollowingmolecularanalyses(Desprésetal., 2007).Amongairsamplersthatoperateusinggravitational/inertial forces,themostcommontypeusedforbioaerosolsisthecyclone, whereparticlesfromtheairaredirectlyassociatedwiththeliquid. Afterconcentratingthemicroorganismsfromtheliquid,molecularbiologicalmethodcanbeappliedformicrobialdiversityanalyses(Maron etal.,2005 ).Electrostaticsamplerscanalsobeusedforbioaerosols, whereparticlesarecollectedaccordingtotheirchargesaftertheyare passedthroughanelectrostatic field(DixkensandFissan,1999).After particlesaredepositedonamediumsuchasaliquid,theycanbeanalyzedbymultiplemoleculartechniques.Oneoftheadvantagesofelectrostaticsamplingisthatwecanchoosethesizeoftheparticlestobe analyzed.Insuchcases,dustparticleinterferencepresentsamajorchallengeinairbornemicroorganismsampling.Dustparticles,whichare largelypolydispersedandheterogeneousinnature,mayinterferewith aerosolcollectionanddetectionmethods(Moonetal.,2009).Theauthorsofthelatterstudysuggestedanewmethod, i.e.,dielectrophoresis, fortheseparationofbiologicalparticlesfromdust.Environmentalfactorssuchastemperature,relativehumidity,andultravioletradiations fromdifferentaerosolsourcesaffectthebehaviorandfateoftheaerosolizedparticles.Inaddition,microorganisms'nature,whetherpathogenicornot;concentration;relativeresistancetostress;andthetype ofparticletheycontainmustbeconsideredwhenchoosingthe bioaerosolsamplingmethod.
Anotherfactorthatcanaffectthechoiceofairsamplingapproachis thenucleicaciddegradation,especiallywhenstudyingRNA.When studyingmetatranscriptomics(theentiretranscriptmaterialofanenvironment)orRNAviruses,itbecomesimportanttogiveparticularcareto RNAdegradation.Indeed,theribonucleases(oftenshortenedtoRNAses)areubiquitousinnatureandarecapableofdegradingRNAinto smallerfragments(Ohtanietal.,1999).Therefore,usingalongsampling
periodcanleadtothedegradationofnucleicacids,especiallyRNA.This phenomenonofRNAdegradationisexacerbatedwhen filtersanddry samplersareused.Turgeonetal.showedthatwhen filterswereused forairsampling,theenzymaticactivityofinfluenzaAH1N1neuraminidasedecreasedcomparedwiththatwhenawetcyclonesamplerwas used.TheyalsoshowedthattheneuraminidaseenzymewasmorestablethaninfluenzaRNAgenomewhen filterswereusedforairsampling (Turgeonetal.,2016).Evidently,ashortersamplingtimetoavoidthe long-termeffectofRNAsesandtheuseofawetsamplershouldbeaprioritywhenlookingforthedetectionandquanti ficationofRNAsegmentsororganisms.Theconditionsandthemechanismsbehindthe RNAdegradationfollowingaerosolsamplingarenotwellunderstood. Unfortunately,totheauthors'knowledge,thereisnoaerosolstudyaddressingRNAviruses'stabilitypriortoRT-PCRandtheexacthalf-lifeof RNAvirusesduringlong-termsampling.
4.Bioaerosolemissionpatternsandsamplingstrategies
Someenvironmentsandhuman-relatedactivitiescanleadtothe generationoflargenumbersofbioaerosolsandnumerousairborne pathogenicagents.Aconcreteexampleisindustrialfacilitiesthathost confinedanimalfeedingoperations(swinebarns,dairyfarms,poultry, etc.),wherethebioaerosolsourcesarenumerousandvarywithtime andspace(Nehmeetal.,2008;Lecoursetal.,2012;Oppligeretal., 2008).Theseconditionsleadtoaconstant,highbioaerosolgeneration inconfinedspaces,andsometimeswithlimitedairchangeratesand subsequenthumanexposure.Despitethehighbioaerosolgeneration rates(atsometimepoints),theairstillrepresentsamuch-dilutedmediumformicroorganismsthanotherenvironments.Incontrasttosolid orliquidmattersuchasapileofcompostoralakewhereparticlesare enclosedina finitespace,therearenophysicalboundariesforaerosolizedparticlesastheyaremoreeasilydispersed(BrownandHovmøller, 2002).Ahigh-volumesamplingregimeisnecessarytoconcentrate bioaerosolcontentandtoincreasetheprobabilityofdiscoveringrare microorganismspresentinthisdilutedbiomass.Thevolumesampled islinkedtotwoparameters:the flowrateandthetime.High-volume samplingcanbeachievedbyahigh flowrateofsamplingforarelatively shortperiodoftimeorbyalow-to-moderate flowrateofsamplingfora longerperiodoftime.Boththescenariosenhancetheoddsofrevealing rareevents.Anexampleillustratingtheimportanceofthehigh-volume samplingforlow-concentrationpathogendetectionwasdemonstrated in2012whenNorovirusGIIRNAwasrecoveredfromairsamplesin healthcarecentersinEasternCanadabyusingthehigh-flowratesamplerCoriolis μ,whichwasoperatedat200L/min(Bonifaitetal.,2015). Thenatureoftheaerosolizationsourceanditsenvironmentalsoaffect thetypeofsamplingstrategypreferred.Forexample,apointsource, suchasacoughorasneeze,inaclosedroomrequiresalowvolumeof airtobesampledtocoverthewholearea.Therefore,itwilltakeless timetorecoverthetotalityofwhatwasemittedbythepointsource
Fig.2. Distributionofthenumberofstudiesusingculture-dependentandculture-independentapproachestodeterminethemicrobialdiversityoverthepast7yearsonascaleof50 randomlyselectedstudies.
Table1 Studiesshowingthelinkbetweentheculture-dependentandculture-independentapproacheswhendeterminingmicrobialdiversityinvariousenvironments.
Sampling environment Culture vs. molecularoutcomeReferences
Bioaerosols (compost)
Areviewonbioaerosolsemittedfrom compostingfacilitieshighlightingthe importanceofusingculture-independent approacheswhenstudyingbioaerosols diversity.Fromthe53mostabundant species,only11weredetectedby culture-dependentmethods
Wéry,2014
time(e.g.,onesampleperdayforaweek)tobetterestimateemissions fromthesourceandevaluateexposure(Wangetal.,2015).
5.Organismpeculiarity:whatarethemainchallenges?
Plants(Cucurbita pepo)
Thenumberofgeneradetectedwith pyrosequencing(Perrottetal.,2013)was higherthanthenumberthatcouldbe cultivated(Nehmeetal.,2008).
Food(sausages)Sequencingtechnologiesgavemore detailedsubpopulationsatthespecieslevel, whichwerehardlyhighlightedthrough culture-dependentapproaches.
Rhizosphere (Thymuszygis)
ThenumberofobservedOTUsdetectedby next-generationsequencingwas3orderof magnitudehigherthanthecultured bacteria.
Soil(mountain)Theisolatedgenera,whichbelongedtothe phylum Firmicutes and Actinobacteria, were fastcultivars,whereasfewmembersof Proteobacteria wereuncultivableevenwith differentcultivationmediaandconditions.
Brewing(rice wine)
Soil (petrochemical plant)
Comparativeanalysisrevealedthatsome speciescouldonlybedetectedusing sequencingtechniqueandsomespecies couldonlybedetectedby culture-dependentmethod.
Only8.2%ofthebacterialoperational taxonomicunitsweresharedbetweenthe culturedependentand culture-independentapproaches.
Food(cheese)Althoughfewisolatescouldbeidentified onlybyculturedependentmethod,the culture-independenttechniqueproduceda completeinventoryofthebacterialspecies encounteredwithintheOscypek ecosystem.
endotracheal tube
Therewaslittlecorrelationbetweenthe resultsobtainedbysequencingandby cultivation.Pyrosequencingidentifieda muchwidervarietyofbacteria.
InsectPyrosequencingrecovered445microbe operationaltaxonomicunits(OTUs)not detectedwithtraditionaltechniques.
HumanskinWhencomparedtotheculturedisolates, culture independentmethodshowed greaterfungaldiversityonallskinsites sampled.
WaterInspiteoftheexpectedmismatchbetween thebacteriatargetedbyculturedependent andculture-independentmethodsthe resultsofthisstudydemonstratethe relevanceofcultivationapproachesto characterizebacterialcommunities.
HumanfecesThediversityindices(Shannon-Weaver) obtainedwithculturetechniqueswere lowerthanthoseobtainedwiththe moleculartechniques,whichindicateda superiorsensitivityofthemolecular technique.
Breast-milkPyrosequencingdemonstratedthat bacterialdiversityinbreastmilkisbyfar higherthanmeasuredby culture-dependentmethods.
Eeversetal., 2015
Fontanaetal., 2016
Pascualetal., 2016
Venkatachalan etal.,2014
Lvetal.,2015
Molecularapproachesforbioaerosolstudywere firstappliedonbacteria(Alvarezetal.,1995;Langeetal.,1997;Orsinietal.,2002).Althoughothersamplecontentcouldinhibitmolecularreactions, molecularapproachesforbacteriaandarchaea(Nehmeetal.,2008; Lecoursetal.,2012)aremorestraightforward,andless fine-tuningis necessary.Whenanalysisofmoldsandvirusesisnecessary,severalobstaclesmustbeovercome.
5.1.Moldsrecoveredfromairinaliquidsample
Stefanietal., 2015
Alegríaetal., 2012
Vandecandelaere etal.,2012
Kautzetal., 2012
Findleyetal., 2013
Vaz-Moreira etal.,2012
Becauseanindividualairbornemicroorganismcontainsanoftenundetectableamountofnucleicacids,concentrationofmicroorganismsis necessarybeforeapplyingmoleculartechniquestoasample.Oneofthe waystorecoverfungifromtheairistouseasamplerwherethemold sporesaresuspendedinaliquid.Theirconcentrationisthenclassically foundbycentrifugingliquidsamplesandresuspendingthepelletsina smallervolume(Vaccarietal.,2006).Centrifugationisacommonly usedtechniqueforconcentratingbacteriaandarchaea(Jacobsenand Rasmussen,1992;CullisonandJaykus,2002;Lucoreetal.,2000)and canbeappliedforseveraltypesofsamplesandenvironmentalaerosol samples(Nehmeetal.,2008;Lecoursetal.,2012;Cayeretal.,2007; Nehméetal.,2009).However,arecentstudydemonstratedthatcentrifugationisnotappropriateformoldsporesandmaterialrecovery(Manuscriptinpreparation).Acomparisonbetweenculture-basedandqPCR methodsbothusingacentrifugationprotocolpriortoDNAextraction ledtoinconsistentresults,withtheculture-basedmethodshowing moreconcentratedmicroorganismsinsamples.Fungiareparticularly difficulttocentrifugeastheyhavemanyparticularitiesincluding charges,hydrophobicity,anddensity,mainlybecauseoftheirspecial structureandthefungalprotein “hydrophobin,” whichmakesthem water-repellentmicroorganisms.Inthisstudy,anewprotocolforconcentratingmoldsporespriortonucleicacidextractionwasdeveloped. Sampleswere filteredusingpolycarbonatemembranesandfrozen (flash-frozen)inabuffersolution,afterwhichthefrozen filterswere pulverizedusingstainlesssteelbeadsandvortexing.Resultsfrom usingthistechniquewerecomparedwiththosefromusingcentrifugationprotocols,whichshowedthatthecollectedDNAfromsporesby usingcentrifugationprotocolswasupto3ordersofmagnitudehigher thanthatcollectedusingthenewlydevelopedin-houseprotocol(Manuscriptinpreparation).Thissimpleand firststepfortherecoveryof moldmaterialfromairsamplesthereforerevealsmajorproblemsand leadstounderestimation.
5.2.Viruses
Molesetal., 2013
Jostetal.,2013
thaninahomogenouslycontaminatedopenarea.Incontrast,large environmentalsourcessuchascompostpiles,wastewatertreatment facilities,andswinebarnsnecessitatelongersamplingtimesandsometimesrequiremultiplesamplescollectedoveranextendedperiodof
Virusesinwatersamples, e.g.,seaanddrinkingwaters,areoftenconcentratedthrough filtrationorultracentrifugationpriortomolecular analyses.Similartothatforbacteriaandfungi,thisstepofconcentration isalsomandatoryforvirusespriortoapplyingmolecularapproachesto studytheminanaerialenvironmentalsample.Incontrasttothecommonthinking,airbornevirusesarerarelyfoundassingleentitiesbut areratherpresentasnumerouscopiesattachedtoparticlesofvarious micrometersizes(Lindsleyetal.,2010;Verreaultetal.,2008).This statementsuggeststhatthestandarduniversalapproachesusedtoenhanceviruses'concentrationarenotapplicabletoairsamples.Because ofthesmallsizeofviruses,theyareusuallyisolatedandconcentrated by filtrationorcentrifugation.Clearly,concentratingthemfromanaerosolsampleisachallengingtaskbecauseoftheirsmallsizeastheymay betrappedindifferentanddifficulttopredictrangesofparticlesizes. However,otherorganismsmayneedtobetreateddifferentlywhenairborne.Evidently,thestressoftheaerosolizationmayleadvirusesto
behavedifferentlycomparedtootherenvironmentalsamples.Thiscan beseeninametagenomeanalysisoftheairborneenvironmentin urbanspaceswherevirusesoutnumberbacteria;however,theirsequenceswereunder-representedthanthoseofbacteria,probablybecausetheapproachusedismoresuitableforbacteriathanviruses(Be etal.,2015).
Anotherlimitingfactorinthedetectionandquantificationofviruses, includingairborneviruses,isthelackofauniversalsequence.Unlikethe 16Sgeneforbacteriaand18Sgenes/ITSregionsformolds,virusesdo notshareequivalentsequencesasnogeneorgenomicregionishomologousacrossallviruses.Consequently,viraldetectioncanonlybe achievedbytargetingaspecificgenusorfamily(Labrieetal.,2013).Anotherwaytodetectabroadrangeofvirusesistousemetagenomics (Posada-Cespedesetal.,2016).Thus,ageneralselectionofRNAor DNAvirusescanbemadebytreatingnucleicacidswithDNasesorRNases.Itisoneofthewaysusedtotargetaspecifictypeofviruses.
5.3.Nucleicacidextractionapproaches
Thenucleicacidextractionprocesscangreatlyaffectthesequencing analysesbyusingeitherPCRorshotgunmetagenomicmethod. Table2 showsseveralexamplesofstudiesthatusedmolecularapproaches,in whichtheextractionprocessplayedamajorroleintheobtainedresults. In2010,Morganetal.compareddifferentDNAextractionkitsandsequencingprotocolsbyusingastandardizedsample.Theyconcluded thatthecombinationofthesetwofactorsconsiderablyaffectedtheobservedrelativeabundancesoforganismsinthesample(Morganetal., 2010).Moreover,airsamplesarefundamentallyoverdilutedcompared tosamplesfromotherenvironments,whichmayrevealrareeventsand couldleadtopotentiallylargerbiases.Asthetargetedorganismsare ofteninlimitedconcentrations,competitionwiththebackgroundcontaminantsisarealthreattotheauthenticityoftheresults.Thisisparticularlytruewhenworkingwithsamplescontainingalowmicrobial biomass.Therefore,usingdifferentnegativecontrolsiscrucialinthis contexttosegregatethebiologicalmaterialfromthesamplesfrom anycontaminantintheextractionkit.Itremainsachallengeto findmolecularbiologykitsthatarecompletelyfreeofresidualnucleicacids. However,ina2014study,thenucleicacidcontaminationbackground wassubtractedfromdatasets,whichledtoacompletelynewinterpretationoftheresults(Salteretal.,2014).
Theextractionprocesscanleadtodifferentnucleicacidrecoveries dependingontheorganism.ForsomeDNAviruses,qPCRmaybe
performedwithoutDNApurificationbecausetheheatshockusedtoactivatetheDNApolymeraseissufficienttoliberatetheDNAfromthe capsidofallDNAphagestested(Qiu,2012).However,comparisonsof theqPCRyieldwithandwithoutDNApurificationhavetobemadefor allDNAvirusesbeforeassumingthatthisstepisnecessary.ForRNAviruses,nucleicacidextractionismandatory.Thechoiceoftheextraction kitandreagentswillalsogreatlyaffecttheextractionyield.Aprevious studycomparedthenucleicacidrecoveriesoftwoRNAvirusesby usingtwodifferentextractionmethods(Qiagen'sQIAampViralRNA MiniKitandInvitrogen'sTRIzol®RLSReagent).RNAextractionwas performedonasolutionof1000plaqueformingunitspermilliliterof purecultureofPhi6andMS2phages(Gendronetal.,2010).Forboth thephages,the firstextractionmethodgaveahigheryieldthanthesecondone.Additionally,theqPCRquantificationforMS2wastwoorders ofmagnitudehigherthanthatforPhi6,evenwhenthesamequantity ofphageswasused.ThisresultmaybeduetotheRNApurification,reversetranscription,and/orqPCRbeinglessefficientforphagePhi6than MS2.TherecoveryoffungalDNAisalsoaffectedbytheextractionprocessasenzymaticlysisandmechanicalagitationoffungalcellwallsmay leadtodifferentqPCRyieldsdependingonthetypeoffungidetected (Fredricksetal.,2005).Morganetal.documentedthebiasesgenerated bynucleicacidextraction(Morganetal.,2010).Insomecases,itwas possibletonormalizetheoutputtocompensateforless-efficientqPCR detectioninthedataanalysis.Theseobservationshighlighttheimportanceofensuringthatsamplesarepreparedusingaprotocolthatisspecifictothetypeoforganismweareworkingwithandtheneedfor cautionwhencomparingstudiesthatusedifferentmethods.
Thereareonlyafewpublished,peer-reviewedstudiesofair metagenomesorwholegenomessequencedfromacomplexenvironmentalsample.Arecentpaperdescribingthemetagenomefoundin urbanair(Beetal.,2015)observedthatthemostabundantsequences arefrombacterialcells.However,itisknownthatvirusesoutnumber bacteriabyafewordersofmagnitudeintheenvironment(Breitbart andRohwer,2005).Virusesarelikelyunderrepresentedinthesequences forthreereasons:theviralentitiesinairsamplesmaybedifficulttocapture,theirgeneticmaterialcouldbedegradedduringthesamplingprocess,andtheirsmallgenomesmakethemlesslikelytobesequenced againstmillionsofothersequences.Althoughthereisanobviousobstacle forconcentratingvirusesinairsamples,thebestapproachforstudying thiscommunityistheisolationofvirus-likeparticlesbysizefractionation andsequencingthemusingmetagenomics.Thus,moreeffortshouldbe madeforincreasingviruses'concentrationinairsamples.
Table2 Examplesofsomestudiesthatshowcasesthepossiblebiasesintroducedbytheextractionapproachesapplyingmolecularmethodstoenvironmentalsamples.
TargetMethodPrincipal findingsofbiases
References
Bacteria-Eukaryota-ArchaeaMetagenomics'TherelativeabundancesoforganismsvariedsignificantlydependingontheDNAextractionand sequencingprotocolutilized. Morgan etal.,2010
BacteriaMetagenomics'& PCR-basedsequencing
BacterialDNAcontaminationinextractionkitscansignificantlyinfluencetheresultsofmicrobiota studiesinlowmicrobialbiomass. Salteretal., 2014
Virus(RNA)QuantitativePCRDifferentrecoveryyieldsdependingontheextractionprotocol. Gendron etal.,2010
FungiQuantitativePCRDifferentrecoveryyieldsdependingontheextractionprotocol.
Fredricks etal.,2005
BacteriaPCR-basedsequencingUnderestimationofsomephylawhenapplyingchemicalcelllysisextractionprotocol Zhanand Guo,2012
BacteriaPCR-basedsequencingThebacterialdistributionvariedsignificantlydependingonextractionmethodsused(beadbeating vs. silicagelcolumns) Starkeetal., 2014
Bacteria-Fungi-ArchaeaQuantitativePCR& PCR-basedsequencing
MajorimpactofDNAextractiononmicrobialcommunitystructureasnoneofthetenextractionkits testedresultedin100%comparablebacterial,archaealandfungalcommunitycomposition quantitativelyandqualitatively. Henderson etal.,2013
Viruses(DNA&RNA)QuantitativePCRDifferentrecoveryyieldsdependingonthelevelofinhibitorspresentinthesampleandonthe extractionkitused Ikeretal., 2013
BacteriaPCR-basedsequencingExtractionmethodswerethesecond-greatestcontributingfactortovariationincommunitystructure. Brettetal., 2015
BacteriaPCR-basedsequencingContaminationcanaffectinterpretationofresultsinmicrobiomeanalysis. Weissetal., 2014
6.Fromtheenvironmenttobioaerosols
Evenifairborneparticlesinanenvironmentareoftenlinkedtoa knownsource,theproportionoforganismspresentinthatsourcemay notbethesameasthatintheair.Thespeciesofmicroorganisms foundinthesourcewilllikelyberecoveredinairsamples,buttheirproportionscangreatlydiffer.Thiswas firstsuggestedbyParkeretal. (Parkeretal.,1983),whoproposedthatrespiratorypathogenssuchas Mycobacterium spp.and Legionella spp.couldbefoundinhigherconcentrationsinaerosolsfromtheenvironment.Astudycomparingbiogas microbialcontentwiththatofananaerobicdigestorsuggestedthat somemicrobialphylaareoverrepresentedintheairthaninthesource (Molettaetal.,2007).Studyofaerosolsincompostfacilities(Veillette etal.,2016;MBarecheetal.,2015 )showedthatproportionsof Proteobacteria incompostpilesweredifferentfromthoseintheairsamples.Somespeciessuchas Methylobacterium wereoverrepresentedin airsamplesthanincompost.Additionally, Legionella wasthemost abundantairbornespeciesinacarcasscompostfacilitybutwasnotdetectedincompostsamples.Thisislikelyduetoitsverylowconcentrationrelativetothoseofotherspecies.Conversely,somespeciessuch as Pseudomonas sp.werepresentinthecompostpilesbutwerenotrecoveredfromairsamples.Theseobservationsconfirmthatenvironmentalsourcescannotbeassumedtomirrorwhatispresentinthe surroundingair.Thisisaconceptknownaspreferentialaerosolization. Thehypothesissuggeststhatsomebacteriamaybepreferentiallyaerosolizedcomparedtoothers.Arecent invitro studyshowedthatsome virulentstrainsof Streptococcussuis werepreferentiallyaerosolized comparedtootherstrains(Gauthieretal.,2016)andthataerosolization ofgram-negative(P.aeruginosa)andgram-positive(S.aureus)bacteria areeithermoreviableorconcentrated(PCR)in invitro aerosols(Perrott etal.,2013).Although invitro studiesareaproofofconcept,further studiesthatincludealargernumberofstrainsandothertypesofbacterianeedtobeconductedtobetterunderstandtheprocessofpreferentialaerosolization.
7.Challengesinthebioinformaticspipeline
Currently,microbialdataareobtainedpredominantlyusing16S rRNAgenesequencingsurveysthatprovideacharacterizationofbacterialandarchaealdiversity.Instudiesusing16SrRNAgenesequencing, thechoiceofprimersetdependsonanumberoffactors,includingcompatibilitywithpreviousstudiesandthespecificitiesoftheprimers (Soergeletal.,2012).16SrRNAgenesequencedatafromdifferentmicrobialenvironmentspresentbioinformatical,statistical,andcomputationalchallenges.ThemostwidelyusedbioinformaticstoolsareQIIME andmothur(Caporasoetal.,2010;Schlossetal.,2009).Bothpackages areopensourceandhaveonlinetutorialsandforumsthatprovidethe usersastep-by-stepanalysisofthe16SrRNAgenesequences.
IntheNGSPCRanalysis,errorsarecommonandaresometimesdifficulttodetect.Chimeras,whicharecausedbytheincompleteextension ofDNAstrandduringamplificationthatresultsinarecombinationbetweenthetwosequences,cancausebiasesindiversityresults,particularlythealphametrics(Haasetal.,2011;Leyetal.,2008).Multiple chimeracheckingsoftwaresareavailable,andtheyoftenhavedifferent filteringmethods.Oneshouldchoosethesoftwarethat fitsadequately totheproject(Haasetal.,2011;Edgaretal.,2011;Wrightetal.,2012; Quinceetal.,2011).
Data filteringisoftenperformedatthesamestageas demultiplexing,whereeachsequenceisassignedtoasampleonthe basisofthebarcodeinformation.Qualitythresholdincludesquality scores,readlength,andthepresenceofambiguousbasecalls.After quality filtering,themicrobialgroupsmustbeidentifiedthroughOTU analyseswheresequencesareclusteredtogetheraccordingtothesequenceidentity.Infact,therearedifferentOTUclusteringalgorithms, andtheuseofthesealgorithmsconsiderablyaffectsthe findingsandinterpretationsofdata.In denovo OTUclustering,sequencesareclustered
intoOTUsbycomparingthemwiththewholedataset,withouttheuse ofareference(SchlossandHandelsman,2005).Incontrast,closed OTUclusteringusesareferencesequencedatabase,whereinthesequencesthatdonotmatchthereferencesequencedatabaseare discarded.Open-referenceOTUclusteringisatwo-stepprocessthat combinesboththeabovealgorithms.First,closed-referenceOTUclusteringisdonefollowedby denovo clusteringofsequencesthatfailto matchthereferencedatabase.Eachoftheclusteringmethodshaspros andcons,anditshouldbechosenwithaparticularattention.Forexample,ifdatasetsthathavesequencesfromdifferentregionsofthe16S rRNAgeneneedtobecombined,theclosedreferencealgorithmshould beused,assequencesfromdifferentregionsofthesame16SrRNAgene wouldclusterintodifferent denovo OTUs.Incontrast,usingtheclosed referencealgorithminsamplesfromanundiscoveredenvironment mayleadtoahighpercentageofthesequencesbeingdiscardeddue toafailuretomatchtoareferencedatabase.
LabelingtheOTUsisthenextstep,whichisperformedusingtaxonomyassignmentalgorithmwithareferencedatabase.Forthe16SrRNA gene,thethreemaindatabasesareGreengenes,RibosomalDatabase Project(RDP),andSILVA(McDonaldetal.,2012;Coleetal.,2009; Quastetal.,2013).Themicrobialdiversityanalysisistypicallydescribed withinsamples(alphadiversity)andbetweensamples(betadiversity). MultiplescriptsfordatavisualizationareavailablethroughQIIME, mothur,andRstatisticsoftware.Aparticularattentionshouldbe givenwhenchoosingtheclassificationandclusteringmethodsdependingonthemetadatainformationavailableaboutthesamples.Forexample,classificationmethodscanbeusedtodeterminewhichtaxadiffer betweenpredefinedgroupsofsamples.However,clusteringmethods donotmakeuseofanypriorknowledgeaboutthesamples.Both methodsusebetween-sampledistancemetrics,andthespecificitiesof eachmethodusedcanaffecttheoutcomeandtheinterpretationof theanalyses.Therefore,itismandatorytouseseveraldifferentways ofclassificationandclusteringtoensurethattheexistenceofclusters isnotdependentononlyonesetofparameters.Finally,statistical modelingcanbeusedtoexplainthevariationsobservedbetweenthe differentsamples.Asmanycovariatescanbeincludedandmanyparameterscanbestatisticallycontrolled,westronglyrecommendthe consultationofastatisticianorabiostatisticsexpertduringtheexperimentaldesignandtheanalysesstages.
Characterizationoffungalcommunitiesisanareathatneedsmore research.Althoughthebioinformaticspipelinemayseemtobethe sameforeukaryoticmarkergenesasthatforbacterialmarkergenes, therearenostandardizedbioinformaticsprotocolsfor16SrRNAgene analyses.Thismaybebecauseofthelackofastandardmarkergene andareferencedatabase.Asmentionedintheintroduction,several markergeneoptionsexist.However,theITSregionisgenerallypreferredforobtaininghightaxonomicresolution.Becauseoftheamplicon lengthlimitationsimposedbythesequencingplatforms,onlyoneofthe twoITSsubregions(ITS1orITS2)canbeusedinasequencingrun.To thebestofourknowledge,therearenorelevantstudiesthatexplored thepotentialofboththesubregionsinthecharacterizationoffungal communitiesinanenvironment.Astrategythatisbeingexploredina bioaerosolprojectusingmultipleenvironmentsusesboththesubregionstodemonstratetheirpotentialandrelevanceforaglobalfungal characterization,therebyresultinginastandardizedmethodology.
ThebioinformaticsworkflowusedfortheITSsequencesdiffers greatlyfromtheoneusedforthe16SrRNAgeneinthealignmentalgorithm,astheITSregionisofdifferentlengthscomparedtothe16SrRNA gene.Therefore,OTUclustering,referencedatabases,taxonomyassignment,diversitymetrics,datavisualization,andstatisticalmodeling shouldbespecifictoITSsequenceanalysesforthecharacterizationof fungalcommunities.TheUNITEdatabaseisoftenusedforITS sequence-basedanalysesoffungalsequences(Abarenkovetal.,2010).
However,the18SrRNAgenecangenerallybeusedtoanalyzeeukaryoticcommunitiesinthesamemanneras16SrRNAgenesare used.TheSILVAdatabasecontainsmanyeukaryoticregionsandcan
beusedforsuchanalyses.However,oneshouldconfirmthattheregion ofthe18Sgeneamplifiedvariesdependingonthetaxastudiedand shouldbeawarethatthe18SrRNAgeneisnotsufficienttocharacterize thefungaldiversityandwilllikelyenduptobeofquestionableutility.
Characterizingtheviromeofanenvironmentrequiresadifferentapproachbecause,asdiscussedearlier,nogeneorgenomicregionishomologousacrossallviruses.Metagenomicsisthesolutionforthe absenceofamplificationprimers.Inaddition,metagenomicdataare alsousedtoportrayfunctionalpotential.Thebioinformaticsworkflow consistsmainlyofprocessingtheNGSreadsbyremovingtheadapter first.Subsequently,low-qualityandless-complexsequencesare subtractedbasedonqualityscores.Specificquality filteringconditions canbeadaptedfordifferentdownstreamanalyses(Bokulichetal., 2013).BacterialandrelatedrRNAreadsareremoved,andtheremaining readsarealignedtoavirusdatabase.Differentalignmentsoftwaresare available. Denovo assemblyalgorithmsreconstructoriginalgenomes presentinthesamplebymergingshortgenomicfragmentsintolonger sequencescalledcontigs.Therearetwomaintypesof denovo assembly programs:Overlap/Layout/Consensusassemblersthataremoresuitable forlongerreadsanddeBruijnGraphAssemblersthataresuitablefor shorterreads.Metagenomicreadscanbetaxonomicallyclassifiedinto similarity-andnonsimilarity-basedmethods.Similarity-basedtaxonomicclassificationsareperformedbyNCBIBLASTsearcheswheresequencesarecomparedtoknowngenomes.However,alargenumber ofsequencesgeneratedfrom denovo assemblyareunknown.Toclassify sequencesonthebasisofthealignmentresults,severalmethodssuchas MEGANcanbeused(Husonetal.,2007).MEGANusesthelowestcommonancestornodeofallblast-matchingsequencesinthephylogenetic tree,whichreducestheriskoffalse-positivematches.Incontrast, nonsimilarity-basedmethodsexplorethecompositionofthegenomes lookingforparameterssuchask-mersandareusefultoclassifysequencesthatdonothaveanyhomologsinreferencedatabases.Many programsareavailabletoperformthiscomposition-basedmethodon viralsequences(McHardyetal.,2007;Mohammedetal.,2011;Wang etal.,2012).ProgramssuchasPHAACS,CatchAll,andUCLUSTcanbe usedonthecontigsspectrageneratedbyCirconspecttoestimateviral diversity(Anglyetal.,2005;Bungeetal.,2012;Anglyetal.,2006).
Althoughthebioinformaticsworkflowsdescribedearlierweredevelopedfordifferentenvironments,manyoftheissuesapplytomicrobialcommunitiesofdifferenthabitats.Therefore,theycanbeappliedto bioaerosolsamplesaswell.Thisreviewencouragesresearchersentering the fieldtousemoleculartoolstocharacterizethemicrobialcontentof bioaerosolsindifferentenvironments.Thisactiveresearchareaneeds morestandardizedprotocolsthatwillhelpbuildabioaerosolsequence catalog,whichwillinturnallowcross-comparativestudiesgloballyand givethis fieldofstudyagreatimpetus.
8.Conclusion
ImprovementsinsequencingtechnologiesledtoadecreaseinDNA sequencingcostsandanincreaseinsequencingspeed.Thesetechnologiesarewidelyusedinclinicalsettingsbutlessinexperimentalsettings. Theapplicationofthesetechnologiestothestudyofbioaerosolsisa necessarynextstep.Itcanbesaidthatthemolecularmethodscanbe easilyappliedtobioaerosolsamples.However,manytechnicaldetails needtobe fine-tunedtoobtainmolecularresultsthatarerepresentative ofthesampledenvironment.Modificationofknownprotocolsneedsto beconsideredateverystepofanexperimenttoensurethattheoptimumconditionsforaspecificsituationarebeingused.Dependingon thetargetgenesand/ororganisms,adjustmentsmayberequired startingwiththeinitialsamplingprocess.Whenexaminingbioaerosols, otherdownstreamstepssuchascellisolationandconcentration,nucleic acidisolation,andestablishmentofdatabasesalsohavetoberetested. Optimizationcansometimesbealongandhazardouspath,butomitting thiscriticalstepwillgreatlydecreasetheaccuracyoftheresults.
Acknowledgments
TheauthorsacknowledgeAmandaKateToperoffforEnglishrevision ofthemanuscript.EBreceivedInstitutderechercheRobert-Sauvéen santéetsécuritédutravailduQuébec(IRSST)scholarship.
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