Quantification of bacterial populations in complex ecosystems using fluorescent in situ hybridization, confocal laser scanning microscopy and image analysis

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ecosystems using fluorescent in situ hybridization, confocal laser scanning microscopy and image analysis

T. Bouchez, P. Dabert, M. Wagner, Jean-Jacques Godon, Renatto Moletta

To cite this version:

T. Bouchez, P. Dabert, M. Wagner, Jean-Jacques Godon, Renatto Moletta. Quantification of bacterial

populations in complex ecosystems using fluorescent in situ hybridization, confocal laser scanning

microscopy and image analysis. Genetics Selection Evolution, BioMed Central, 2001, 33 (Suppl1),

pp.S307-S318. �hal-02671752�

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Original article

Quantication of bacterial populations

in complex ecosystems using uorescent

in situ hybridization, confocal laser

scanning microscopy and image analysis

Théodore Bouchez

a,c

,Patrick Dabert

a,∗

, Michael Wagner

b

,

Jean-Jacques Godon

a

,René Moletta

a

Laboratoiredebiotechnologiedel'environnement,Institutnationaldelarecherche

agronomique,AvenuedesÉtangs,11100 Narbonne,France

b

TechnischeUnivertitätMünchen,Arcisstr. 16, D-80290München,Germany

c

ENGREF,19avenueduMaine,75015Paris,France

Abstract Aprocedureforquanticationofdistinctbacterialpopulationsinaggre-

gatedecosystemswasdeveloped.Itisbasedonuorescentinsituhybridizationcou-

pledwithconfocal-laser-scanningmicroscopyandsurfacemeasurementofhybridized

bacteria byimage analysis. The proportion ofa targetedbacterial species was ob-

tained by comparison between the surface hybridized by a specic probe and the

surfacehybridizedwithageneralbacterialreferenceprobe. Theaccuracyofthere-

sults obtainedwas evaluated by directvisual countingon thesame sets of images.

Theanalyticaluncertainty oftheimage analysisprocedurewas determinedandde-

pendedonthethresholdvaluesselectedbytheoperatorforcuttingbetweensignaland

backgrounduorescence. Thenumberofeldstobeanalyzedfor reliablequantica-

tionwasalsodetermined. Thisprocedureallows aquanticationoftheproportions

ofbacterialspeciesinaggregatedbacterialecosystemswhichgivesmoreaccuratere-

sultsthanvisualcountingbecauseofthelargenumberofbacteriacounted.Moreover,

sinceocstructuresarepreserved,italsogivesinformationaboutthedistributionof

bacterial populations in the oc materialwhichmight reveal bacterial interactions

withinthecommunity.

uorescentin situ hybridization/bacterial quantication/image

analysis/activated sludge

Résumé Quantication des populations bactériennes des écosystèmes

complexespar hybridation insitu en uorescence,microscopie lasercon-

focale et analysed'image. Uneprocéduredequanticationde populationsbac-

tériennes agrégées a étédéveloppée. Elle repose surl'analyse d'images obtenues à

∗

Correspondence andreprints

(3)

l'aide d'un microscope laserconfocal aprèshybridationin situ enuorescence. La

proportiond'uneespècebactériennecibleestobtenueparcomparaisonentrelasur-

facehybridéeparunesondenucléiquespéciqueetlasurfacehybridéeparunesonde

générale bactérienne deréférence surunesérie d'images données. L'exactitude des

résultats obtenusa étéévaluéeparcomptagemanuelsurles mêmeséries d'images.

L'incertitude de la procédure d'analyse d'image dépend de la taille de l'intervalle

choisi par l'opérateur comme étant représentatif des seuils de niveau de gris entre

signal positif etbruit de fond. Il est également possible de déterminerle nombre

dechamps nécessaire àprendre encomptepour obtenirunequanticationvalable.

La procédure développée permet de quantier les proportions des espèces bactéri-

ennes présentes dansun écosystème agrégé (ocs, biolms...). Étant donné qu'un

grandnombredebactériesestprisencompte,les résultats obtenussontplusprécis

que ceux obtenus parcomptagemanuel. Deplus, les structuresbactériennesétant

préservées, il est possible d'obtenir des informations concernant la répartition des

espècesauseinduoc. Lanaturedecetterépartitionpeutnousaideràcomprendre

lesinteractionsbactériennesauseinduoc.

hybridationin situ enuorescence /microscopie laserconfocal /

quanticationbactérienne /analysed'images /boues activées

1. INTRODUCTION

Ecological studies on bacterial ecosystems were longrestricted to the cul-

tivable fraction of microorganisms. We know that today only 0.1% to 15%

of the bacteria present in acomplex ecosystem, such as soil, sediments, sea-

wateroractivated sludges, arecultivable [3]. This methodological limitation

hashinderedsignicantadvancesinmicrobialecologyofcomplexecosystems.

Nowadays, techniques based on 16S rRNA sequences have considerably ex-

tended our knowledge in the eld. These cultivation-independent techniques

are commonlygroupedunder thedesignation ofrRNA approach". They en-

compassacharacterizationstep,byPCR-amplicationandsequencingofrDNA

molecules presentin anenvironmental sample, and a monitoringstep, either

by the use of sequence-specic DNA separation techniques (DGGE, SSCP,

T-RFLP...) orbytheuseof16SrRNAtargeted-oligonucleotideprobes. Inthe

latterstrategy,FluorescentInSitu Hybridization(FISH)isparticularlysuited

to ecological studies since it enables the direct visualization of the targeted

populationwithin itsenvironment. Itthusprovidesusefulinformationonthe

spatialorganizationandontheparticularlocationofthedierentspeciesinthe

ecosystem[7,9]. However,untilnow, FISHhasnotallowedrapid andreliable

countingofaggregatedbacterialpopulations.

Because of the aggregated nature of activated sludge, direct microscopic

countingofthenumberof probe-targetedbacteriaafterFISHwasalwaysdif-

cultandtedious. Quanticationhadeither tobedonemanuallybycounting

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orusingdisintegrationtechniques,whichcandamagethesample[1]. Toover-

cometheselimitationsweproposedaprocedurebasedonthecomputeranalysis

ofimagesofhybridizedbacterialsurfaces. Theobjectiveof theprocedure was

to quantifythe surfaceareaof bacteriahybridizedby aspecic probetarget-

ing the microorganisms of interest against the total surface area of bacteria

hybridizedwith ageneralbacterialprobe. Theprocedurewasdevelopedona

confocallaserscanningmicroscope.

2. MATERIAL ANDMETHODS

2.1. Samplexation

Activatedsludgesampleswereretrievedfromlaboratory-scalenitrifyingre-

actorstreatingsyntheticwastewater. Thesereactorshavebeendescribedelse-

where [4]. Sampleswere xedbyadditionof4%paraformaldehydephosphate

bueredsolutionaspreviouslydescribed[6]exceptthatthesamplecentrifuga-

tionstepswerereplaced bygentleltration onnylon0.2 mltersto preserve

ocstructure.

2.2. Fluorescentin situ hybridization

Fluorescentinsituhybridizationwith16SrRNAtargetedprobesandsubse-

quentwasheswereperformedonslidesaccordingtoconventionalprotocols[6].

Forthequanticationprocedure,samplesweresimultaneouslyhybridizedwith

a specic probe of interest labeled with Cy3 and a general bacterial probe

EUB338 labeled with Cy5. After hybridization, the slides were air dried

andmountedwithCitiuoranti-fadingAF1solution(CITIFLUORLtd,UK).

Observations were performedon aconfocal laser scanningmicroscope (Zeiss,

LSM510)equippedwithimageanalysissoftware(LSM510forWindowsNT4.0).

Foreachprobepair,randomlyselectedmicroscopiceldsof1

µ

^m^thickness^were

recorded with a40X objectiveusing the543and 633 nmlaser for excitation

ofCy3 andCy5, respectively. Typically,10to 20microscopiceldswerecap-

turedwithconstantmicroscopeparametersandgain. Imagesweredigitizedin

1024 × 1024

^pixels,⁸ ^bits^gray. Ârticial^blue ând ^red^colors^wereâpplied ^to

Cy5andCy3hybridized bacteria,respectively.

The probesused in thisstudy and theirrecommendedhybridizationstrin-

gency conditionsaresummarized in TableI.These probeswerepurchasedas

HPLC-puried, Cy3 or Cy5-labeled, oligonucleotide derivatives (Interaktiva,

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ableI.FluorescentlylabeledrRNA-targetedprobesusedinthisstudyandrecommendedhybridizationconditions. ProbenameProbeSequenceTargetedrRNA%formamideTargetedorganisms

5 0 − 3 0

moleculeinthe[Ref.] andhybridization E.colipositionbuer EUB338GCTGCCTCCCGTAGGAGT16S(339-355)$Mostbacteria[2] Alf968TGGTAAGGTTCTGCGCGT16S(968-986)35AlphasubclassofProteobacteria [8] GAM42aGCCTTCCCACATCGTTT23S(1027-1043)35GammasubclassofProteobacteria [6] CF319aTGGTCCGTGTCTCAGTAC16S(319-336)35Cytophaga-Flexibactercluster [9] ACAATCCTCTCCCATACTCCTA16S(652-669)35GenusAcinetobacter[10] NEU23aCCCCTCTGCTGCACTCTA16S(653-670)40Halophilicandhalotolerantmembersofthe genusNitrosomonas[11] CTETTCCATCCCCCTCTGCCG16S(659-676)40UsedasacompetitorforNEU23a [11] S-

∗

-Ntspa-1026-a-A-18AGCACGCTGGTATTGCTA16S(1026-1043

◦

)20GenusNitrospira;Nitrospiramoscoviensis andaliatedclones[5] NIT3CCTGTGCTCCATGCTCCG16S(1030-1047)40GenusNitrobacter[12] ForprobeEUB338,anyformamideconcentrationbelow70%issuitable. probeS-

∗

-Ntspa-1026-a-A-18doesnottargetNitrospiramembersrelatedtoN.marina.

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Figure 1. Determination of the threshold values for surface area measurement of

thebacterialpopulations. Top. Zoominofadigitizedimageofanitrifyingactivated

sludgesamplesimultaneouslyhybridizedwiththeCy3-labeledNEU23aprobespecic

of the Nitrosomonas group(in red)and the Cy5-labeled EUB338 generalbacterial

probe (inblue). Bottom. Greylevelproleobtained across thered lane drawnon

theimage.

3. RESULTS

3.1. Determinationof the thresholdvalues for surface area

measurementofthe bacterial populations

Sludgesampleswere spottedonaslideandsimultaneouslyhybridizedwith

the Cy3-labeled NEU23aprobespecic to theNitrosomonas groupof micro-

organisms andthe Cy5-labeled EUB338generalbacterialprobe. Microscopic

elds were captured and digitized. A zoom in of a digitized image is shown

in Figure 1 top. The non-Nitrosomonas bacteria appeared in blue, the Ni-

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generalbacterialprobeEUB338andthespecicNEU23aprobe. Wecanseethe

typicaltightlyaggregatedmicrocoloniesformedbytheNitrosomonas bacteria.

Thecritical stepofthesurfaceareameasurementprocedure wasobviously

thedetermination ofthe thresholdvaluesthat should be usedto cutbetween

bothpositivesignals (Cy3and CY5 labeledprobes) and thebackground u-

orescence. Rather than arbitrarily selecting a given value, possible intervals

for threshold valueswere determined. On a set of 3 to 5images, alane was

drawnacrossthe image and a proleof intensity of the detectedsignals was

obtainedasshowninFigure1bottom. Bycomparisonbetweentheimageand

thegraylevel intensitiesrecorded, aconservativethreshold value,that gave

alarge surfaceareaofhybridized bacteria, anda stringent threshold value,

that minimized the surfacearea of hybridized bacteria, were determined. In

Figure 1, the conservative threshold valuesfor the blue and the red colors

wereset at 20 and100 gray levels, respectively. The stringent valuesfor the

blue and the redcolors were set at 50 and 170graylevels, respectively. The

surfaceareaofthehybridizedbacteriawas measuredusingthefourestimated

threshold values. The four possible surfacearea-ratios(red-100/blue-20;red-

100/blue-50;red-170/blue-20andred-170/blue-50)werecalculated. Themean

valueofthefourcorrespondingsurfacearea-ratioswastakentobethenalre-

sult(Tab.II).Thestandarderrorbetweenthedierentratiosobtainedonthe

sameimagewastakenasanestimateoftheuncertaintyoftheimageanalysis

procedure.

Foragivensampleandthesamehybridizationexperiment,weobservedthat

the threshold values between dierent images were almost constant. These

manuallydeterminedthresholdvalueswerethereforeappliedtoalltheimages

ofthesameseriesbyanautomatedsoftwarecommand.

3.2. Numberof eldsto beanalyzed

Thenumberofmicroscopiceldsrequiredfor statisticallyreliablequanti-

cationcanbedetermined. Itdependsontheregularityofthedistributionofa

particular species in theocmaterial. An exampleisgiven in Figure2using

asetofimages(400xmagnication)takenfromasludgesamplehybridizedsi-

multaneouslywithprobesCy3-NEU23aandCy5-EUB338. For20microscope

eldstherelativeabundanceofNEU23a-positiveNitrosomonas cellswascom-

puted by surfacearea-ratiomeasurements. After analysis of ten microscopic

elds, stabilization of the mean value for Nitrosomonas abundance was ob-

served(Fig.2A).DespitethehighlyirregulardistributionofNitrosomonas sp.

microcolonies within the ocs, thestandard deviation of their relativeabun-

dance also tended to stabilize after examination of 10-12 microscopic elds

(Fig. 2A). Similar results were obtained for quantication of other probe-

dened bacterial populations (data not shown). For some bacterial popula-

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ableII.SurfaceareameasurementoftheNitrosomonaspopulation. NEU23a/EUB338surfacearea-ratios(%)Analyticalerror(%) FieldsThresholdsThresholdsThresholdsThresholdsMeanStandardStandard 170/20170/50100/20100/50byelddeviationerror eld119.822.024.327.023.32.71.3 eld279.681.784.788.583.63.31.7 eld351.456.662.367.759.56.13.1 eld459.062.866.370.264.64.12.1 eld588.691.494.196.692.73.01.5 eld667.776.085.094.180.79.94.9 eld760.362.765.368.564.23.01.5 eld896.199.8103.9108.3102.04.62.3 eld988.890.892.995.492.02.51.2 eld1078.982.185.589.183.93.81.9 eld1143.247.251.155.149.24.42.2 eld1276.880.484.087.682.24.02.0 Mean68.171.374.778.473.14.32.1 error:standarddeviation/

√ n u m b er of m eas ur es effectued

.

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Figure 2. Determinationofthe numberofeldsto beanalyzed. Plot, againstthe

numberofmicroscopicelds,ofthemeanvalue()andthestandarddeviation()

of the surface area-ratio : (a) of cells hybridized with probe NEU23a to that of

cellshybridizedwiththebacterialprobeEUB338. (b)ofcellshybridizedwithprobe

CF319atothatofcellshybridizedwiththebacterialprobeEUB338.

meanvalueandofthestandarddeviationwasachievedwithfewerelds indi-

cating amoreequaldistributionof these microorganismsin theocmaterial

(Fig.2B).Examinationof12microscopiceldswasconsideredto besucient

forsubsequentquanticationexperiments.

3.3. Accuracy ofthe surface area-ratio methodofcounting

Toevaluatetheaccuracyofthesurfacearea-ratiomethodforquantifyingthe

abundanceofabacterialpopulation,wecomparedtheresultsobtainedbysur-

faceareameasurementswiththoseobtainedbydirectmanualcounting. Since

allthenitrifyingpopulationsmonitored(Nitrosomonas,NitrobacterandNitro-

spira)formedverydenseaggregates,itwasnotpossibletocounttheirnumbers

accurately. We were therefore unable to performthis comparison on nitrify-

ing populations. The comparison was made on a sludge sample hybridized

with theCy3-labeledACA probespecic of thegenus Acinetobacter and the

EUB338probe. A G=1000magnicationwasuseto allowvisual countingof

thebacteriainfourmicroscopicelds. Ofthe3630bacteriacountedthatwere

hybridizedwith theprobeEub338,453(12.5%)werealsohybridizedwith the

ACAprobe. Onthesamesetofpictures,application ofthesurfacearea-ratio

method gaveusaratioof13.9%withastandarddeviationof2.4%depending

onthedierentvaluesofthresholdused. Themanualcountingttedwellwith

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3.4. Distributionof the dierentmicroorganisms in the oc

material

Thesurfacearea-ratiotechniquewasusedto evaluatetheproportionofthe

dierent bacterialpopulationspresentin anautotrophicnitrifyingreactoraf-

ter stabilization of the performance of the system. Around 10,000 bacterial

cellsweretakenintoaccountforeacheldanalyzed. Atthetimeofsampling,

the ecosystemwascomposed essentiallyof nitrifyingbacteria(Nitrosomonas,

Nitrobacter, Nitrospira) which added up to 97.9% of the microbial commu-

nity hybridized area. Among them, the ammonia oxidizing bacteria Nitro-

somonas seemedto bethemajorones withabout70%of thehybridizedbac-

terialarea(Tab.III).Thenitriteoxidizingbacteria,representedbyNitrobacter

andNitrospira,correspondedto20.8and7.3%ofthehybridizedbacterialarea,

respectively.

The dispersion coecients varied from one species to another, indicating

forexamplethatthenitrifyingbacteriaweremoreunequallydistributed than

membersofthegammasubclassofProteobacteriaormembersoftheCytophaga-

Flexibactercluster. Tworeasonscouldbegiventoexplainthisdierence. First,

qualitativeobservation oftenreportedthat thenitrifyingbacteriawere aggre-

gatedin clusters within theocmaterial. Second, theprobeused in thecase

ofthegammaProteobacteria andmembersfromtheCytophaga-Flexibacter are

moregeneralthanthe genus-specicprobeused fornitrifyingbacteria. They

could thus hybridizeto a potentially morediverse population. This tends to

give amore equaldistributionfor the total population monitoredwith these

probes.

4. CONCLUSIONS

The procedure presented here is a semi-automated treatment of digitized

images of activated sludge samples hybridized with bacterial rRNA targeted

uorescentprobes. Itquantiestheproportionsofthedierentbacterialpop-

ulationsoftheecosystemandgivesinformationabouttheirlocationanddistri-

butionwithintheocmaterial. Itismorerapidandmoreprecisethanmanual

countingbecause of the large number of cells taken into account. The uo-

rescentin situ hybridization lasts about ve hours overall, and thecomplete

analysis of onesamplelasted about onehour, includingthecapture of about

40images(20eldswithtwodyes) andtheimage analysisprocess. Assuming

that acellhasameanapparentsurfaceof1

µ

^m

²

^,^eachmicroscopiceld ana- lyzed(400

×

⁾^containedât^least^5,000^bacteria,^so^that^with¹²êlds ânalyzed

atleast60,000bacterialcellswereanalyzedforeachquanticationexperiment.

This procedure can be used to compare and monitor the microbial popu-

lations of complexecosystems [4]. It should beemphasized howeverthat the

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ableIII.Quanticationofthedierentbacterialsubgrouppresentintheocofanitrifyingreactor. PopulationanalyzedNumberofMeanMeanVarianceDispersioncoecient eldsanalyzedValueStandarderror(%)(%) (%)(%) Nitrosomonas1269.82.23.95.6 Nitrobacter1220.81.31.36.4 Nitrospira127.30.20.811.3 AlphasubclassofProteobacteria1425.21.11.45.6 GammasubclassofProteobacteria124.00.30.12.0 Cytophaga-Flexibactercluster141.50.40.021.3 thatthepercentagesadduptomorethan100%becauseNitrobacteraremembersofthealpha-subclassofProteobacteria.

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the same parameters. Manual counting givesthe proportion of the targeted

bacterialpopulationin cellnumbers,while thearea-ratioprocedure givesthe

proportion of the bacterial areaoccupied by this population on a given oc

section. Whenthedepth ofthesectionanalyzedis keptconstantandthemi-

croscopicelds analyzedarechosenrandomly, thearea-ratiomethod givesan

estimateofthebacterialbiovolumeproportionoccupiedbyonegivenbacterial

population. As bacterialsizes are not constant from onespecies to another,

thisproportiondoesnotcorrespondtotherelativenumericalabundanceofthe

targetedbacteria. Thisisparticularlytrueifthesamesamplefeaturesawide

range ofbacterial sizes (e.g.,small single cellscompared to largelamentous

bacteria). Forexample,Nitrobacter andNitrospira aremuchsmallerthanNi-

trosomonas andforthesamehybridizedarea-ratiotheircellnumbersareprob-

ablythree to fourtimes higherthanthat ofNitrosomonas. Whenpopulation

dynamicsareanalyzedin accordancewithconcomitantactivitymeasurements

oftheecosystem,itisnotclear,however,whatbetterreectstheactivityofa

givenmicrobialpopulation: thenumberofcellsorthebiovolumeoccupied by

thisspecicpopulation. Finally,withinthesamebacterialpopulation,thecell

biovolumemaychangewithenvironmental parametersand/or thephysiologi-

cal stateofthecell. This longlist ofrecommendationsshould notdiscourage

use of this veryeective tool since, compared to other molecular destructive

tools,italwaysallowsavisualcheckofthecell'sstatein thesamples.

TheprobeS-*-Ntspa-1026-a-A-18wasoriginallydesignedtotargetactivated

sludge clones aliatedto Nitrospiramoscoviensis. However,as theattempts

madetoisolatethesebacteriawereunsuccessful,theirinvolvementinthenitri-

cationprocesswasnotclearlydemonstrated[5]. Inourreactor,operatedunder

strictlyautotrophicnitrifyingconditions,themaintenanceofbacteriahybridiz-

ingwiththisprobesuggestsarealimplicationoftheNitrospiramoscoviensis-

relatedpopulationsin thenitrifyingactivityoftheecosystem.

Theextensionofthis procedureto conventionalepiuorescencemicroscopy

isunderway. Comparedtoconfocallaserscanningmicroscopy,themajorprob-

lem is the thickness of the samples which results in signals originating from

aboveandbelowthefocusplaneandinterfereswiththesurfaceareameasure-

ment. Samplesmustbesubjectedtoadispersiontreatmentsothatthecellsare

disposedinasinglelayeronthemicroscopicslide. Thesurfacearea-ratiomea-

surementtechniquecanthen beapplied to these partially dispersed samples.

However, as the initial structures are not preserved, it is no longer possible

to obtain information on the distribution of the bacterial species in the oc

material.

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