The Ecological Dynamics of Fecal Contamination and Salmonella Typhi and Salmonella Paratyphi

18 Pages·2015·1.54 MB·English

RESEARCHARTICLE
The Ecological Dynamics of Fecal
Salmonella
Contamination and Typhi and
Salmonella
Paratyphi A in Municipal
Kathmandu Drinking Water
AbhilashaKarkey1☯,ThibautJombart2☯,AlanW.Walker3,4,CorinneN.Thompson5,6,
AndresTorres7,SabinaDongol1,NgaTranVuThieu5,DuyPhamThanh5,DungTranThi
Ngoc5,PhatVoongVinh5,AndrewC.Singer8,JulianParkhill3,GuyThwaites5,6,
BuddhaBasnyat1,NeilFerguson2,StephenBaker5,6,9*
1 OxfordUniversityClinicalResearchUnit,PatanAcademyofHealthSciences,Kathmandu,Nepal,2 MRC
CentreforOutbreakAnalysisandModelling,DepartmentofInfectiousDiseaseEpidemiology,Schoolof
PublicHealth,ImperialCollegeLondon,London,UnitedKingdom,3 TheWellcomeTrustSangerInstitute,
Hinxton,Cambridgeshire,UnitedKingdom,4 MicrobiologyGroup,TheRowettInstituteofNutritionand
Health,UniversityofAberdeen,Aberdeen,UnitedKingdom,5 TheHospitalforTropicalDiseases,Wellcome
TrustMajorOverseasProgramme,OxfordUniversityClinicalResearchUnit,HoChiMinhCity,Vietnam,
6 CentreforTropicalMedicine,OxfordUniversity,Oxford,UnitedKingdom,7 GrupodeInvestigación
OPENACCESS
CienciaeIngenieríadelAguayelAmbiente,FacultaddeIngeniería,PontificiaUniversidadJaveriana,
Citation:KarkeyA,JombartT,WalkerAW, Bogotá,Colombia,8 NERCCentreforEcologyandHydrology,Wallingford,Oxfordshire,UnitedKingdom,
ThompsonCN,TorresA,DongolS,etal.(2016)The 9 TheLondonSchoolofHygieneandTropicalMedicine,London,UnitedKingdom
EcologicalDynamicsofFecalContaminationand
SalmonellaTyphiandSalmonellaParatyphiAin ☯Theseauthorscontributedequallytothiswork.
*[email protected]
MunicipalKathmanduDrinkingWater.PLoSNegl
TropDis10(1):e0004346.doi:10.1371/journal.
pntd.0004346
Abstract
Editor:JohnA.Crump,UniversityofOtago,NEW
ZEALAND
OneoftheUNsustainabledevelopmentgoalsistoachieveuniversalaccesstosafeand
Received:June29,2015
affordabledrinkingwaterby2030.ItislocationslikeKathmandu,Nepal,adenselypopu-
Accepted:December9,2015 latedcityinSouthAsiawithendemictyphoidfever,wherethisgoalismostpertinent.Aiming
Published:January6,2016 tounderstandthepublichealthimplicationsofwaterqualityinKathmanduwesubjected
weeklywatersamplesfrom10sourcesforoneyeartoarangeofchemicalandbacteriologi-
Copyright:©2016Karkeyetal.Thisisanopen
accessarticledistributedunderthetermsofthe calanalyses.Weadditionallyaimedtodetecttheetiologicalagentsoftyphoidfeverand
CreativeCommonsAttributionLicense,whichpermits longitudinallyassessmicrobialdiversityby16SrRNAgenesurveying.Wefoundthatthe
unrestricteduse,distribution,andreproductioninany
majorityofwatersourcesexhibitedchemicalandbacterialcontaminationexceedingWHO
medium,providedtheoriginalauthorandsourceare
guidelines.Furtheranalysisofthechemicalandbacterialdataindicatedsite-specificpollu-
credited.
tion,symptomaticofhighlylocalizedfecalcontamination.Rainfallwasfoundtobeakey
DataAvailabilityStatement:Allrelevantdatafor
driverofthisfecalcontamination,correlatingwithnitratesandevidenceofS.TyphiandS.
thisstudyareavailablewithinitssupporting
informationfiles,andthesequencedataisavailable ParatyphiA,forwhichDNAwasdetectablein333(77%)and303(70%)of432watersam-
attheEuropeanNucleotideArchiveunderStudy ples,respectively.16SrRNAgenesurveyingoutlinedaspectrumoffecalbacteriainthe
AccessionNumberERP004371/SampleAccession
contaminatedwater,formingcomplexcommunitiesagaindisplayinglocation-specifictem-
numberERS373486.
poralsignatures.OurdatasignifythatthemunicipalwaterinKathmanduisapredominant
Funding:TJisfundedbytheMedicalResearch
vehicleforthetransmissionofS.TyphiandS.ParatyphiA.Thisstudyrepresentsthefirst
CouncilCentreforOutbreakAnalysisandModelling
andtheNationalInstituteforHealthResearch- extensivespatiotemporalinvestigationofwaterpollutioninanendemictyphoidfeversetting
HealthProtectionResearchUnitforModelling
Methodology.FundingforAWW,JPand454
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004346 January6,2016 1/18
BacterialContaminationofDrinkingWaterinKathmandu
pyrosequencingwasprovidedbytheWellcomeTrust andimplicateshighlylocalizedhumanwasteasthemajorcontributortopoorwaterquality
(098051).AWWandTheRowettInstituteofNutrition intheKathmanduValley.
andHealth,UniversityofAberdeen,receivecore-
fundingsupportfromtheScottishGovernmentRural
andEnvironmentalScienceandAnalysisService
(RESAS).SBisaSirHenryDaleFellow,jointly
fundedbytheWellcomeTrustandtheRoyalSociety AuthorSummary
(100087/Z/12/Z).Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto Aimingtounderstandtheecologyofmunicipaldrinkingwaterandmeasurethepotential
publish,orpreparationofthemanuscript. exposuretopathogensthatcausetyphoidfever(SalmonellaTyphiandSalmonellaParaty-
CompetingInterests:Theauthorshavedeclared phiA)inKathmandu,Nepal,wecollectedwatersamplesfrom10watersourcesweeklyfor
thatnocompetinginterestsexist. oneyearandsubjectedthemtocomprehensivechemical,bacteriologicalandmolecular
analyses.WefoundthatKathmandudrinkingwaterexhibitslongitudinalfecalcontamina-
tioninexcessofWHOguidelines.Thechemicalcompositionofwaterindicatedsite-spe-
cificpollutionprofiles,whichwerelikelydrivenbylocalizedcontaminationwithhuman
fecalmaterial.WeadditionallyfoundthatSalmonellaTyphiandSalmonellaParatyphiA
couldbedetectedthroughouttheyearineverywatersamplinglocation,butspecifically
peakedafterthemonsoons.Amicrobiotaanalysis(amethodforstudyingbacterialdiver-
sityinbiologicalsamples)revealedthewatertobecontaminatedbycomplexpopulations
offecalbacteria,whichagainexhibitedauniqueprofilebybothlocationandtime.This
studyshowsthatSalmonellaTyphiandSalmonellaParatyphiAcanbelongitudinally
detectedindrinkingwaterinKathmanduandrepresentsthefirstmajorinvestigationof
thespatiotemporaldynamicsofdrinkingwaterpollutioninanendemictyphoidsetting.
Introduction
Enteric(typhoid)feverisaseveresystemicinfectionandacommoncauseofcommunity
acquiredfebrilediseaseinmanylow-incomecountriesinAsiaandAfrica[1].Theinfectionis
triggeredbytheingestionofthebacteriaSalmonellaTyphi(S.Typhi)andSalmonellaParatyphi
A(S.ParatyphiA).BothS.TyphiandS.ParatyphiAarehumanrestrictedpathogens(they
havenoknownanimalreservoir)andisitacknowledgedthattheyaretransmittedthrough
contaminatedfoodandwaterorviacontactwithfecalmatterfromacuteorchronically
infectedindividuals[1].However,thepredominantrouteofinfectionhasneverbeenrigor-
ouslyinvestigatedinanendemicsettingoutsideaconventionalcase/controlstudydesign[2,3].
TyphoidfeverisacommoninfectioninKathmandu(thecapitalcityofNepal)andourpre-
viouslygeneratedserologicaldataimpliesthatthelocalpopulationhaslongitudinalexposure
tobothoftheseendemicpathogens[3,4].Furtherstudies,generatedthroughinvestigatingthe
spatiotemporaldynamicsoftyphoidfeverinKathmandupredictedthatbothS.TyphiandS.
ParatyphiAaremorelikelytobetransmittedthroughcontaminatedwaterthanviahuman-to-
humantransmissioninthissetting[5,6].Significantly,wefoundthattyphoidfevercasescluster
inareaswithahighdensityofurbanwatersources,whicharegravitydrivenand,therefore,
rationallylocatedatlowerelevations.ThevariousurbanwatersourcesinKathmanduaremost
commonlyintheformofsunkenwells(asfoundinmanyurbanandruralsettingsinlower-
incomecountries),pipedsuppliesintolargecommunalholdingtanksorthemoretraditional
stonewaterspouts(hitis/dhungedharas)[7].Theiconicstonespoutsarecommonacrossthe
Nepalesecapitalandthewaterflowintothesesacredlocationsisgravity-dependent(Fig1a),
replenishedbyrainfallandsnowmeltfromthesurroundingHimalayanMountains.Natural
soft-rockaquifersactasreservoirsforgroundwaterandultimatelytheuntreatedwaterenters
thestonespoutsfromtheaquifersthroughaseriesofancientporousundergroundchannels.
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BacterialContaminationofDrinkingWaterinKathmandu
Fig1.ThephysicalandchemicalpropertiesofwaterfromtensamplinglocationsinKathmandu,Nepal.a)Photographofchildcollectingwaterfroma
traditionalstonewaterspout(dhungedhara)inKathmandu.b)AmapshowingthelocationofthetensamplinglocationsinKathmandu.Thelocationsare
color-codedcorrespondingtootherfiguresandthesiteofPatanhospital(thehealthcarefacilityforthosewithtyphoidfever)ishighlightedbytheHsymbol.
(SeeTable1andBakeretal.[6]formoredetailsregardingthesamplinglocationsandthemappedarea).c)Scaledprincipalcomponentsanalysisofthe
physicalandchemicalpropertiesofwatersamplesfromthetensampledlocations,showingthefirsttwoprincipalcomponents(PC1-2).Individualwater
sampleareidentifiedbydotscoloredaccordingtotheirsamplinglocation(seeFig1a).Inertiaellipsesindicatetheoveralldistributionofeachwatersource.
Labeledarrowsindicatetherelativecontributionsofthevariablestotheprincipalcomponents,withlongerarrowsreflectinglargercontributions.The
screeplotofeigenvalues(inset)indicatestheamountofvariationcontainedinthedifferentprincipalcomponents,withPC1andPC2indicatedinblack.
doi:10.1371/journal.pntd.0004346.g001
Ourpreviousanalysisspecificallyidentifiedlocationsaroundthesestonespoutsarehotspots
forS.TyphiandS.ParatyphiAinfections[5,6].
Hypothesizingthatthelocalwater,particularlythewateraccessedviathestonespouts,isa
substantialpublichealthriskfortyphoidfeverandotherentericinfectionsinKathmandu,we
aimedtolongitudinallyassessbacterialcontamination,thechemicalcompositionandtheeco-
logicaldynamicsofentericbacteriainthewatersupplyinthislocation.Toaddressthishypoth-
esis,andfocusingonwatersourcesaccessedbythelocalpopulation,weeklywatersamples
werecollectedoveraone-yearsamplingperiodfromtenlocationsandsubjectedtovarious
physical,chemical,microbiologicalandmolecularanalyses.
Methods
Watersamplinglocations
Thewatersourcesforthisstudywerethetenmostcommonlyusedwatersources(identified
byquestionnaire)lyingwithinapreviouslyidentifiedtyphoidfeverhotspotinLalitpur,
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BacterialContaminationofDrinkingWaterinKathmandu
Kathmandu[6].TheselectedlocationswereGPSlocatedusinganeTrexlegend(Garmin)and
consistedoffivestonespouts,threesunkenwellsandtwopipedsupplies.Thelocationof
thesewatersourcesareshowninFig1banddescribedindetailinTable1.Dailyrainfalldata
fromKathmanduAirportwasprovidedbytheNepaleseDepartmentofHydrologyandMete-
orology(http://www.dhm.gov.np/)andaggregatedintoweeksforthepurposesoftheanalysis
presentedhere.
Collectionofwatersamplesforanalysis
Waterwascollected(whenpermittedbywaterflow),fromallofthe10locationsonceperweek
overoneyearfromMay2009toApril2010.Fromeachofthesourcesmid-flowwatersamples
werecollectedintwosterilebottlesinvolumesof1Land500ml.Fromthestonespoutsand
thepipedsupply,thestopperwasasepticallyremovedandfreeflowingwaterwasallowedto
flowdirectlyintothesterilebottle.Forwells,asterilizedsteelbucket(bleachedandwashed
withautoclavedwaterpriortouse)wasloweredintothewelluntilitwaspartiallysubmerged,
thebucketwasthenremovedandthewaterwaspouredintothebottles.Allthebottleswere
labeledwiththesourcecode,dateandtimeofcollectionofthesamples.Afterrecordingthe
watertemperaturethebottlesweretransportedtothelaboratoryatambienttemperatureand
wereprocessedwithinonehourforphysical,chemicalandmicrobiologicalanalysis.
Physicalandchemicalanalysisofwatersamples
TheKathmanduWaterEngineeringLaboratory(http://www.sodhpuch.com/water-
engineering-training-centre-p.)performedallchemicalandphysicalanalysesfollowingtheir
standardoperatingproceduresforinternationalwaterquality.Themeasuredvariableswere
pH(HannapHmeter,calibratedwithpH4,pH7andpH9buffers),temperature(Hannadigital
thermometerwithprobe),conductivity(Hannaconductivitymeter),color(PerkinElmer’s
LAMDA650UVspectrophotometerat270nm)turbidity(NEPHELOstarPlusnephlometer),
hardness(EDTAtitration),totalalkalinityasCaCO (methylorange),chloride(argentometric
3
titration),ammonia(nesslerisation),totalnitrate(PerkinElmer’sLAMBDA650UVspectro-
photometerat275nm),totalnitrite(PerkinElmer’sLAMDA650UVspectrophotometerat
275nm),andtraceelementsandheavymetals(AtomicAbsorptionSpectrophotometric(AAS)
method).AllvariableswererecordedonthedayofsamplingandcomparedtoWHOguide-
linesforwaterquality[8].
MostProbableNumber(MPN)methodformeasuringthermotolerant
coliforms
Amodifiedmostprobablenumber(MPN)methodwasusedtoassessthemicrobiologicalqual-
ityofthewater,specificallycoliformcontamination[9,10].Briefly,fiveten-foldserialdilutions
weremadefromeachwatersamplebyinoculating1mlofundilutedwatersampleinto9mlof
MacConkeybroth(Oxoid,UK).Thiswascontinueduntiladilutionof1x10−5.Atotalof30
tubes(fivetubesforeachdilution)werepreparedforeachsample.Theinoculatedbrothswere
incubatedat44°Cfor48hoursforthecultureofthermotolerantcoliforms.Afterincubation,
eachtubewasexaminedandthosethatwerepositive(productionofacidandgas)were
counted.Thenumberofpositiveandnegativetubesineachofthesethreesetswasnotedin
orderandthesedatawereusedtoestimatethecoliformcontentusingafive-tubeMPNtable.
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BacterialContaminationofDrinkingWaterinKathmandu
Table1. Watersamplinglocationsinthisstudy.
Location Source Latitude Longitude Depthto Source Treatment Additional Samples 16SrRNA
type water(m) protection collected/weeks geneanalysis
1 Stone N27° E85° Groundlevel Shallow Untreated None 40/53 No
spout 40.482 19.693 aquifer
2 Stone N27° E85° Groundlevel Shallow Untreated None 38/53 Yes
spout 40.461 19.693 aquifer
3 Stone N27° E85°19.51 Groundlevel Shallow Untreated None 51/53 No
spout 40.426 aquifer
4 Sunken N27° E85° 8.4 Shallow Untreated Meshcovered 51/53 No
well 40.454 19.665 aquifer
5 Sunken N27° E85° 7.1 Shallow Untreated Meshcovered 50/53 Yes
well 40.503 19.660 aquifer
6 Sunken N27° E85° 8.1 Shallow Untreated Meshcovered 50/53 No
well 40.545 19.719 aquifer
7 Piped N27° E85° Groundlevel Delivery Irregular Locked 51/53 No
supply 40.422 19.645 truck chlorination container
8 Piped N27° E85° Groundlevel Delivery Irregular Locked 50/53 No
supply 40.457 19.698 truck chlorination container
9 Stone N27° E085° Groundlevel Shallow Untreated None 21/53 No
spout 40.607 19.540 aquifer
10 Stone N27° E085° Groundlevel Shallow Untreated None 30/53 No
spout 40.607 19.540 aquifer
doi:10.1371/journal.pntd.0004346.t001
Directandenrichmentplatingforentericbacteriainwater
Todetectthepresenceofentericbacteriawithpathogenicpotential(e.g.Salmonellae,Shigellae,
Vibrionaceae,andE.coli)10,20,50,100and500μlofundilutedwaterwasdirectlyplatedonto
Xyloselysinedeoxycholate(XLD)andMacConkeyagarplates.Theplateswereincubatedat
37°Covernightandthenobservedforgrowth.ToincreasethelikelihoodofculturingSalmonel-
laeandShigellae,100mlofundilutedwaterwasfilteredthroughamembranefilterwithapore
size0.45μm(Whatman,GELifeSciences,PA,USA)usingasterilesyringe.Thefilterpaper
wasremovedusingsterileforcepsandplacedin90mloftypticsoyabroth(Oxoid,UK).The
soyabrothbottleswereagitatedusingavortextodisplacetheorganismsonthemembraneand
incubatedfor18hoursat37°C.Afterovernightincubation1mlofthepre-enrichmentculture
wastransferredto10mlofselenitebroth.Further,1mlofthepre-enrichmentculturewas
transferredto10mlofRappaport-VassiliadisBroth(RVB).Theincubatedovernightbrothwas
thenplatedontoXLDandMacConkeyagarplates.Theplateswereincubatedovernightat
37°Candthenobservedforgrowth.ForthedetectionofVibrionaceae,1mloftheundiluted
watersamplewasdilutedin9mlofalkalinepeptonewater.Thesuspensionwasthenincubated
overnightat37°CandthenplatedontoMacConkey,XLDandthiosulphate-citrate-bilesalts
sucrose(TCBS)agar.
Bacterialidentification
Thecolonymorphologiesincludingtheform,size,surfaceappearance,texture,color,elevation
andmarginofallindividualcolonieswererecordedfromMacConkeyandXLDplates.Ofspe-
cialinterestwerecoloniesthatwerecircular,withanentiremarginandslightlyraisedelevation
thatwerenon-lactosefermentingonbothplates,withorwithouttheproductionofhydrogen
sulphideontheXLDplate.Individualcolonieswiththeaforementionedcharacteristicswere
isolatedandplatedonnutrientagarandincubatedat37°Cfor24hours.Isolatedcolonies
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BacterialContaminationofDrinkingWaterinKathmandu
obtainedonthenutrientagarplateswerethensubjecttoAPI20EtestingtoidentifyEnterobac-
teriaceaeandothernon-fastidiousGram-negativerods.
TotalDNAextractionfromwatersamples
TotalDNAfromallwatersampleswasextractedusingtheMetagenomicDNAIsolationKit
forWater(EpicentreBiotechnologies,WI,USA).Watersampleswerecentrifugedat1,000
rpm(HettichZentrifugen,EBA21,Germany)for5minutestoremovelargedebrisandthen
decantedintosterilecontainers.Aftercentrifugation,100mlofthecentrifugedwaterwasfil-
teredthroughapre-sterilizedfilterwithaporesizeof0.45μm(Whatman,GELifeSciences,
PA,USA).Usingsterileforcepsandscissorsthemembranewasremovedfromthefilterappa-
ratusandcutintofourpieces.Thecutfilterswerethenplacedina50mlsterileconicaltube
withtheuppersurfaceofthefilterfacinginwards.Onemilliliteroffilterwashbuffercontaining
0.2%Tween-20wasaddedtothefilterpiecesinthetubestoremoveorganismsonthefiltersur-
face.Thetubewasagitatedathighspeedforapproximately2minuteswithintermittentbreaks.
Thecellsuspensionwastransferredtoacleanmicro-centrifugetubeandcentrifugedat14,000
Xg(ThermoFischerScientific,IECMicroCL17,Germany)for2minutestopelletthecells.
Thesupernatantwasdiscarded.Thecellpelletwasre-suspendedin300μlofTEbuffer,and
2μlofready-lyselysozymesolutionand1μlofRNAseAwereaddedandmixedthoroughly.
Thetubewasincubatedat37°Cfor30minutesandthen300μlof2Xmeta-lysissolutionsand
1μlofProteinaseKwereaddedtothetubeandthoroughlymixedbyvortexing.Toensurethat
allthesolutionwasatthebottomofthetube,thetubewaspulsecentrifuged.Thetubeswere
thenincubatedat65°Cfor15minutes.Thesolutionwascooledtoambienttemperatureand
placedonicefor5minutes.350μlofMPCproteinprecipitationreagentwasaddedtothetube
andmixedthoroughlybyvortexingvigorouslyfor10seconds.Thedebriswaspelletedbycen-
trifugationfor10minutesat14,000Xg(ThermoFischerScientific,IECMicroCL17,Ger-
many)at4°C.Thesupernatantwastransferredtoacleanmicro-centrifugetubeandthepellet
wasdiscarded.Tothesupernatant,570μlofisopropanolwasaddedandmixedbyinverting
thetubemultipletimes.TheDNAwaspelletedbycentrifugationfor10minutesat14,000Xg
(ThermoFischerScientific,IECMicroCL17,Germany)at4°C.Theisopropanolwasremoved
andthesamplewasbrieflypulsecentrifugedandanyresidualliquidwasremovedwithoutdis-
turbingthepellet.Tothepellet500μlof70%ethanolwasaddedwithoutdisturbingthepellet.
Thetubewasthencentrifugedfor10minutesat14000Xg(ThermoFischerScientific,IEC
MicroCL17,Germany)at4°C.EthanolwasremovedwithoutdislodgingtheDNApelletand
thesamplewasbrieflypulsecentrifugedandanyresidualfluidwasremovedwithoutdisturbing
thepellet.Thepelletwasthenairdriedfor8minutesatambienttemperaturebeforebeing
resuspendedin100μlofnucleicacidfreesterilewater(EpicentreBiotechnologies,WI,USA).
Real-timepolymerasechainreactiontodetectS.TyphiandS.Paratyphi
Ainwatersamples
QuantitativeReal-timePCRwasperformedonallextractedDNAtodetectDNAsequences
specificforS.TyphiandS.ParatyphiAaspreviouslydescribed[11].Primerandprobe
sequenceswereasfollows;S.Typhi;ST-Frt5'CGCGAAGTCAGAGTCGACATAG3',ST-Rrt
5'AAGACCTCAACGCCGATCAC3',ST-Probe5'FAM-CATTTGTTCTGGAGCAGGCT
GACGG-TAMRA3';S.ParatyphiA;Pa-Frt5'ACGATGATGACTGATTTATCGAAC3',Pa-
Rrt5'TGAAAAGATATCTCTCAGAGCTGG3',Pa-Probe5'Cy5-CCCATACAATTTCA
TTCTTATTGAGAATGCGC-BHQ53'.Briefly,5μlofenvironmentalDNAextractions(as
abovefrom100mlofwaterandresuspendedin100μlofnucleicacidfreesterilewater)
wasusedasthetemplateforeachexperiment,i.e.5μlequatedto5mlofwatersample.
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BacterialContaminationofDrinkingWaterinKathmandu
QuantificationwasperformedusingstandardcurveswhereplasmidDNAcarryingthetarget
sequencesweredilutedin10-foldserialdilutionsrangingfrom100to105plasmidcopies
perμl;standardcurvesforassessingS.TyphiandS.ParatyphiAcopynumberwerecon-
structedbyplottingtheCtvalueagainsttheplasmidDNAcopynumber.
16SrRNAgeneamplificationandsequencing
Forthe16SrRNAgenesurveying,variableregions3to5(V3–V5)ofthe16SrRNAgenewere
PCRamplifiedfromthewaterDNAextractions.Theprimersusedwereasdescribedpreviously
[12],seeS1Tableforthefullbarcodeandprimersequences(30nucleotidesfor454adaptor,12
nucleotidesforuniquerecognition(tag)and18nucleotidestoamplifythespecificV3–V5
region)usedforeachsampleinthepresentstudy.TheconditionsofPCRwereasfollows:1U
ofAccuPrimeTaqDNAPolymeraseHighFidelity(Invitrogen,Carlsbad,CAUSA),200mM
offorwardandreverseprimer,2μloftemplateenvironmentalDNAina20μlreaction.The
reactionwascycledfor1x94°Cfor2minutes,andthen20x(94°Cfor30seconds,53°Cfor30
secondsand68°Cfor2minutes).EachsamplewasPCRamplifiedonfouroccasions,theresult-
ingampliconswerepooledandthenethanolprecipitatedbeforeresuspensionin20μlofTE.
The16SrRNAgeneampliconswereshippedtoTheWellcomeTrustSangerInstituteand
pooledtogetherintoanequimolarmastermix,asmeasuredbyaQubitfluorometer(Invitrogen,
Carlsbad,CA,USA),priortosequencingonaGSFLXTitanium454machine(RocheDiagnos-
tics,Oakland,CAUSA)usingtheLib-Lkit.Theresultingsequencedataisavailableatthe
EuropeanNucleotideArchiveunderStudyAccessionNumberERP004371/SampleAccession
numberERS373486.Sequencedatawasprocessedusingthemothursoftwarepackage(http://
www.mothur.org/),followingapreviouslydescribedprotocol[12].Thisremovedpoorquality
reads,andgeneratedtaxonomicclassificationsforeachOperationalTaxonomicUnit(OTU).
Followingthesefilteringsteps326,155sequencesremained(rangeof1to7,396sequencesper
sample).
Statisticalanalysisofbacterialcontaminationdata
Wefirsttestedforgeographicdifferencesinbacterialassays,usingthenon-parametricMAN-
OVAimplementedinthepackageade4[13]fortheRsoftwaresuite[14].9,999randomper-
mutationsofthedatawereusedtoassessstatisticalsignificanceofPillai’sstatistic[15]and
computetheassociatedp-value.Afterrulingoutthepresenceofgeographicdifferences
betweensamples,datawereaggregatedacrosslocationsbycomputingaverageweeklyprofiles,
fromwhichtemporaltrendsweremorestraightforwardtoinvestigate.Asbacterialassaysmay
eachcapturedifferentaspectsofwatercontamination,thesedataweresubjectedtoacentered
PrincipalComponentAnalysis(PCA)[13],whichweusedtoderivealatentvariable(thefirst
principalcomponent,PC1)ascorrelatedaspossibletoallthedifferentassays[16]andthere-
forereflectingtheextentofbacterialcontamination.PCAisideallysuitedtoderivesynthetic
variables,whichcapturetheessentialtrendsofvariationinquantitativeorbinarydata,andis
thusreadilyapplicabletowaterqualitydataincludingphysicalchemicalproperties,bacterial
assaysandmeta-genomicvariation.
ThetemporaltrendsinPC1werevisualizedusingggplot2[17]andmodeledusingacubic
splineofsamplecollectiondates.Fivebreakpointswereusedinthemodelastheygavethebest
visualfit,andnoothernumberofbreakpointsledtosignificantlybettermodels.Thismodel
wascomparedtoamodelwherePC1wasconstantintimeusingaclassicalANOVAcompar-
ingtheresidualvariancesofthetwomodels.
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BacterialContaminationofDrinkingWaterinKathmandu
Statisticalanalysisofchemicalpollutiondata
Asforbacterialassays,thevariouschemicalpropertiesmeasuredcapturedpotentiallydifferent
aspectsofwaterpollution.PCAwasusedtoidentifythemaintrendsofvariationamongst
watersamples[18,19].Becausemeasurementsweremadeusingdifferentunitsandhadinher-
entlydifferentscalesofvariation,acenteredandscaledPCAwasused[20].Missingdatawere
replacedbytheaverageofthecorrespondingvariables,asiscustomaryinPCA[20].Resultsof
thisfirstPCAweredrivenbyanoutlier,whichturnedouttobeasampleofexceptionallypoor
waterquality.Insuchcases,becausePCAfindslinearcombinationsofvariableswithmaxi-
mumvariance,thepresenceofanoutliermayconcealotherinterestingstructures[20].Asa
consequence,thissamplewasremovedinasecondPCA,theresultsofwhichareshowninFig
2.DifferencesinwaterchemistryofthewatersourcesweretestedusingthesameMANOVA
procedureusedinbacterialassaysanalysis.
Spatiotemporaldynamicsofbacterialcompositionfrom16SrRNAgene
data
16SrRNAgenesurveydataconsistingof93samplesand11,212OTUswerefirsttransformed
intocompositionaldata[21],sothateachsamplewastransformedintoacompositionof
OTUsfrequenciessummingto1.Thistransformationensuresthatfurtheranalysiswillonly
reflectdifferencesintaxacomposition,andnotinabsolutequantitiesofsequencedDNA.The
P
Gini-Simpsonindexwascomputedforeverysampleas1(cid:1) p2wherep istherelativefre-
i i i
quencyofOTUiinthesample.AcenteredPCAwasusedtoanalyzethemetagenomicprofiles,
retainingthefirstfiveprincipalaxesastheyexpressedmostofthestructuredvariationinthe
data(Fig3a).Theproportionofthetotalvariationrepresentedbythejthprincipalaxiswas
computedasλ/∑ λ (Fig3b).ThecontributionofanOTUitoaprincipalaxisUdefinedbya
j j j
vectorofloadings[u ,u ,...,u ]wascomputedasu2,whichisjustifiedbythefactthatU
1 2 11,212 i
hasanormof1(thus_U_2=∑u2=1).TheOTUscontributingmosttotheretainedprinci-
i i
palaxesweredefinedasOTUswithcontributionsgreaterthanagiventhreshold(Fig3c).Dif-
ferentsetsofOTUsweredefinedusingthresholdsof1%,2%,5%,10%,20%,30%,40%,and
50%,whichresultedinretentionofbetween4and14OTUs.Athresholdof10%wasretained
asitallowedforconservingessentiallyallofthevariationofthe7principalaxeswithonly10
OTUs,whichstillrepresented80%ofthetotalvariationinthedata.
DiscriminantAnalysisofPrincipalComponents(DAPC,[22,23])wasappliedtothe16S
rRNAgenedatatoidentifycombinationsofOTUsthatdifferedmostbetweenstonespouts
andwell.Whileoriginallydevelopedforgeneticmarkersdata,thismethodhassincebeen
appliedtovariousothertypesofdata,including16SrRNA(e.g.[24,25]).Cross-validationwas
usedtoassesstheoptimalnumberofPCAaxestoretaininthepreliminarydimension-reduc-
tionstep,using100independentreplicatesforeachnumberofretainedPCAaxesand70%of
thesamplesastrainingset.Thesameanalyseswererepeatedtoinvestigatepossibledifferences
acrossthethreelocations.
ResultsandDiscussion
ThechemicalandphysicalpropertiesofKathmandudrinkingwater
Asummaryofthechemicalandbacterialdatageneratedforeachofthe10samplinglocations
(432watersamples)ispresentedinTable2(totaldataavailableinS1Dataset).Wefirstly
assessedthephysicalqualitiesandchemicalcompositionofthewatersamplesandcompared
thesedatatoWHOguidelines(Table2)[8].Thechemicalanalysesofthewatersamplessigni-
fiedthatseveralsourceshadconcentrationsofironandammoniainexcessofWHOguidelines,
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BacterialContaminationofDrinkingWaterinKathmandu
Fig2.ThequantificationofcoliformsandDNAfromSalmonellaserovarsTyphiandParatyphiAinwatersamplesfromtenlocationsin
Kathmandu,Nepal.a)BoxplotsoftheMPNcounts(log10CFU/ml)aggregatedbywatersourcestype(SS,stonespout;SW,sunkenwell;PS,pipedsupply)
andwithineachindividuallocation(numberedonx-axis).Boxesandhorizontallinesrepresenttheinterquartilerangesandmedians,respectivelyand
whiskersrepresent90%ofdatarange.ThemedianMPNmeasurementsinwaterfromstonespouts;sunkenwellsandpipedsuppliesweresignificantly
different;asdeterminedbyKruskalWallistest(p<0.001;shownbytheasterisk).b)ScatterplotsshowingthenumberofcopiesofS.Typhi(redcircles)andS.
ParatyphiA(bluecircles)genetargetscalculatedtobeineachwatersample(byrealtimePCRamplificationandquantificationusingastandardcurve)
againsttheweekofsampling.TheredandblueshadedlinesshowthebestfitthroughtimeofgenecopyquantificationtrendsforS.TyphiandS.ParatyphiA,
respectively.c)Principalcomponentsanalysisoftheweeklypresence/absenceprofilesofS.TyphiandS.ParatyphiAgenetargetsinwatersamples,
shadedbyweeklyrainfall(seekey).Timeisrepresentedonthex-axis.Thefirstprincipalcomponent,representingfluctuationsinS.TyphiandS.ParatyphiA
DNArelativeabundance,isplottedonthey-axis.
doi:10.1371/journal.pntd.0004346.g002
butnotably,onlynitratelevelsandturbidityconsistentlyexceededWHOrecommendationsin
alllocations.Thesefinding,withrespecttonitratewerebroadlyconsistentwithprevioussingle
timepointobservationsinthislocation[26,27].Furtherinvestigationidentifiedsignificant
differencesinthechemicalprofilesofthewaterfromthevariouslocations(non-parametric
MANOVA:Λ =0.250,p=1×10−4with9,999permutations).Thesedisparitiesinchemical
pillai
compositionscouldbesummarizedusingaPCA,withwaterfromtwoofthesunkenwells
(locations4and6(Table1andFig1b))formingdistinctclusterswithindependentchemical
signatures(Fig1c).Theseprofileswerecharacterizedbyconsistentlyhighconcentrationsof
ironandammoniaandgreaterturbidityatlocation4andgreaterconductivity,hardness,chlo-
ridesandnitratesatlocation6(Fig1c).Distinctively,oneofthepipedwatersupplies(location
8)hadconsistentlylowerchemicalcontaminationindicesthanallothersources(Fig1cand
Table2).Thedifferencesinchemicalcompositionbetweenlocations,confirmedbypairwise
comparisonsbetweenthesamplinglocations(Wilcoxonranktest,allp-values<0.05with
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004346 January6,2016 9/18
BacterialContaminationofDrinkingWaterinKathmandu
Fig3.16SrRNAgenesurveyingofgastrointestinalbacterialtaxafoundinstonespoutandwellwatersamplesinKathmandu,Nepal.a)Screeplot
showingtheeigenvaluesofthePCAof16SrRNAgenedata,withretainedaxesinbluecorrespondingto80%ofthevariationin519identifiedOTUsfrom
fecalbacterialfamilies(Bacteroidaceae,Clostridiaceae,Enterobacteriaceae,Erysipelotrichaceae,Lachnospiraceae,Lactobacillaceae,Prevotellaceae,
RuminococcaceaeandVeillonellaceae)identifiedin93watersamplesfromlocation2(stonespout)andlocation5(sunkenwell).Themaingraphonly
representsthefirst30eigenvalues(fullgraphprovidedininset).b)DiversityrepresentedbytheOTUswithlargecontributionstotheretainedPCAaxes.PCA
axesarerepresentedonthex-axis,withawidthproportionaltothecorrespondingdiversity(eigenvalue).They-axisrepresentstheamountofdiversity
retainedbyretainingonlyOTUswithcontributionsofatleast1%(6taxa),5%(5taxa),50%(4taxa)or75%(2taxa),indicatedbydifferingshadesofblue.The
totalsurfaceofagivencolorisproportionaltothefractionofthetotaldiversityrepresentedbythissetoftaxa.Thereddashedlineidentifiesthesetof6
retainedtaxaplottedinFig3c,representing76%ofthetotalvariationintheentiredata.c)OTUcompositionofthewatersamples,showingtherelative
frequenciesofthesixmoststructuringfecalbacterialtaxaidentifiedinFig3b(Enterobacteriaceae(OTUs00024,00185and01479),Bacteroidaceae
(OTU00979),Clostridiaceae(OTU00263),andPrevotellaceae(OTU2149)).Theirrelativeabundance(y-axis)isrepresentedthroughtimeinlocation2(stone
spout)andlocation5(sunkenwell),thetwolocationswiththegreatestestimatedcoliformcontaminationbyMPN.Emptybarscorrespondtomissingorfailed
samples.d)ADiscriminantAnalysisofPrincipalComponents(DAPC)ofthe16SrRNAgeneidentifyingcombinationsofthe519gastrointestinalOTUs
differingthemostbetweenthestonespout(blue)andthesunkenwell(red).TheOTUsexhibitingthegreatestvariationbetweentheselocationswere
OTU00263;Clostridium,OTU00185;Enterobacteriaceae,OTU00979;BacteroidesandOTU00024,Enterobacteriaceae).
doi:10.1371/journal.pntd.0004346.g003
Bonferronicorrection),suggestwatermineralizationandimplicatecontaminantssuchasvehi-
cleexhaustgases[28,29]andpoorwastehandlingsystemsasthekeydriversofpoorwater
qualityintheselocations[30].
OfthedifferentchemicalpollutantsobservedinKathmandudrinkingwater,thesustained
contaminationofwaterbynitritesandnitrateswasthemostalarming.Nitritesandnitrates
canbeintroducedintothewaterthrougharangeofprocessesincludingsurfacewaterinfiltra-
tion,industrialpollution,agriculturalfertilizerrun-offandtheleakageofseweragesystems
[26,31].Sustainedexposuretonitratescanleadtoarangeofnon-communicablediseases
includingmethemoglobinemia,gastrointestinalcancer,bladderandovariancancers,andmay
expeditetypeIIdiabetes,thyroidhypertrophyandrespiratorytractinfections[32].Nitrate
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