Monitoring waterborne pathogens in surface and drinking waters : are water treatment plants (WTPs) simultaneously efficient in the elimination of enteric viruses and fecal indicator bacteria (FIB)?
Abstract
Monitoring the quality of water is a requisite to prevent outbreaks related to waterborne diseases, predominantly caused by pathogens like enteric viruses, usually transmitted via the fecal-oral route. This study aimed to survey a group of enteric viruses (Enterovirus, Norovirus genogroups I and II, and hepatitis A virus) in two surface water sources of drinking water, also intending to evaluate the extent of their elimination in the two water treatment plants (WTPs) involved in drinking water production. Correlations between these viruses and fecal indicator bacteria (FIB) were also evaluated. Positive samples for viral RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR) and quantified, in genomic copies per liter (gc/L) of sampled water. Viral RNAs were detected in 14 out of 27 samples of surface water, and 21 out of 36 samples of drinking water, NoV II having been the most frequently detected in both (0–78.6 gc/L and 0–12.5 gc/L, respectively). Both WTPs showed variable efficacies in the elimination of viral RNA. Only one correlation was found with FIB, between NoV II and intestinal enterococci. These results recommend the monitoring of enteric viruses over time and their inclusion in the mandatory analysis of water quality.
Full text
Water2020,12,2824;doi:10.3390/w12102824www.mdpi.com/journal/water Article MonitoringWaterbornePathogensinSurfaceand DrinkingWaters.AreWaterTreatmentPlants(WTPs) SimultaneouslyEfficientintheEliminationof EntericVirusesandFecalIndicatorBacteria(FIB)? DanielSalvador1,2,3,*,MariaFilomenaCaeiro2,FátimaSerejo1,PauloNogueira1,RuiNeves Carneiro3andCéliaNeto3 1InstitutodeSaúdeAmbiental,FaculdadedeMedicinadaUniversidadedeLisboa,AvenidaProf.Egas Moniz,EdifícioEgasMoniz,Piso0,AlaC,1649‐028Lisboa,Portugal;fatim[email protected](F.S.); [email protected].pt(P.N.) 2CentrodeEstudosdoAmbienteedoMar(CESAM),DepartamentodeBiologiaVegetal,Faculdadede CiênciasdaUniversidadedeLisboa,EdifícioC2‐Piso4,CampoGrande,1749‐016Lisboa,Portugal; [email protected].pt 3DireçãodeLaboratórioseControlodaQualidadedaÁgua(LAB)daEmpresaPortuguesadasÁguas Livres(EPAL),AvenidadeBerlim,15,1800‐031Lisboa,Portugal;[email protected](R.N.C.);[email protected] (C.N.) *Correspondence:daniel.salva[email protected] Received:8September2020;Accepted:8October2020;Published:11October2020 Abstract:Monitoringthequalityofwaterisarequisitetopreventoutbreaksrelatedtowaterborne diseases,predominantlycausedbypathogenslikeentericviruses,usuallytransmittedviathefecal‐ oralroute.Thisstudyaimedtosurveyagroupofentericviruses(Enterovirus,Norovirusgenogroups IandII,andhepatitisAvirus)intwosurfacewatersourcesofdrinkingwater,alsointendingto evaluatetheextentoftheireliminationinthetwowatertreatmentplants(WTPs)involvedin drinkingwaterproduction.Correlationsbetweenthesevirusesandfecalindicatorbacteria(FIB) werealsoevaluated.PositivesamplesforviralRNAwererecurrentlyfoundbyreversetranscription quantitativePCR(RT‐qPCR)andquantified,ingenomiccopiesperliter(gc/L)ofsampledwater. ViralRNAsweredetectedin14outof27samplesofsurfacewater,and21outof36samplesof drinkingwater,NoVIIhavingbeenthemostfrequentlydetectedinboth(0–78.6gc/Land0–12.5 gc/L,respectively).BothWTPsshowedvariableefficaciesintheeliminationofviralRNA.Onlyone correlationwasfoundwithFIB,betweenNoVIIandintestinalenterococci.Theseresults recommendthemonitoringofentericvirusesovertimeandtheirinclusioninthemandatory analysisofwaterquality. Keywords:drinkingwater;entericviruses;fecalindicatorbacteria;FIB;humanhealth;RT‐qPCR; surfacewater;waterquality;watertreatment 1.Introduction Thewaterusedbyhumansocietiescanhavedifferentorigins,suchassurfacewaterfromrivers anddamreservoirs.TheWorldHealthOrganization(WHO)estimatesthat785millionpeopledonot haveabasicwatersupplyservice,counting144millionpeoplewhoaredirectlydependentonsurface water[1].In2025,halfofthepopulationwillliveinareasofwaterstress[1].Themonitoringofthe qualityofwaterisapermanentrequisitetoprevent,amongotherpossiblethreatstohumanhealth, outbreaksrelatedtowaterbornediseases[2,3].Healthrisksassociatedwiththeuseofsurfacewater
Water2020,12,28242of18 includeinfectiousdiseasespredominantlycausedbyentericpathogens,mainlybacteriaandviruses [4,5]. Therearemorethan200entericvirusesinnature,distributedamongseveralvirusfamilies: Adenoviridae,Astroviridae,Caliciviridae,Hepeviridae,Picornaviridae,andReoviridae[3,6,7].Inthe GuidelinesforDrinkingWaterQuality,WHO(2017)reportedthatvirusesfromthesefamilieshave amoderatetohighimpactonhumanhealth,highlightingthefollowingviruseswithRNAgenomes: Enterovirus,hepatitisAandEviruses,Norovirus,Rotavirus,andSapovirus[8].Recently,Enterovirus, Norovirus,andhepatitisAviruswereclassifiedasemergingagentsbytheUSAEnvironmental ProtectionAgencyandincludedintheContaminantCandidateList4‐CCL[9]. Thepresentstudytargetsthelastthreevirusesreferredtoabove,includedtwovirusfamiliesof entericRNAviruses,whichareconsideredthemostdiversegroupofpathogensforhumansand animals[5,10]. VirusesfromthegenusEnterovirusbelongtothefamilyPicornaviridae,whichcomprisessingle‐ strandedpositive‐senseRNAviruseswithanonenvelopedsmallicosahedralcapsid[11].Enterovirus includes12specieswithnumerousvirusisolates,distributedacrosstheglobe[11].IntheUnited States,enterovirusescausemorethan10millioninfectionsandseveralthousandhospitalizationsper year[12].InPortugal,astudybasedon625fecalsamples,detected22.9%ofsampleswerepositive fortheseviruses[13].Mostinfectionsareasymptomaticbutcanalsotaketheformofafebrileillness thatlastsafewdays,oftenaccompaniedbysymptomsassociatedwithupperrespiratorytractillness. However,theycancausemoreseriousdiseasessuchaspoliomyelitis,paralyticdisease,meningitis, myocarditis,andhand,foot,andmouthdisease[12,14].Enterovirusesweredetectedinsurfacewater intheUSA[15],Japan[16],Netherlands[17],Hawaii[18],SouthAfrica[19],France[20],andinother countries[10],aswellasindrinkingwaterinColombia[21]. HepatitisAvirus(HAV)istheuniqueserotypeofthespeciesHepatovirusA[22],oneoutthenine speciesofthegenusHepatovirus,alsobelongingtothePicornaviridaefamily.AccordingtotheWHO, HAVscausedaround7134deathsworldwidein2016[23].InPortugal,hepatitisAisamandatory reportingdisease,with599casesreportedin2017[24].Mostinfectionsaresilentinchildrenunder theageoffive.Inolderchildrenandadults,themostcommonsymptomsaredarkurine,diarrhea, abdominalpain,fever,nausea,jaundice,andanorexia[3,5].Thisviruswasdetectedinsurfacewater intheUSA[25],Brazil[26],Spain[27],Uganda[28],andalsoinothercountries[10];aswellasin drinkingwaterinColombia[21]. ThegenusNorovirus,belongingtotheCaliciviridaefamily,alsocomprisesnonenveloped icosahedralviruseswithapositive‐sensesingle‐strandedRNAgenome.Thisgenusincludesseveral isolatesfromauniquespecies(Norwalkvirus)[29].Theyaredistributedinsixgenogroups(GIto GVI);however,onlyisolatesfromgenogroupsI,II,andIVinfecthumans[10].Humannoroviruses arethemostcommonetiologicalagentlinkedtogastroenteritisoutbreaksaswellastheleadingcause ofnon‐bacterialgastroenteritis[5].AccordingtotheCentersforDiseaseControlandPrevention (CDC),norovirusescauseapproximately200,000deathsperyearworldwideand70,000,ormore,are amongchildrenindevelopingcountries[30].InPortugal,astudycarriedoutin2015,involving580 stoolsamplesfrompatientshospitalizedforacutediarrheain13hospitals,found11.6%ofsamples werepositivefortheseviruses[31].Inmostcases,infectionbynorovirusescausesmildsymptoms, suchasdiarrhea,vomiting,nausea,abdominalcramps,headache,fever,andmusclepain[3].These virusesweredetectedinsurfacewaterinFinland[32],Norway[33],Netherlands[34],Japan[16],and alsoinothercountries[10];aswellasindrinkingwaterinFinland[35]andSpain[36]. Entericvirusesareusuallytransmittedtohumansviathefecal‐oralroute,byperson‐to‐person contact,aerosols,ingestionofcontaminatedfood,andwater,thisonerecognizedasanimportant transmissionvehicle[3,14].Thesevirusesarereleasedinlargenumbersinthefecesofcarriersduring infection(105to1011virusparticlespergramofstool)[7].Theyareverycontagiousandcansurvive intheenvironmentforlongerperiodsthanbacteria,havingbeenreportedthattheycanremain infectiousfor120daysinfreshwater[3,7,8,37].Astheyhaveveryrobustcapsids,theyaremore resistanttodisinfection,temperaturechanges,andlowpH,thanbacteria[3,37–39].Theirelimination iscomplexinwatermatrices,requiringtheuseofcombinedprocessesinwatertreatmentplants
Water2020,12,28243of18 (WTP)[37,40].Particularlyindrinkingwater,wherethesevirusesareusuallyfoundinlow concentrations,theirdetectionisanothertechnicalchallenge[41]. Despitetheindisputableimportanceofentericviruses,thecurrentPortugueselegislationdoes notincludethesepathogensinthelistofthemicrobiologicalparametersrequiredtocontrolthe qualityofthedrinkingwater.Themicrobialindicatorsoffecalcontaminationincludedinthatlistare fecalindicatorbacteria(FIB),comprisingcoliformbacteria(Escherichiacoliandothercoliforms),and intestinalenterococci[42].AlthoughFIBhavebeenconsideredeffectiveindicatorsofthepresenceof otherpathogens,thisassociationhasbeenquestioned[43–45]. Inthiscontext,ourstudyconductedinPortugalaimedtoevaluatethepresenceofEnterovirus, Norovirus(GIandGII),andHAV,innaturalwatersampledintwosurfacesources(ariverandadam reservoir)feedingtwoWTPs,whoseefficacyineliminatingviralRNAwasaddressedby simultaneoussamplingofdrinkingwaterattheoutletofbothWTPs;asamplingpointlocatedinthe waterdistributionnetworkwasincluded,asoutsidecontrol.Thisstudyalsointendedtoextendthe evaluationtothedegreeofcorrelationoftheentericviruseseventuallydetected,withFIBandsome physical‐chemicalcharacteristicsofthesampledwater. 2.MaterialsandMethods 2.1.SamplingSitesofSurfaceWaterandDrinkingWater Thesurfacewatersamplescamefromtwobodiesofwater:ariverandadamreservoir.They weresampledintwolocations:attheabstractionsiteinthecaseoftheriver,andtheinletoftheWTP inthecaseofthedamreservoir.Theriverhasalengthof1007km,itsnaturalwaterisusedfor hydroelectricenergyproduction,irrigation,fishingactivity,navigability,andhumansupply[46]. Thedamreservoirhasamaximumcapacityof1,095,000dam3,anditisasourceofwaterfor2million people,whichrepresentsaboutonefifthofthePortuguesepopulation;itswaterisusedforenergy production,recreationaluse,andhumansupply[46].Thetwowaterbodiesbelongtothesame districtandthesamehydrographicbasinwithatotalareaof3964.56km2[46].Whilethewaterfrom thedamreservoirhasanoriginindependentoftheriver,theriverreceivesthewaterdischargedfrom thedamreservoir,atalocationupstreamfromthesamplingsiteincludedinthissurvey.Theareas surroundingthedamreservoirarecharacterizedbytheexistenceofforest,agriculturalfields, dispersedhumanpopulations,agro‐livestockunits(mainlyraisingpigs),andsomeindustries.The areasurroundingthesamplingsiteintheriverischaracterizedbytheexistenceofagreatextension ofagriculturalfields,agro‐livestockunits(raisingpigs,cows,goats,andsheep),andindustries[47]. Themonitoringofthewaterinpreviousyearsshowedahigherlevelofbacterialandchemical contaminationintheriverthaninthewaterofthedamreservoir(unpublisheddata). ThewaterfromtheriveranddamreservoirwastreatedatWTP_RandWTP_D,respectively. TheWTPsuseddifferenttreatmentschemes,asdescribedinSalvadoretal.(2020)[48],themain differencesbeinginthefirstpre‐oxidationstep,carriedoutwithozoneinWTP_Randwithchlorine inWTP_D;andtheinclusionofanadsorptionstepwithactivatedcarbon,inWTP_R.BothWTPs includeafinaldisinfectionwithchlorine.Thesamplingofdrinkingwateroccurredattheoutletof thetwoWTPsandatonerepresentativesiteinthewaterdistributionnetworkfedbyboth. 2.2.SampleCollection ThewatersamplingcampaignwascarriedoutbetweenJanuaryandDecember2019,usually consistingofonesamplingpermonth.Onthesameday,sampleswerecollectedforthe detection/quantificationofentericviruses,quantificationofFIB,andphysical‐chemical characterization.Concerningthesamplingintendedforentericvirusdetection,thereweresome exceptions:biweeklysamplingattheriverandWTP_Rsitesinsomemonthsandabsenceofsampling atthedamreservoirandWTP_DsitesinJulyandAugust(Figure1).Largevolumesofwaterwere collectedatthesamplingsites(Figure1)andconcentratedbyfiltrationthruNanoceramfilters (Argonite;Sanford,Florida,USA)setupinhousingchambers[49].Thefilteredvolumesfollowed Method1615(EPA/600/R‐10/181)[50],butweresubjectedtovariations,mainlydependingonthe
Water2020,12,28244of18 occurrenceofmembraneclogging.Theaveragevolumesofwaterconcentratedwere243.2Lforthe river,790.0Lforthedamreservoir,andlargelyhigherforthedrinkingwatercollectedatthedifferent sites:1490.0LforWTP_R,1120.0LforWTP_D,and1717.8Lforthesamplingpointinthenetwork distribution(Figure1).FordetectionandquantificationofFIB,avolumeof1Lwascollectedina sterilepolyethylenecontainer(Labinox,Esmoriz,Portugal)withsodiumthiosulfatetoinactivate chlorine.FordeterminationofpH,250mLofwaterwascollectedinglasscontainers.Thecollected samplesweretransportedrefrigeratedtothelaboratory.Determinationsoftemperatureandchlorine werecarriedoutatthesamplingsite. Figure1.Volumesofwatersampledin2019atthefivesamplingsitesundersurvey,aimedat detectionandquantificationofentericviruses.(A)Riveranddamreservoir(n=27).(B)WTP_R, WTP_D,andpointinthewaterdistributionnetwork(n=36). 2.3.DetectionandQuantificationofEntericViruses’GenomicRNAs MostoftheproceduresfollowedinthisstudywereperformedasdescribedinSalvadoretal. (2020)[48].Briefly,theNanoceramfilters(Argonite;Sanford,Florida,USA)withthesamplesof concentratedwaterwereelutedwithbeefextract(BDBioscience;FranklinLakes,NewJersey,USA) andtheelutedsolutionsweresubjectedtoanorganicflocculationprocessfollowedbycentrifugation andfiltrationthrough0.22‐μmpore‐sizeAcrodiscSyringefilters(PALLCorporation;AnnArbor, Michigan,USA).Theresultingvolumeswerealiquotedandkeptat−70°C,untiluse.Inasecondstep, thawedsamplesofabout20mLwereappliedtoVivaspinconcentrators(Sartorius;Goettingen, Germany)andcentrifugedat8000gand4°Cfor6h,untilavolumeoflessthan1mLwasachieved ineachsample.ThefinalconcentratesweresubjectedtoRNAextractionandpurification,usingthe viralQIAampRNAMinikit(Qiagen;Hilden,Germany),accordingtothemanufacturerʹs instructions. RT‐qPCRreactionswereperformedonaStepOnePlusthermocycler(AppliedBiosystems;Foster City,California,USA).EnterovirusgenesigAdvancedKit(PrimerdesignTMLtd;ChandlerʹsFord,
Water2020,12,28245of18 UnitedKingdom)wasusedfordetectionandquantificationofEnterovirusinreactionmixturesof20 μLcontaining5μLofextractedRNA.ForHAVandNoVtheCeeramToolsHepatitisAKit, CeeramToolsNorovirusGIKitandCeeramToolsNorovirusGIIKit(allpurchasedfrombioMérieux; Marcy‐lʹEtoile,France)wereusedinreactionmixturesof25μLcontaining5μLofextractedRNA. TheamplificationconditionsaredescribedinTable1.Enterovirus,NoVI,NoVII,andHAVgenomic RNAswerequantifiedbystandardcurveswithfivepoints,constructedwith1:10serialdilutionsof standardRNAsacquiredinStandardkitsfromCeeramTools(bioMérieux;Marcy‐lʹEtoile,France). EachRT‐qPCRresult,expressedingenomiccopies(gc)perfivemicrolitersofRNA,istheaverage valueoftwoindependentamplificationsandwasconvertedintogenomiccopiesperliterofcollected sample(gc/L).Onlyresultsthatmetthequalityrequirementsspecifiedbythekitsreferredabove werevalidated.OnlysampleswithCq(cyclequantification)valuesbelow40wereconsidered positive. Table1.RT‐qPCRconditionsfordetectionandquantificationofentericviruses. StepTimeTemperatureNumberofCycles Enterovirus ReverseTranscription10min55°C1 Enzymeactivation2min95°C1 Denaturation10seg95°C50 Datacollection1min60°C NoV (NoVIandNoVII) andHAV ReverseTranscription10min45°C1 Enzymeactivation10min95°C1 Denaturation15seg95°C45 Datacollection45seg60°C 2.4.QuantificationofFecalIndicatorBacteria(FIB) 2.4.1.ColiformBacteriaandE.Coli TheprocedureforquantifyingcoliformbacteriaandE.coliinsurfacewaterwasbasedonthe internationalstandardmethodISO9308‐2[51]usingtheColilerttestwithQuanti‐Tray(IDEXX Laboratories,Westbrook,Texas,USA),at36°C.Theresultswereexpressedinthemostprobable numberper100mL(MPN/100mL).TheprocedureforquantifyingcoliformbacteriaandE.coliin drinkingwaterwasbasedontheinternationalstandardmethodISO9308‐1:2014[52].Briefly,a100‐ mLaliquotofwatersamplewasfilteredthrougha0.45μmcellulose‐estermembrane(PallLife Sciences;Westborough,USA)placedoveraplatewithmediumCCA(ChromogenicColiformAgar) (OXOIDLimited;WadeRoad,UnitedKingdom)thatwasincubatedat36±2°Cfor21±3h.Positive β‐D‐galactosidasecolonies(pinkorred)werepresumedtobecoloniesofcoliformbacteria.These colonieswerethenculturedonnon‐selectivenutrientagar(YeastExtractAgar,OXOIDLimited; WadeRoad,UnitedKingdom)at36±2°Cfor21±3h.Aftertheincubationtime,thecytochromec oxidasetest(AbtekBiologicalsLtd;Liverpool,UnitedKingdom)wasperformed.Theoxidase negativecolonieswereconfirmedascoliformbacteriaandpositiveβ‐D‐galactosidasecolonies(dark blueorviolet)werecountedasE.coli.Theresultswereexpressedincolonyformingunitsper100mL (cfu/100mL). 2.4.2.FecalColiforms QuantificationoffecalcoliformsinsurfacewatersampleswasperformedusingtheColilerttest withQuanti‐Tray(IDEXXLaboratories;Westbrook,Texas,USA)at44.5°C,followingthe manufacturerʹsinstructions.TheresultswereexpressedinMPN/100mL.Thequantificationoffecal coliformsindrinkingwatersampleswasperformedbyfiltrationofa100‐mLaliquotthrougha0.45 μmcellulose‐estermembrane(PallLifeSciences)thatwasplacedonaplatewithLaurylculture medium(MLSA,OXOIDLimited;WadeRoad,UnitedKingdom)andincubatedat44±0.5°Cfor21 ±3h.Yellowandorangecoloniesweretransferredtonon‐selectivenutrientagarmedium(Yeast
Water2020,12,28246of18 ExtractAgar,OXOIDLimited;WadeRoad,UnitedKingdom)andincubatedat36±2°Cfor21±3h. Afterincubation,thecytochromecoxidasetest(Merck;Darmstadt,Germany)wasperformed.The negativeoxidasecolonieswereincubatedinDEV‐Fluorocult(Biogerm;Maia,Portugal)culture mediumat44±0.5°Cfor21±3h.Afterthisperiod,fecalcoliformswerequantifiedasthosethat changedthemediumcolortoyellow,duetolactosefermentation.Theresultswereexpressedin cfu/100mL. 2.4.3.IntestinalEnterococci Theprocedureforquantifyingintestinalenterococciwasbasedontheinternationalstandard methodISO7899‐2[53].A100‐mLwatersamplewasfilteredthrougha0.45μmcellulose‐ester membrane(PallLifeSciences).ThemembranewasplacedoveraplatewithselectiveSlanetzand Bartleyagarmedium(VWRChemicals;Radnor,Pennsylvania,USA),andincubatedat36±2°Cfor 44±4h.Membranescontainingred,brown,orpinkcoloniesweretransferredtobileesculinagar medium(Merck;Darmstadt,Germany)andincubatedat44±0.5°Cfor2h.Blackcolonieswiththe capacitytoreduce2,3,5‐triphenyltetrazoliumchloridetohydrolaseesculinwerecountedaftertwo hoursasintestinalenterococci.Theresultswereexpressedincfu/100mL. 2.5.DeterminationofPhysical‐ChemicalParameters Thephysical‐chemicalparametersincludedinthisstudy(temperatureandtotalchlorine concentration)weredeterminedinsituatthesamplingmoment.Thevaluesoftemperaturewere obtainedusingModelE905000thermometers(Amarell‐Electronic;Kreuzwertheim,Germany).The determinationoftotalchlorineconcentrationindrinkingwatersampleswasperformedbymolecular absorptionphotometry,usingaportablephotometer(Palintest;Gateshead,UnitedKingdom)[54]. ThepHwasdeterminedatthelaboratoryina785DMPTitrinoequipment(Metrohm;Herisau, Switzerland)[55]. 2.6.StatisticalAnalyses DataprocessingandstatisticalanalysiswereperformedusingMicrosoftExcel2017(Microsoft Inc.,Redmond,WS,USA)andIBMSPSSStatisticsVersion26(IBMCorporation;Armonk,NewYork, USA).Spearmancorrelationcoefficientwasusedtoevaluatethecorrelationsinvolvingthefollowing parameters:entericvirusesdetected,microbiologicaldata,andphysicochemicaldata.The correlationswereclassifiedasstrong(r>0.90),moderate(0.40<r<0.89)orweak(r<0.39)[5]. 3.Results 3.1.DetectionandQuantificationofEntericViruses Insurfacewatersamples,viralRNAsweredetectedin14(51.9%)outofatotalof27samples: 9/17intheriverand5/10inthedamreservoir(Table2).Oftheninepositivesamplescollectedinthe river,NoVIRNAwasthemostfrequentlydetected,at137.0gc/Linonesample,andatconcentrations notgreaterthan11.6gc/Lintheotherfive.NoVIIRNAwasdetectedat19.6gc/Linonesampleand at2.4gc/Lorlessintheotherfoursamples,andEnterovirusRNAwasonlydetectedonce,at4.4gc/L. Ofthefivesamplespositivefromthedamreservoir,fourwerepositiveforNoVIIRNA,detectedat 78.6gc/Linonesampleandat2.0gc/Lorbelowintheothers.EnterovirusRNAwasdetectedinone sample,at0.2gc/L(Table2). Regardingthedrinkingwatersamples,viralRNAsweredetectedin21(58.3%)outofatotalof 36samples,distributedamongthethreesamplingpoints,asfollows:9/17atWTP_R,6/10atWTP_D, and7/9atthedistributionnetwork(Table2).AtWTP_R,onlyNovIIRNAwasdetected(nine samples),withaconcentrationthatvariedbetween0.1and9.7gc/L.AtWTP_D,NovIIRNAwas detectedinfivesamples,atamaximumconcentrationof7.9gc/L,andHAVRNAinonesample,at 0.1gc/L.Atthesamplingpointinthedistributionnetwork,sevensampleswerepositiveforNoVII RNA,detectedatconcentrationsrangingbetween0.1and12.5gc/L(Table2).
Water2020,12,28247of18 3.2.FecalIndicatorBacteria(FIB)QuantifiedinWaterSamples Fromthe24surfacewatersamplesanalyzed,coliformbacteriaweredetectedinall(100.0%), fecalcoliformsin18(75.0%),E.coliin17(70.8%),andintestinalenterococciin16(66.7%).Thefecal contaminantsundersurveywerenotdetectedinthethreesamplingpointsofdrinkingwater. Coliformbacteriaweredetectedthroughoutthesamplingcampaign,inconcentrationsof644– 2714MPN/100mLintheriverand13–148MPN/100mLinthedamreservoir(Figure2).Fecal coliformsweredetectedintheriver,throughoutthe12monthsofthesamplingcampaign,at concentrationsof9–291MPN/100mL,whilstinthedamreservoirtheyweredetectedinsixofthe twelvemonths,atamaximumconcentrationof11MPN/100mL(Figure2).E.coliwasdetectedinthe river,throughoutthesamplingcampaign,at291MPN/100mLinJanuaryandunder50MPN/100mL intheothermonths.Inthedamreservoir,E.coliwasdetectedinfivemonths,atconcentrations rangingfrom1to7MPN/100mL(Figure2).Intestinalenterococciwerealsodetectedduringthe12 monthsofthesamplingcampaign,intheriverat12–57cfu/100mLandduringfourmonthsinthe damreservoir,atconcentrationsbelow7cfu/100mL(Figure2). Figure2.Microbiologicalcharacterizationofthesurfacewatercollectedduringthe2019sampling campaign(n=96).Thegraphsrepresentthevariationinconcentrationvaluesfromthefourgroups ofFIBevaluated(coliformbacteria,E.coli,fecalcoliforms,andintestinalenterococci)registered throughoutthesamplingcampaign,inthesurfacewatersourcesundersurvey(RiverandDam reservoir).
Water2020,12,28248of18 Table2.Detectionandquantificationofentericvirusesinsurfacewater(n=27)anddrinkingwater(n=36)sampledin2019. Sampling RiverDamReservoirWTP_RWTP_DPointintheDistributionNetwork EnteroNoVINoVIIHAVEnteroNoVINoVIIHAVEnteroNoVINoVIIHAVEnteroNoVINoVIIHAVEnteroNoVINoVIIHAV gc/Lgc/Lgc/Lgc/Lgc/L J anuaryNDNDNDND0.2NDNDNDNDNDNDNDNDNDNDNDNDND12.5ND February(A)NDND0.4ND––––NDND2.6ND–––––––– February(B)ND3.619.6NDNDND2.0NDNDND9.7NDNDND7.90.1NDNDNDND March(A)4.40.2NDND––––NDNDNDND–––––––– March(B)ND5.3NDNDNDNDNDNDNDNDNDNDNDNDNDND–––– AprilND10.62.4NDNDND0.8NDNDNDNDNDNDND1.4NDNDNDNDND MayNDNDNDNDNDND1.4NDNDND1.5NDNDND4.5NDNDND4.6ND J uneNDND0.5NDNDND78.6NDNDND0.6NDNDND0.9NDNDND0.1ND J ulyNDNDNDND––––NDND0.2ND––––NDND0.7ND August(A)NDNDNDND––––NDNDNDND––––NDND0.2ND August(B)NDNDNDND––––NDND0.2ND–––––––– September (A)ND0.4NDND––––NDND0.1ND––––NDND0.1ND September(B)ND2.00.8NDNDNDNDNDNDNDNDNDNDND0.9ND–––– October(A)ND137.0NDND––––NDND0.2ND–––––––– October(B)NDNDNDNDNDNDNDNDNDND0.1NDNDNDNDNDNDND0.1ND NovemberNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND–––– DecemberNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND–––– (A):samplingperformedinthefirsthalfofthemonth;(B):samplingperformedinthesecondhalfofthemonth;Entero:Enterovirus;HAV:hepatitisAvirus;NoVI: NorovirusgenogroupI;NoVII:NorovirusgenogroupII;gc/L:genomiccopiesperliterofsampledwater,basedontheaveragevalueoftwoindependentRT‐qPCR results;ND:RNAnotdetected;‐:absenceofsampling.
Water2020,12,2824;doi:10.3390/w12102824www.mdpi.com/journal/water 3.3.EvaluationoftheEffectivenessoftheWTPsintheReductionofEntericVirusesandFIB EnterovirusandNoVIRNAs,detectedinsamplesfromtheriver,werenotdetectedinthewater samplescollectedattheoutletofWTP_R.ResultsconcerningNoVIIRNAvariedfromtotal eliminationtoabsenceofeliminationandslight“increments”werealsodetected;HAVRNAwasnot detectedinbothwatermatrices(Figure3,riverandWTP_R). AlthoughEnterovirusRNAwasdetectedinonesamplefromthedamreservoir,itwasnotfound inthedrinkingwatercollectedattheoutletofWTP_D.NoVIIRNAevidenceda98.9%decreaseafter treatmentinasinglesample,andnodecreaseorslightincrementsintheremainingsamples;HAV RNA,notpreviouslydetectedindamreservoirsamples,wasdetectedinadrinkingwatersampleof WTP_D,at0.1gc/L(Figure3,damreservoirandWTP_D). Althoughpresentinthetwosurfacewatersourcessurveyed,FIBwerenotdetectedindrinking water. Figure3. VariationintheNoVIIRNAconcentrationthroughoutthe2019samplingcampaign,inRiverand WTP_R(n=34)(firstgraph)andinDamreservoirandWTP_D(n=20)(secondgraph).Eachconcentrationvalue, ingc/L,istheaverageoftwoindependentRT‐qPCRresults. 3.4.DeterminationofPhysical‐ChemicalParametersintheWatersSurveyed BetweenJanuaryandDecember,24samplesofsurfacewaterand36samplesofdrinkingwater werecollectedforanalysesoftemperature,pH,andtotalchlorineconcentration(Figure4).Thewater temperaturesvariedbetween12.5and23.9°ContheriverandWTP_R,between11.6and18.9°Cin thedamreservoirandWTP_D,andbetween12.5and21.0°Catthesamplingpointinthewater distributionnetwork(Figure4).ThepHwassimilaralongthesamplingcampaignontheriverand WTP_R(7.4–8.0and7.1–8.0,respectively)whileonthedamreservoirandWTP_Danincreasewas detectedafterthetreatment(6.7–7.5and7.8–8.1,respectively);atthepointinthedistribution network,thepHofthedrinkingwatersampleswassimilaroverthe12months(7.7–8.1)(Figure4). Thesurfacewatersunderstudydidnotcontainchlorine.Theaveragevaluesofthetotalchlorine concentrationwere1.1mg/LCl₂inthewatersampledatWTP_R,1.0mg/LatWTP_D,and0.64mg/L atthesamplingpointinthewaterdistributionnetwork(Figure4).
Water2020,12,282416of18 30. Lopman,B.;Atmar,R.;Baric,R.;Estes,M.;Green,K.;Glass,R.;Hall,A.;Iturriza‐Gomara,M.;Kang,C.; Lee,B.;etal.Globalburdenofnorovirusandprospectsforvaccinedevelopment.Cent.Dis.ControlPrev. 2015,1–46. 31. Costa,I.;Mesquita,J.R.;Veiga,E.;Oleastro,M.;Nascimento,M.J.S.SurveillanceofnorovirusinPortugal andtheemergenceoftheSydneyvariant,2011–2013.J.Clin.Virol.2015,70,26–28, doi:10.1016/j.jcv.2015.07.002. 32. Maunula,L.;Söderberg,K.;Vahtera,H.;Vuorilehto,V.P.;VonBonsdorff,C.H.;Valtari,M.;Laakso,T.; Lahti,K.Presenceofhumannoro‐andadenovirusesinriverandtreatedwastewater,alongitudinalstudy andmethodcomparison.J.WaterHealth2012,10,87–99,doi:10.2166/wh.2011.095. 33. Grøndahl‐rosado,R.;Yarovitsyna,E.;Trettenes,E.;Myrmel,M.;Robertson,L.J.AOneYearStudyonthe ConcentrationsofNorovirusandEntericAdenovirusesinWastewaterandASurfaceDrinkingWater SourceinNorway.FoodEnviron.Virol.2014,6,232–245,doi:10.1007/s12560‐014‐9161‐5. 34. Lodder,W.J.;deRodaHusman,A.M.PresenceofNorovirusesandOtherEntericVirusesinSewageand SurfaceWatersinTheNetherlands.Appl.Environ.Microbiol.2005,71,1453–1461, doi:10.1128/AEM.71.3.1453. 35. Kukkula,M.;Maunula,L.;Silvennoinen,E.OutbreakofViralGastroenteritisDuetoDrinkingWater ContaminatedbyNorwalk‐likeViruses.J.Infect.Dis.1999,180,1771–1776. 36. Blanco,A.;Guix,S.;Fuster,N.;Fuentes,C.;Bartolomé,R.;Cornejo,T.;Pintó,R.M.;Bosch,A.Norovirusin BottledWaterAssociatedwithGastroenteritisOutbreak,Spain,2016.Emerg.Infect.Dis.2017,23,1531–1534. 37. Gall,A.M.;Mariñas,B.J.;Lu,Y.;Shisler,J.L.WaterborneViruses:ABarriertoSafeDrinkingWater.PLoS Pathog.2015,11,1–7,doi:10.1371/journal.ppat.1004867. 38. Goyal,S.M.;Cannon,J.L.TheMolecularVirologyofEntericViruses.InVirusesinFoods;Springer: Berlin/Heidelberg,Germany,2006;pp.471–503.ISBN978‐3‐319‐30721‐3. 39. Rzezutka,A.;Cook,N.Survivalofhumanentericvirusesintheenvironmentandfood.FEMSMicrobiol. Rev.2004,28,441–453,doi:10.1016/j.femsre.2004.02.001. 40. Guerrero‐Latorre,L.;Gonzales‐Gustavson,E.;Hundesa,A.;Sommer,R.;Rosina,G.UVdisinfectionand flocculation‐chlorinationsachetstoreducehepatitisEvirusindrinkingwater.Int.J.Hyg.Environ.Health 2016,219,405–411,doi:10.1016/j.ijheh.2016.04.002. 41. Yeh,H.Y.;Hwang,Y.C.;Yates,M.V.;Mulchandani,A.;Chen,W.DetectionofhepatitisAvirusbyusinga combinedcellculture‐molecularbeaconassay.Appl.Environ.Microbiol.2008,74,2239–2243. 42. RepúblicaPortuguesa.Decreto‐Lein.152/2017.DiáriodaRepública,2017:Lisboa,Portugal. 43. Espinosa,A.C.;Arias,C.F.;Snchez‐Colán,S.;Mazari‐Hiriart,M.Comparativestudyofentericviruses, coliphagesandindicatorbacteriaforevaluatingwaterqualityinatropicalhigh‐altitudesystem.Environ. HealthAGlob.AccessSci.Source2009,8,1–10,doi:10.1186/1476‐069X‐8‐49. 44. Teixeira,P.;Costa,S.;Brown,B.;Silva,S.;Rodrigues,R.;Valério,E.QuantitativePCRdetectionofenteric virusesinwastewaterandenvironmentalwatersourcesbytheLisbonmunicipality:Acasestudy.Water 2020,12,544,doi:10.3390/w12020544. 45. Zhu,H.;Yuan,F.;Yuan,Z.;Liu,R.;Xie,F.;Huang,L.;Liu,X.;Jiang,X.;Wang,J.;Xu,Q.;etal.Monitoring ofPoyanglakewaterforsewagecontaminationusinghumanentericvirusesasanindicator.Virol.J.2018, 15,1–9,doi:10.1186/s12985‐017‐0916‐0. 46. SNIRHRecursosHídricos.Availableonline:https://snirh.apambiente.pt/(accessedon20May2020). 47. APAPlanosdeOrdenamentodeAlbufeiras.Availableonline: https://apambiente.pt/index.php?ref=16&subref=7(accessedon20July2020). 48. Salvador,D.;Neto,C.;Benoliel,M.J.;FilomenaCaeiro,M.Assessmentofthepresenceofhepatitisevirus insurfacewateranddrinkingwaterinPortugal.Microorganisms2020,8,761, doi:10.3390/microorganisms8050761. 49. Ikner,L.A.;Gerba,C.P.;Bright,K.R.ConcentrationandRecoveryofVirusesfromWater:AComprehensive Review.FoodEnviron.Virol.2012,4,41–67,doi:10.1007/s12560‐012‐9080‐2. 50. Fout,G.S.;Brinkman,N.E.;Cashdollar,J.L.;Griffin,M.;McMinn,B.R.;Rhodes,E.R.;Varughese,E.A.; Karim,M.R.;Grimm,A.C.;Spencer,S.K.;etal.Method1615—MeasurementofEnterovirusandNorovirus OccurrenceinWaterbyCultureandRT‐qPCR.U.S.EnvironmentalProtectionAgency:Cincinnati,United StatesofAmerica,2014,pp.1–105. 51. ISO.ISO9308‐2:2012.Availableonline:https://www.iso.org/standard/52246.html(accessedon15May 2020).
Water2020,12,282417of18 52. ISO.ISO9308‐1:2014.Availableonline:https://www.iso.org/standard/55832.html(accessedon18May 2020). 53. ISO.ISO7899‐2:2000.Availableonline:https://www.iso.org/standard/14854.html(accessedon18May 2020). 54. StandardMethodsfortheExaminationofWaterandWastewater.Chlorine(Residual).Availableonline: https://www.standardmethods.org/doi/abs/10.2105/SMWW.2882.078(accessedon1June2020). 55. StandardMethodsfortheExaminationofWaterandWastewater.pHValue.Availableonline: https://www.standardmethods.org/doi/abs/10.2105/SMWW.2882.082(accessedon1June2020). 56. Prevost,B.;Lucas,F.S.;Goncalves,A.;Richard,F.;Moulin,L.;Wurtzer,S.Largescalesurveyofenteric virusesinriverandwastewaterunderlinesthehealthstatusofthelocalpopulation.Environ.Int.2015,79, 42–50,doi:10.1016/j.envint.2015.03.004. 57. Pang,X.;Qiu,Y.;Gao,T.;Zurawell,R.;Neumann,N.F.;Craik,S.;Lee,B.E.Prevalence,levelsandseasonal variationsofhumanentericvirusesinsixmajorriversinAlberta,Canada.WaterRes.2019,153,349–356, doi:10.1016/j.watres.2019.01.034. 58. Elmahdy,M.E.I.;Fongaro,G.;Magri,M.E.;Petruccio,M.M.;Barardi,C.R.M.Spatialdistributionofenteric virusesandsomaticcoliphagesinaLagoonusedasdrinkingwatersourceandrecreationinSouthern Brazil.Int.J.Hyg.Environ.Health2016,219,617–625,doi:10.1016/j.ijheh.2016.07.009. 59. PordataValordaProduçãoAgrícola:TotalePorTipo.Availableonline: https://www.pordata.pt/Municipios/Valor+da+produção+agrícola+total+e+por+tipo‐956(accessedon26 March2020). 60. Tandukar,S.;Sherchand,J.B.;Id,D.B.;Sherchan,S.P.;Malla,B.;Shrestha,R.G.;Haramoto,E.Presenceof HumanEntericViruses,Protozoa,andIndicatorsofPathogensintheBagmatiRiver,Nepal.Pathogens 2018,7,38,doi:10.3390/pathogens7020038. 61. Megchún‐García,J.V.;Landeros‐Sánchez,C.;Soto‐Estrada,A.;delRefugioCastañeda‐Chávez,M.; Martínez‐Dávila,J.P.;Nikolskii‐Gavrilov,I.;Galaviz‐Villa,I.;Lango‐Reynoso,F.TotalColiformsand EscherichiacoliinSurfaceandSubsurfaceWaterfromaSugarcaneAgroecosysteminVeracruz,Mexico.J. Agric.Sci.2015,7,110–119,doi:10.5539/jas.v7n6p110. 62. Onyango,A.E.;Okoth,M.W.;Kunyanga,C.N.;Aliwa,B.O.MicrobiologicalQualityandContamination LevelofWaterSourcesinIsioloCountyinKenya.J.Environ.PublicHealth2018,2018,1–10, doi:10.1155/2018/2139867. 63. Olasoji,S.O.;Oyewole,N.O.;Abiola,B.;Edokpayi,J.N.Waterqualityassessmentofsurfaceand groundwatersourcesusingawaterqualityindexmethod:Acasestudyofaperi‐urbantowninsouthwest, Nigeria.Environments2019,6,23,doi:10.3390/environments6020023. 64. Yuan,T.Aconsecutivecentrifugalmethodforconcentrationofhumanentericvirusesinwatersamples. Arch.Virol.2016,161,3323–3330,doi:10.1007/s00705‐016‐3031‐4. 65. Haramoto,E.;Kitajima,M.;Hata,A.;Torrey,J.R.;Masago,Y.;Sano,D.;Katayama,H.Areviewonrecent progressinthedetectionmethodsandprevalenceofhumanentericvirusesinwater.WaterRes.2018,135, 168–186,doi:10.1016/j.watres.2018.02.004. 66. DaSilva,A.K.;LeSaux,J.C.;Parnaudeau,S.;Pommepuy,M.;Elimelech,M.;LeGuyader,F.S.Evaluation ofremovalofnorovirusesduringwastewatertreatment,usingreal‐timereversetranscription‐PCR: DifferentbehaviorsofgenogroupsIandII.Appl.Environ.Microbiol.2007,73,7891–7897, doi:10.1128/AEM.01428‐07. 67. Wurtzer,S.;Prevost,B.;Lucas,F.S.;Moulin,L.Detectionofenterovirusinenvironmentalwaters:Anew optimizedmethodcomparedtocommercialreal‐timeRT‐qPCRkits.J.Virol.Methods2014,209,47–54, doi:10.1016/j.jviromet.2014.08.016. 68. Gregory,J.B.;Litaker,R.W.;Noble,R.T.Rapidone‐stepquantitativereversetranscriptasePCRassaywith competitiveinternalpositivecontrolfordetectionofenterovirusesinenvironmentalsamples.Appl. Environ.Microbiol.2006,72,3960–3967,doi:10.1128/AEM.02291‐05. 69. Fout,G.S.;Cashdollar,J.L.;Varughese,E.A.;Parshionikar,S.U.;Grimm,A.C.EPAMethod1615. MeasurementofEnterovirusandNorovirusOccurrenceinWaterbyCultureandRT‐qPCR.I.Collection ofVirusSamples.J.Vis.Exp.2016,107,1–13,doi:10.3791/52067. 70. Seo,M.;Lee,H.;Kim,Y.RelationshipbetweenColiformbacteriaandwaterqualityfactorsatweirstations intheNakdongRiver,SouthKorea.Water2019,11,1171,doi:10.3390/w11061171.
Water2020,12,282418of18 71. Addo,M.G.;Woods,C.A.;Andoh,L.A.;Obiri‐Danso,K.Effectofvaryingstoragetemperaturesonthe microbiologicalqualityofstreetsoldwaterintheKumasimetropolis,Ghana.Res.J.Microbiol.2016,11,56– 63,doi:10.3923/jm.2016.56.63. ©2020bytheauthors.LicenseeMDPI,Basel,Switzerland.Thisarticleisanopenaccess articledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution (CCBY)license(http://creativecommons.org/licenses/by/4.0/).