GLOBAL WATER PATHOGEN PROJECTPART TWO. INDICATORS AND MICROBIAL SOURCE TRACKING MARKERSGENERAL AND HOSTASSOCIATED BACTERIALINDICATORS OF FAECALPOLLUTIONValerie HarwoodUniversity of South FloridaTampa, United StatesOrin ShanksUnited States Environmental Protection AgencyCincinnati, United StatesAsja KorajkicUnited States Environmental Protection AgencyCincinnati, United StatesMatthew VerbylaSan Diego State UniversitySan Diego, United StatesWarish AhmedCommonwealth Scientific and Industrial Research OrganisationBrisbane, AustraliaMercedes IriarteUniversidad Mayor de San SimonCochabamba, Bolivia
Copyright:This publication is available in Open Access under the Attribution-ShareAlike 3.0 IGO (CC-BY-SA 3.0 IGO)license ). By using the content of this publication, the usersaccept to be bound by the terms of use of the UNESCO Open Access use-ccbysa-en).Disclaimer:The designations employed and the presentation of material throughout this publication do not imply theexpression of any opinion whatsoever on the part of UNESCO concerning the legal status of any country,territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Theideas and opinions expressed in this publication are those of the authors; they are not necessarily those ofUNESCO and do not commit the Organization.Citation:Harwood, V., Shanks, O., Koraijkic, A., Verbyla, M., Ahmed, W. and Iriate, M. 2017.General and hostassociated bacterial indicators of faecal pollution. In: J.B. Rose and B. Jiménez-Cisneros, (eds) Global WaterPathogen Project. http://www.waterpathogens.org (A.Farnleitner, and A. Blanch (eds) Part 2 Indicators andMicrobial Source Tracking Markers) ators Michigan StateUniversity, E. Lansing, MI, knowledgements: K.R.L.(http://www.agroknow.com)Young,Last published: February 19, 2018ProjectDesigneditor;WebsiteDesign:Agroknow
General and host-associated bacterial indicators of faecal pollutionSummaryFaecal indicator bacteria (FIB) are used worldwide towarn of faecal and sewage contamination and associatedhuman health risk due to an increased probability of thepresence of waterborne pathogens. Ideally, FIB are nonpathogenic, and include bacteria such as thermotolerant(faecal) coliforms, Escherichia coli, enterococci,Bifidobacteria Bacteroidales, and Clostridium perfringens.These FIB are widely distributed in the faeces of humans,and most animals. Their levels in sewage and faeces arehigh enough that they can usually be detected when faecalcontamination is present in surface waters. Current use ofFIB in regulatory settings is reviewed in this chapter, aswell as their ecology, persistence, and density in faeces,sewage, soil/sediments, biosolids and sewage sludge(primary and secondary). Furthermore, the benefits andlimitations of using FIB as indicators of sewage and otherfaecal contamination in developed, developing, andemerging regions with a variety of climates are discussed.Although FIB have served as useful sentinels ofcontaminated water for many decades, changing needs inwater quality management and better understanding of FIBecology have revealed several shortcomings, includingextended persistence or replication in environmentalhabitats, and greater survival through wastewatertreatment and disinfection systems than some pathogens.The ubiquitous distribution of FIB across different animalpollution sources, which is quite useful for assessingdrinking water quality, becomes problematic for manysurface water quality applications. The faecal pollutionsource frequently assumes a greater importance incontaminated surface waters because mitigation strategiesand human health risk differ greatly depending upon theparticular type of human and/or animal input involved. Thefield of microbial source tracking (MST) offers a diverse setof methodologies designed to identify human and otherfaecal contamination sources. This chapter discusses MSTmethods designed to identify bacteria that are associatedwith human waste, as well as methods targeting waste fromruminant, porcine, and avian animal groups. In addition,the roles of method standardization, data acceptancecriteria, and emerging technologies are explored.1.0 Introduction to Faecal Indicator Bacteriaand Host-Associated BacteriaFaecal indicator bacteria (FIB) are members of themicrobial community of the gastrointestinal tract of mostanimals (including humans), and can be released into theenvironment in faeces, sewage, sludge, and other types ofwaste. The presence of FIB in environmental waters is awarning signal of faecal pollution, indicating the potentialpresence of pathogens. Ideally, FIB should not bepathogenic to minimize the health risk to analysts (e.g.WHO, 2004); however, some FIB groups are pathogenic(e.g. E. coli O157:H7), and many are opportunisticpathogens, such as Enterococcus faecium (a member of theenterococci group). However, even high FIB levels do notalways correspond to increased human health risk. FIB aremembers of bacterial groups or taxa that are ubiquitous inhuman and other animal faeces, and therefore provide littleor no information about specific contamination source(s). Incontrast, host-associated bacteria are closely linked to aparticular animal group, and therefore can be used toindicate probable contamination sources, which is the basisof the emerging science field of microbial source tracking(MST). This chapter covers FIB and host-associatedbacteria and their use for waste and water qualitymanagement. Faecal indicator organisms other thanbacteria are covered in the chapters entitled “General andhost-associated bacteriophage indicators of faecalpollution” and “Human and animal enteric viral markers fortracking the sources of faecal pollution”; while bacterialpathogens are covered in Part Three, Section II.FIB are highly prevalent in the faeces of humans andmost other animals and are easily enumerated by culturemethods. High levels are considered to indicate faecalcontamination; however, many of these bacteria can surviveand even grow in permissive environments with elevatednutrients, shielding from sunlight, and low pressure frompredation, e.g. sediments, compost, sewage sludge,biosolids, and soil (Solo-Gabriele et al., 2000; Zaleski et al.,2005). Decades of research have led to the realization thatnumerous shortcomings are associated with FIB,particularly for surface water quality assessmentapplications (Harwood et al., 2005).The distribution of FIB in the gastrointestinal tract ofmany host species is, however, advantageous for a broadoverview of faecal pollution levels in surface waters, andoffers minimal impediments to the assessment of solidwaste and wastewater treatment. FIB are useful fordetecting breaches and inadequate treatment in drinkingwater distribution systems, as potable water should containno FIB. However, their suitability for assessing surfacewater safety for recreational use can sometimes beconfounded due to variable human health risks posed bythe presence of non-human faecal sources (Soller et al.,2010; 2014). Furthermore, as FIB provide no informationabout a particular contamination source, they can havelimited usefulness for preventing and remediating pollutioninputs (Harwood et al., 2014). Host-associated faecalmicroorganisms, including bacteria, are used in MSTapplications to provide information about faecal pollutionsources in water (i.e. human faeces versus the faeces ofdifferent animals).The objectives of this chapter are to (i) briefly describe thetaxonomy, physiology, and ecology of FIB and hostassociated bacteria, (ii) review the occurrence andpersistence of these bacteria in faeces, wastewater, andsewage sludge, (iii) provide an overview of detection andquantification methods, and (iv) discuss future directionsfor their use in practice and regulatory settings.1.1 Description and Taxonomy of Faecal IndicatorBacteriaFIB are a taxonomically and phylogeneticallyheterogeneous collection of microorganisms which aredefined by characteristics that allow for their selective3
General and host-associated bacterial indicators of faecal pollutiondetection and quantification. Total coliforms,thermotolerant (faecal) coliforms, E. coli, and enterococciare used routinely for regulatory purposes throughout theworld. Some of the methods approved by regulatoryagencies and other standardizing bodies, e.g. the AmericanPublic Health Association (Standard Methods), the UnitedStates Environmental Protection Agency, and theInternational Organization for Standardization (ISO) areshown in Table 1. Tables 2 and 3 contain FIB water qualityregulations in various water types based from manycountries and organisations, including the European Union,the United States, and the World Health Organization.Several genera of strictly anaerobic faecal bacteria(Bacteroides, Bifidobacterium, and Clostridium) are alsoinhabitants of the gastrointestinal tract of humans andother warm-blooded animals, and they each have certaincharacteristics that make them useful indicators of faecalcontamination as well.Table 1. Summary of methods for detecting and quantifying general faecal indicator bacteriaTarget OrganismorGroup ofOrganismsIdentifiersMethod TypeExamples ofStandardized Methodsand Test KitsReferencesTotal coliformsGrowth at 35 0.5 CLactose fermentationAcid productionNegative oxidase enzymeactivityβ-galactosidase enzymeactivityStandard Methods 0221B;Presence/AbsenceIDEXX Colilert and QuantiMost Probable NumberTrayAPHA, 2012Total coliformsGrowth at 35 0.5 CLactose fermentationAcid productionNegative oxidase enzymeactivityβ-galactosidase enzymeactivityMembrane Filtration Standard Methods 9222B,Colony Forming Units 9222C; French Norm NF(CFUs)T90-414APHA, 2012;AFNOR 1985ThermotolerantcoliformsGrowth at 44.5 0.2 CLactose fermentationAcid productionNegative oxidase enzymeactivityβ-galactosidase enzymeactivityStandard Methods 9221E;Presence/AbsenceIDEXX Colilert and QuantiMost Probable NumberTrayAPHA, 2012ThermotolerantcoliformsGrowth at 44.5 0.2 CLactose fermentationAcid productionNegative oxidase enzymeactivityβ-galactosidase enzymeactivityE. coliGrowth at 44.5 CLactose fermentationAcid productionNegative oxidase enzymeactivityβ-glucuronidase enzymeactivityE. coliGrowth at 44.5 CLactose fermentationAcid productionNegative oxidase enzymeactivityβ-glucuronidase enzymeactivityE. coliIdentification of uidA genevia qPCRIdentification of theEC1531 sequence via FISHMembrane FiltrationColony Forming Units(CFUs)Standard Methods 9222Dand 9222EISO 9308-2, 9308-3;Presence/AbsenceIDEXX Colilert; Hach KitMost Probable Number Method 8091; AquagenxCompartment Bag TestMembrane Filtration US EPA Method 1603; ISOColony Forming Units9308-1; Hach Kit (m(CFUs)ColiBlue24 broth)MolecularNRaAPHA, 2012ISO, 1998; ISO,2012;Stauber et al. 2014USEPA, 2006;ISO, 2014Chern et al., 2009;Noble et al., 2010;Langendijk et al.19954
General and host-associated bacterial indicators of faecal pollutionEnterococci andFaecal streptococciGrowth in azide dextrosemedia within 48 hoursβ-D-glucosidase enzymeactivityCulture (MPN)Enterococci andFaecal streptococciGrowth in azide dextrosemedia within 48 hoursβ-D-glucosidase enzymeactivityMembrane FiltrationColony Forming Units(CFUs)Enterococci andFaecal streptococciIdentification of theEntero1a gene via qPCRMolecularUS EPA Methods 1609 and1611Ludwig andSchleifer 2000;Noble et al.; 2010Bacteroides spp.Identification of theGenbac3 gene via qPCRIdentification of thesequence between primersBac32F and Bac708R viaendpoint PCRMolecularUS EPA Method B,EPA-822-R-10-003Bernhard and Field2000; Dick andField , 2004Bifidobacteriumspp.Identification of colonyforming units (CFUs) onMembrane FiltrationBIM-25 media, YN-6, YN-1, Colony Forming UnitsBeerens, BFM or HBSA(CFUs)media.NRMara and Oragui,1983; Munoa andPares, 1988;Beerens, 1990;Nebra and Blanch,1999Bifidobacteriumspp.Identification of theBifidobacterium gene viaqPCRIdentification of the BIF164sequence via FISHMolecularNRGueimonde et al.,2004;Langendijk et al.,1995Clostridium spp.Chromogenic CPChromoSelect AgarIdentification of colonyforming units (CFUs)on m-CP agarPresence/AbsenceMost Probable NumberISO 6461-1;ISO, 1986Clostridium spp.Chromogenic CPChromoSelect AgarIdentification of colonyforming units (CFUs)on m-CP agarMembrane FiltrationColony Forming Units(CFUs)ISO 6461-2ISO, 1986Clostridium spp.Identification of the Cperfgene via qPCRIdentification of the HIS150sequence via FISHNRSivaganesan et al.,2010;Langendijk et al.,1995MolecularISO 7899-1ISO, 1998Standard Methods 9230BISO, 1998; USEPA,and 9230C; ISO 7899-2;2006; APHA, 2012US EPA Method 1600aNR: Not reportedTable 2. Summary of general faecal indicator bacteria norms, regulations, and standards in wastewater,surface, recreational and marine watersAreaGlobalRegulatory UseMaximum Limit for Faecal IndicatorBacteriaWastewater,excreta,Does not specify a maximum limit for faecalgreywater use in indicator bacteria; instead recommends theagriculture anduse of microbial risk assessmentaquacultureGuideline, Norm, or StandardReferenceWorld Health OrganizationGuidelines for the Safe Use ofWastewater, Excreta andGreywaterWHO, 20065
General and host-associated bacterial indicators of faecal pollutionAreaRegulatory UseMaximum Limit for Faecal IndicatorBacteriaGuideline, Norm, or StandardReferenceBoliviaEffluentdischarge to theenvironmentFaecal coliforms:1000 MPN/100mLLaw 1333 – Law of theEnvironmentMMAyA, 1992Regulation/GM/No. 0013:Classifying domestic watercourses in order to protecttheir qualityBrazilianMinistry ofHealth, 1976BrazilChinaDomesticwater coursesClass 1 Waters(domestic use with little or no treatment):Discharge of treated effluent not permittedClass 2 Waters (domestic use after conventionaltreatment; irrigation of horticulture or fruitingplants; primary contact recreation):Total coliforms: 5,000/100mLin 80% of at least 5 monthly samples Faecalcoliforms: 1,000/100mLin 80% of at least 5 monthly samplesClass 3 Waters (domestic use after conventionaltreatment; protection of fish and other flora andfauna; use by wildlife for drinking):Total coliforms: 20,000/100mLin 80% of at least 5 monthly samples Faecalcoliforms: 4,000/100mLin 80% of at least 5 monthly samplesClass 4 Waters (domestic use after heavytreatment; navigation; scenic purposes;industrial use, irrigation and less demandinguses):No faecal indicator limits specifiedWastewater from hospitals:Faecal coliforms:50 MPN/L (Class 1);1,000 MPN/L (Class 2);5,000 MPN/L (Class 3)Wastewater from hospitals withtuberculosis units:Faecal coliforms:100 MPN/L (Class 1);500 MPN/L (Class 2);1,000 MPN/L (Class 3)Wastewaterdischarge to theenvironmentNational Standards of thePeople’s Republic of China:Integrated WastewaterDischarge Standard(GB 8978-1996)Unrestricted irrigation (crops consumedNorms for the Study and Designraw, sports fields, and public green spaces):of Potable Water Systems andWastewater useFaecal coliforms: 1,000/100mLEcuadorthe Deposition of Wastewaterfor irrigationRestricted irrigation (crops not consumedfor Populations Greater thanraw):1,000 InhabitantsFaecal coliforms: no limit specifiedSalvadoran Norm: Water,WastewaterElTotal coliforms: 10,000 MPN/100mLWastewater Discharged to adischarged toSalvadorFaecal coliforms: 2,000 MPN/100mLReceiving Water Body (NSOthe environment13.49.01:09)WastewaterHonduras discharged tothe environmentFaecal coliforms: 5,000/100mL*MPN method preferred but membranefiltration acceptedJapanMarine andfreshwatersourcesCategory AA Rivers and Lakes:Total coliforms: 50 MPN/100mLCategory A Rivers, Lakes, and CoastalBathing Waters:Total coliforms: 1,000 MPN/100mLFishery Class 1 Coastal Waters:70 MPN/100 mLCategory B Rivers:Total coliforms: 5,000 MPN/100mLKenyaSources ofdomestic waterE. coli: 1/100mLChineseEnvironmentalProtectionAgency, 1996IEOS, 1992CONACYT,2009Technical Norm for theDischarge of Wastewater toERSAPS, 1996Receiving Waters and SanitarySewers (Agreement No. 058)Environmental QualityStandards Regarding WaterPollutionJapanEnvironmentAgency, 1986Environmental Managementand Co-ordination (WaterQuality) RegulationsRepublic ofKenya, 20066
General and host-associated bacterial indicators of faecal pollutionRegulatory UseMaximum Limit for Faecal IndicatorBacteriaGuideline, Norm, or StandardReferenceKenyaEffluentdischarge to theenvironmentE. coli: 1/100mLTotal coliforms: 30/100mLEnvironmental Managementand Co-ordination (WaterQuality) RegulationsRepublic ofKenya, 2006KenyaTotal coliforms:1,000 MPN/100mL(unrestricted irrigation)Wastewater use200 MPN/100mLin agriculture(irrigation of public lawns such as hotellawns with which the public may have directcontact)Environmental Managementand Co-ordination (WaterQuality) RegulationsRepublic ofKenya, landsPapuaNewGuineaRecreationalwatersFaecal coliforms: 1/100mLTotal coliforms: 500/100mLEnvironmental Managementand Co-ordination (WaterQuality) RegulationsRepublic ofKenya, 2006Wastewaterdischarged tothe environmentand wastewaterreuse inagricultureFor discharge to water bodies or to land(irrigation):Faecal coliforms (monthly average): 1,000 MPN/100mLFaecal coliforms (daily average): 2,000 MPN/100mLFor discharge to land only (irrigation):Helminth eggs: 1 egg/L (unrestricted irrigation) or 5 eggs/L (restricted irrigation)Official Norms to Establish theMaximum Permissible Limits forContaminants in WastewaterDischarged to National Waters(NOM-001-ECOL-1996)CONAGUA,1997Marine Water QualityRegulationsRepublic 1992Sanitationdischarge tomarine watersFaecal coliforms: 200/100mLMarine andfreshwatersourcesClass AA Waters and Class 1 Groundwater:Total coliform (median of 10 samples):70/100mLTotal coliform: 230/100mL(any one sample)Class A/B Waters and Class 2 Groundwater:Faecal coliform:200/100mL(geometric mean of 10 samples)Faecal coliform: 400/100mL(any one sample)Class AA/A Waters (Palau):Enterococci: 33/100mL(geometric mean of 5 samples)Enterococci: 60/100mL(any one sample)Class AA and Shellfish Waters (MarshallIslands):Enterococci:7/100mL(arithmetic mean of 5 samples)Class A Waters (Marshall Islands):Enterococci:35/100mL(arithmetic mean of 5 samples)Marine andfreshwatersourcesFreshwater:Faecal coliforms:200/100mL(median of 5 samples)Seawater:No regulations for faecal indicator bacteriaRepublic ofMarshallChapter 2401-11. Marine andIslandsFresh Water QualityEnvironmentalRegulationsProtectionMarine Water QualityAuthority,Regulations (Marshall Islands)1992;Repuclic ofPalau, 1996Environment (Water QualityCriteria) RegulationPapua NewGuineaConsolidatedLegislation,20067
General and host-associated bacterial indicators of faecal pollutionAreaSri LankaTurkeyUKRegulatory UseMaximum Limit for Faecal IndicatorBacteriaGuideline, Norm, or StandardTreatedWastewaterDischarge to Inland Surface Waters:Faecal coliforms: 40 MPN/100mL (max)Discharge on Land for Irrigation:Faecal coliforms: 40 MPN/100mL (max)Discharge to Marine Coastal Areas:Faecal coliforms: 60 MPN/100mL (max)Sri LankanMinistry ofNational Environmental Act, No. Environmentand Natural47 of 1980Resources,2008TreatedWastewaterDischarge to Class I Waters:Total coliforms: 100 MPN/100mLFaecal coliforms: 10 MPN/100mLDischarge to Class II Waters:Total coliforms: 2,000 MPN/100mLFaecal coliforms: 200 MPN/100mLDischarge to Class III Waters:Total coliforms: 10,000 MPN/100mLFaecal coliforms: 2,000 MPN/100mLClassification “Excellent” (95th percentile oflog10 densities):Enterococci: 200 CFU/100mLE. coli: 500 CFU/100mLClassification “Good” (95th percentile oflog10 densities):Inland BathingWatersEnterococci: 400 CFU/100mLE. coli: 1,000 CFU/100mLClassification “Sufficient” (90th percentile oflog10 densities):Enterococci: 330 CFU/100mLE. coli: 900 CFU/100mLUKCoastal BathingWatersClass
INDICATORS OF FAECAL POLLUTION Valerie Harwood University of South Florida Tampa, United States Orin Shanks United States Environmental Protection Agency Cincinnati, United States Asja Korajkic United States Environmental Protection Agency Cincinnati, United States Matthew Verbyla San Diego State University San Diego, United States Warish Ahmed
For the purification of viral RNA and DNA and bacterial DNA from animal whole blood, serum, plasma, other body fluids, swabs and washes, and tissue IndiSpin Pathogen Kit (cat no SP54104), formerly QIAamp cador Pathogen Mini Kit (50) IndiSpin Pathogen Kit (cat no SP54106), formerly QIAam
2 Activities for AP* Biology POGIL 1. In Model 1 a pathogen (virus, bacteria, foreign protein, parasite) has entered the bloodstream of an individual. Draw the symbol that represents the pathogen. 2. One response of the human immune system is endocytosis of a pathogen by a phagocyte (a type of white blood cell).
Chapter 4 Appendix B: Secondary BSI Guide). Notes: 1. If a patient meets both LCBI 1 and LCBI 2 criteria, report LCBI 1 with the recognized pathogen entered as pathogen #1 and the common commensal as pathogen #2. 2. No additional elements (in other words, no sign or symptom such as fever) are needed to meet LCBI 1
Important definitions * Vector- intermediate host carrying a pathogen Mechanical transmission - pathogen does not replicate in vector; accidental spread (i.e., legs) Biological transmission - pathogen replicates inside vector and transmitted to hosts by excretion
REPORT TO PARTICIPANTS IN . THE AOAC LABORATORY PROFICIENCY TESTING PROGRAM . PATHOGEN FREE MICROBIOLOGY PROGRAM . 1.0 Introduction. Test materials for the Pathogen Free Microbiology Program were shipped to participants on May 6, 2019. Each laboratory was given a site identification number in order to maintain confidentiality.
Slide 1: Bloodborne Pathogen Training for School Staff December 1991- Occupational Safety & Health Administration (OSHA) in the U.S. Department of Labor issued regulations regarding bloodborne pathogen (BBP) occupational exposure to reduce risk for workers. In Ohio, public employees are subject to Public Employee Risk Reduction Program (PERRP).
The following points must be covered in Bloodborne Pathogen and Universal Precautions training and therefore ALL employees should KNOW: Foodborne Diseases and Outbreaks Where to access a copy of the OSHA Bloodborne Pathogen Standard (Law) with explanation Where they can obtain a copy of their employer's exposure control plan and
species independently by two different lab groups in 2002. The first of these reports (Crous et al., 2002), documented a new species of fungus infecting boxwoods in New . and clothing, if not properly sanitized, can vector pathogen propagules inadvertently to healthy plants and non-threatened areas. The life cycle for the pathogen is rather .