GLOBAL WATER PATHOGEN PROJECT PART TWO. INDICATORS AND .

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