Predictors of Enteric Pathogens in the Domestic Environment from

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Predictors of Enteric Pathogens in the Domestic Environment from Human and Animal Sources in Rural Bangladesh Erica R. Fuhrmeister, Ayse Ercumen, Amy J Pickering, Kaitlyn M. Jeanis, Mahaa Ahmed, Sara Brown, Benjamin F. Arnold, Alan E. Hubbard, Mahfuja Alam, Debashis Sen, Sharmin Islam, Mir Himayet Kabir, Laura H. Kwong, Mahfuza Islam, Leanne Unicomb, Mahbubur Rahman, Alexandria B Boehm, Stephen P. Luby, John M. Colford, and Kara L. Nelson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07192 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

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Predictors of Enteric Pathogens in the Domestic Environment from Human and Animal Sources in Rural Bangladesh Erica R. Fuhrmeister1, Ayse Ercumen2,3, Amy J. Pickering4, Kaitlyn M. Jeanis 1, Mahaa Ahmed 1, Sara Brown1, Benjamin F. Arnold2, Alan E. Hubbard2, Mahfuja Alam5, Debashis Sen5, Sharmin Islam5, Mir Himayet Kabir5, Laura H. Kwong6, Mahfuza Islam 5, Leanne Unicomb5, Mahbubur Rahman5, Alexandria B. Boehm6, Stephen P. Luby7, John M. Colford Jr. 2, Kara L. Nelson1* 1Department

of Civil and Environmental Engineering, University of California, Berkeley, California, 94720, United States 2School of Public Health, University of California, Berkeley, California 94720, Unites States 3Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, North Carolina, United States 4Civil and Environmental Engineering, Tufts University, Medford, Massachusetts, 02153, United States 5Infectious Disease Division, International Centre for Diarrhoeal Disease Research Bangladesh, Dhaka, 1212, Bangladesh 6Department of Civil and Environmental Engineering, Stanford University, Stanford, California, 94305, United States 7Woods Institute for the Environment, Stanford University, Stanford, California 94305, United States Correspondence to: [email protected]

For submission as an original research article to Environmental Science & Technology

Abstract: 223 Main Text: 5833 Figures: 900 Tables: 600 Total: 7556


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Abstract

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Fecal indicator organisms are measured to indicate the presence of fecal pollution, yet the

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association between indicators and pathogens varies by context. The goal of this study was to

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empirically evaluate the relationships between indicator Escherichia coli, microbial source

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tracking markers, select enteric pathogen genes and potential sources of enteric pathogens in

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600 rural Bangladeshi households. We measured indicators and pathogen genes in stored

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drinking water, soil and on mother and child hands. Additionally, survey and observational data

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on sanitation and domestic hygiene practices were collected. Log10 concentrations of indicator E.

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coli were positively associated with the prevalence of pathogenic E. coli genes in all sample types.

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Given the current need to rely on indicators to assess fecal contamination in the field, it is

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significant that in this study context indicator E. coli concentrations, measured by IDEXX Colilert-

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18, provided quantitative information on the presence of pathogenic E. coli in different sample

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types. There were no significant associations between the human fecal marker (HumM2) and

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human-specific pathogens in any environmental sample type. There was an increase in the

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prevalence of Giardia lamblia genes, any E. coli virulence gene, and the specific E. coli virulence

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genes stx1/2 with every log10 increase in the concentration of the animal fecal marker (BacCow)

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on mothers’ hands. Thus, domestic animals were important contributors to enteric pathogens in

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these households.

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Introduction

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In low- and middle-income countries diarrheal illness is a leading cause of morbidity and

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mortality.1 In Bangladesh, 6% of the 129,000 deaths in children under five in 2013 were attributed

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to diarrheal diseases.2 Diarrheal illness results from exposure to fecal pathogens which can be

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transmitted from feces to a new human host through a variety of environmental pathways,

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including fingers, fields, flies, fluids, and food, described in the F-diagram.3 Recent additions to

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the F-diagram stress the importance of animal hosts by expanding fecal sources to include feces

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from livestock, free-roaming animals and synanthropic rodents.4 While it is well known that

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enteric pathogens are transmitted through these pathways, few studies have measured

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pathogens to characterize exposure from different animal reservoirs.

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There is a high potential for zoonotic enteric disease transmission in low- and middle- income

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countries where animal husbandry is a primary source of income.5 For example, in Bangladesh

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raising livestock such as cows, goats and chickens results in animals roaming freely within the

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home environment.6 Close proximity to domesticated animals can lead to human exposure to

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livestock feces. Many pathogens can be transmitted from animal feces to human hosts and result

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in diarrheal illnesses.5 Of the four most common etiological agents of moderate to severe

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diarrhea in children 0-11 months in Bangladesh, two (Cryptosporidium and Campylobacter) are

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known to have important animal reservoirs.7,8

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Measuring enteric pathogens in the environment can help identify reservoirs and potential

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exposure pathways, which can better inform design of strategies to reduce human exposure.


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However, there are many different fecal pathogens from humans and animals capable of causing

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disease, making it infeasible to measure them all, especially given that most are difficult to

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measure in the environment due to their low concentrations in environmental matrices and

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costly and complex methods of detection.9 Therefore, fecal indicators are commonly used to

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indicate the presence of fecal contamination, which may contain pathogens.9 Thermotolerant

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coliforms and Escherichia coli are recommended indicators of drinking water quality by the World

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Health Organization.10 Other fecal indicators such as Bacteroidales are used for identifying

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sources of fecal contamination through microbial source tracking (MST).11 However, the degree

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to which indicators and pathogens co-occur in environmental samples varies depending on their

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concentrations in the original fecal source (pathogen concentrations depend on the infection

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status of the human or animal whereas indicator organism concentrations are expected to

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remain more stable) and the relative transport and die-off/growth rates of organisms once they

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are in the environment. Additionally, correlations between indicator organisms and pathogens

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in natural environments, such as soil and surface waters, are impacted by the concentrations of

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naturally occurring E. coli in the environment, often referred to as “naturalized” E. coli. Thus, the

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relationship of indicator organisms and specific pathogens in or on different environmental

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matrices (e.g., water, soil, hands, fomites, food) varies spatially and temporally depending on

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these factors.12 Nonetheless, a better empirical understanding of the relationship between

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indicators and pathogens in specific environmental reservoirs and contexts in low- and middle-

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income countries may improve the ability to estimate human health risk and identify fecal

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sources and exposure pathways of greatest concern. Furthermore, pinpointing dominant sources


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and reservoirs will help improve the design of targeted interventions that can reduce exposure

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to fecal pathogens, mitigating the burden of diarrheal illnesses.13,14

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The aim of this study was to measure fecal indicators and select human pathogen genes in

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different reservoirs in the domestic environment to: 1) determine the association between

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concentration of indicator E. coli and presence of pathogen genes; and 2) use microbial source

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tracking and observations of animal feces in compounds to investigate potential human and

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animal sources of detected pathogen genes.

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Methods

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We selected multiple types of microorganisms that are leading causes of diarrheal illness in

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developing countries and have a range of host specificity between humans and animals. The

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selected targets were indicators (E. coli, human-associated Bacteroidales-like gene, and non-

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human animal-associated Bacteroidales gene) and pathogen genes (enteropathogenic E. coli

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(EPEC), enteroaggregative E. coli (EAEC), shiga-toxin producing E. coli (STEC), enteroinvasive E.

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coli (EIEC), enterotoxigenic E. coli (ETEC), norovirus GII, Giardia lamblia, and Cryptosporidium

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spp.).We measured these targets in different reservoirs in the domestic environment within rural

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Bangladeshi compounds - stored water, soil, and mother and child hands (Figure 1).

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We reviewed current literature to determine which of the selected pathogens had the potential

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to originate from both human and animal reservoirs or only human reservoirs. Pathogens

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associated with both human and animal feces include Cryptosporidium, Giardia lamblia, EPEC,

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STEC and ETEC.15–17 Humans serve as a reservoir of typical EPEC, while animals such as pigs and


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chickens can serve as reservoirs of atypical EPEC.17,18 Though typical and atypical EPEC can be

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differentiated by the presence of the gene bfpA, this study only measured eaeA and will therefore

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not distinguish between these strains.19 STEC is characterized by the presence of shiga toxin

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genes (stx1 or stx2) and is known to come from ruminants.17 Enterohemorrhagic E. coli (EHEC) is

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a common type of STEC that can also carry the eaeA gene found in EPEC.17,20 ETEC is found in

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both humans and animals but species-specific adhesion factors confer host specificity.8 We were

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unable to differentiate ETEC with human adhesion factors in this study because the enterotoxins

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we measured, st1b and lt1, can be found in both humans and animals.21–24 Previous studies have

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isolated Giardia lamblia and Cryptosporidium spp. in many fecal sources and identified closely

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related isolates from humans and animals.15,25 On the other hand, norovirus GII, EAEC and EIEC

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are associated with primarily human sources, largely based on genetic analysis of pathogens in

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human and animal fecal samples.17,26,27 While EAEC is also found in animals, the gene we

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detected, aggR, has been identified in EAEC isolated from humans but not animals.27,28 EIEC has

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also been found in primates, which are unlikely to be important contributors to fecal pollution in

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the study communities.17 It should be noted that the associations between pathogens and

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possible hosts in Figure 1 are based on current scientific understanding and evidence. Zoonotic

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pathogen transmission is difficult to demonstrate and we are limited by the methods that have

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been employed and the pathogens, virulence genes, locations, and animal hosts that have been

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investigated.

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Study Setting and Design

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600 households were randomly selected from those enrolled in the sanitation and control arms

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(300 households per arm) of the WASH Benefits randomized controlled trial in rural Bangladesh,


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described elsewhere.29 One study household was selected per compound and compounds were

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usually relatives living in adjacent households surrounding a central courtyard. Compounds were

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clustered in groups of eight to reduce spillover effects and for the ease of community promoters

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delivering the interventions. Clusters were geographically pair-matched and then randomly

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allocated to different intervention arms.30 Study households were from the rural Gazipur,

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Mymensingh, Tangail and Kishoreganj districts. WASH Benefits enrolled and followed households

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with a pregnant woman. All study households contained a young child (aged 9-44 months) at the

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time of our visit. In this same subset of households, another analysis in preparation was

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conducted to investigate the impact of the sanitation intervention on the prevalence of pathogen

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genes and microbial source tracker markers.31 Another related study investigated how the

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presence of domestic animals impacted concentrations of indicator E. coli in reservoirs in a subset

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of households from the control arm.32

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Data Collection

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During household visits between March and October 2015, trained field staff from the

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International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) collected

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environmental samples, interviewed the female caregiver of young children regarding household

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practices on handling of animal feces, and observed the number and type of animal fecal piles in

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compound courtyards. Field surveyors also recorded the number and type of animals owned by

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the household as reported by the caregiver.

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Sample Collection


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We collected approximately 720 stored drinking water samples, 720 soil samples, 720 mother

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hand rinses, and 360 child hand rinses from 600 study households. 176 of households in the

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control and 156 of households in the sanitation arms were sampled twice, approximately four

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months apart to assess the impact of season. Hand rinse samples were collected by mothers

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placing their left hand into a sterile Whirlpak bag (Nasco, Modesto, CA) filled with 250 mL of

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distilled water. The hand was massaged from the outside of the bag for 15 seconds, followed by

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15 seconds of shaking. The same procedure was repeated with the right hand in the same bag.

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To collect child hand rinse samples, respondents placed their child’s hand into a separate

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Whirlpak bag and followed the same procedure. Soil samples were collected from a 30 x 30 cm2

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area as close to the house entrance as possible by scraping the top layer of soil within a stencil

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into a sterile Whirlpak bag using a sterile disposable plastic scoop. The sample area was scraped

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both vertically and horizontally. Stored water samples were collected by asking mothers to

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provide a glass of water as they would give it to their child under five. The provided water was

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poured into a sterile Whirlpak bag. All samples were transported to the icddr,b field laboratory

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on ice and processed within 12 hours of collection.

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Indicator E. coli Enumeration and Detection of Pathogenic Genes

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E. coli were enumerated (100 mL sample volumes) using IDEXX Colilert-18 (IDEXX Laboratories,

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Westbrook, Maine). Trays were incubated at 44.5 °C for 18 hours.33 To prepare for IDEXX analysis,

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hand rinse samples were diluted 1:2 with sterile distilled water in Whirpak bags while stored

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water was analyzed undiluted. 20 g of soil was homogenized with 200 mL of distilled water and

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diluted 1:104. Soil was dried at 110° C for 24 hours to determine moisture content. Each lab

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technician processed one blank (sterile distilled water) for E. coli enumeration per day.


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Pathogenic E. coli genes were identified using previously published methods.20,34 All samples

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positive for E. coli as determined by IDEXX Colilert-18 were archived for subsequent pathogenic

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E. coli gene analysis. Broth from up to 20 positive large wells was aseptically extracted from IDEXX

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trays, composited and centrifuged. Pellets were treated with 10x the pellet volume (≈ 0.1 mL) of

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RNAlater (Qiagen, Germantown, MD), stored at -80 °C and transported to UC Berkeley at room

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temperature. Each lab technician analyzed one lab blank per week for the archiving process by

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archiving wells from IDEXX trays incubated with sterile distilled water. Pellets were stored at -80

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°C upon arrival at UC Berkeley. DNA was extracted from bacteria pellets using the DNeasy Blood

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and Tissue Kit (Qiagen, Germantown, MD). Multiplex reactions were used to detect seven E. coli

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virulence genes indicative of five possible pathotypes of E. coli: EAEC (aggR), EPEC or EHEC

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(eaeA), STEC (stx1/ stx2), EIEC (ipaH), ETEC (lt1/st1b) (Table S1). PCR products were run on 2%

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agarose gels at 110 V for 30 min. Additional information on DNA extraction and PCR protocols is

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available in the supporting information.

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Filtration and Nucleic Acid Extraction for qPCR Targets

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Laboratory technicians pre-processed samples in the field laboratory by filtering 50 mL of hand

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rinse samples and up to 500 mL of stored water (range: 100-500 mL) through a 0.45 m HA filter

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(Millipore, Burlington, MA) to capture bacteria and viruses. 0.5 mL of 2.5M MgCl2 was added to

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every 50 mL of sample to increase retention of viruses.35 Filters were treated with 0.5 mL of

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RNAlater, stored at -80 °C and transported to UC Berkeley at room temperature. Once per day,

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each lab technician processed a lab blank by filtering 5 mL of sterile distilled water with 0.5 mL

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of MgCl2, followed by the addition of RNAlater. Five grams of soil were weighed and stored at -


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80 °C until transport at room temperature to UC Berkeley in accordance with a USDA soil import

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permit (PPQ 525). Filter and soil samples were stored at -80 °C at UC Berkeley. DNA and RNA

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were extracted from filters and 0.25 grams of soil samples using modified Mobio PowerWater

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and PowerViral (both now Qiagen, Germantown, MD) protocols, described in previous studies

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and in the supporting information.36,37 An extraction blank was included in each batch of 23

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samples. Extraction efficiencies for DNA and RNA were determined in a subset of samples using

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Pseudomonas syringae pv. phaseolicola (pph6) and MS2 (Table S2).

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qPCR Assays

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Samples were analyzed for norovirus GII, Giardia lamblia, Cryptosporidium spp. genes, and

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microbial source tracking markers using quantitative PCR (Table S3). Specifically, to detect human

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fecal contamination we used the HumM2 assay which targets a gene for a hypothetical protein

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in human-associated Bacteroidales-like microorganisms.38,39 We obtained a research license

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(#864-15) from the U.S. Environmental Protection Agency to use the patented HumM2 assay. To

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detect non-human, animal-associated fecal contamination we used the BacCow assay which,

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although designed to target the 16S rRNA of Bacteroidales associated with ruminants, has been

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shown to detect Bacteroidales in cows, ducks, goats, and chickens but not humans in rural

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Bangladesh.36,40 The microbial source tracking markers were previously evaluated for sensitivity

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and specificity in rural Bangladesh.36 BacCow was sensitive but not specific to ruminant feces and

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HumM2 performed the best out of all tested human-associated assays (HumM2, HF183 and

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BacHum), although it also amplified in the feces of chickens and goats.36

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Approximately 100 stored water, soil and mother and child hand rinse samples were processed

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for norovirus GII, Giardia and Cryptosporidium genes. Among this subset, 0) compared to those with no fecal piles. For

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chicken/non-chicken poultry feces the prevalence ratio represents the prevalence of pathogen

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genes in households with > 5 fecal piles compared to those with ≤ 5 fecal piles. Cow patties for

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cooking were differentiated from cow feces because they were formed into cakes and dried in

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the sun. Exposures (feces of different animals) were screened against outcomes (pathogen

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genes) in bivariate models and those with a p-value

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