This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Article pubs.acs.org/est Animal Feces Contribute to Domestic Fecal Contamination: Evidence from E. coli Measured in Water, Hands, Food, Flies, and Soil in Bangladesh Ayse Ercumen,*,† Amy J. Pickering,‡,○ Laura H. Kwong,§ Benjamin F. Arnold,† Sarker Masud Parvez,∥ Mahfuja Alam,∥ Debashis Sen,∥ Sharmin Islam,∥ Craig Kullmann,⊥ Claire Chase,⊥ Rokeya Ahmed,# Leanne Unicomb,∥ Stephen P. Luby,∇ and John M. Colford, Jr.† † School of Public Health, University of California, Berkeley, California 94720, United States ‡ Civil and Environmental Engineering, Tufts University, Medford, Massachusetts 02153, United States See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. § Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States ∥ Infectious Disease Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, 1212, Bangladesh ⊥ Water Global Practice, World Bank, Washington, D.C. 20433, United States Downloaded via 138.220.206.73 on March 12, 2019 at 14:22:12 (UTC). # Water Global Practice, World Bank, Dhaka, 1207, Bangladesh ∇ Infectious Diseases & Geographic Medicine, Stanford University, Stanford, California 94305, United States ABSTRACT: Fecal-oral pathogens are transmitted through complex, environmentally mediated pathways. Sanitation interventions that isolate human feces from the environment may reduce transmission but have shown limited impact on environmental contamination. We conducted a study in rural Bangladesh to (1) quantify domestic fecal contamination in settings with high on-site sanitation coverage; (2) determine how domestic animals affect fecal contamination; and (3) assess how each environmental pathway affects others. We collected water, hand rinse, food, soil, and fly samples from 608 households. We analyzed samples with IDEXX Quantitray for the most probable number (MPN) of E. coli. We detected E. coli in source water (25%), stored water (77%), child hands (43%), food (58%), flies (50%), ponds (97%), and soil (95%). Soil had >120 000 mean MPN E. coli per gram. In compounds with vs without animals, E. coli was higher by 0.54 log10 in soil, 0.40 log10 in stored water and 0.61 log10 in food (p < 0.05). E. coli in stored water and food increased with increasing E. coli in soil, ponds, source water and hands. We provide empirical evidence of fecal transmission in the domestic environment despite on-site sanitation. Animal feces contribute to fecal contamination, and fecal indicator bacteria do not strictly indicate human fecal contamination when animals are present. ■ INTRODUCTION Fecal-oral pathogens are transmitted from feces to new hosts diarrhea.3,4 Other transmission pathways remain understudied even though these could present major sources of fecal through complex, environmentally mediated pathways. The exposure. For example, complementary foods for young complexity arises from a multitude of transmission pathways, a children contain FIB in low-income country settings5 and broad diversity of pathogens, the influence of environmental child diarrhea has been linked to food contamination.6 High conditions and interactions between the environment and FIB levels are found on hands in low-income countries7 and human behavior. In the absence of effective sanitation and handwashing interventions reduce self-reported diarrhea.8 Flies sewerage facilities that isolate human feces from the environ- are known to carry human pathogens9,10 and fly control ment, human fecal organisms can spread into fields and programs have successfully reduced diarrheal disease.11 FIB and ambient waters. These are subsequently transported by fomites pathogens have been detected in soil12 and geophagia has been and vectors (e.g., hands, flies) into drinking water and food as associated with diarrhea, markers of environmental enteric well as ingested through mouth contact with contaminated dysfunction, and stunting in young children.13,14 However, it hands and objects or geophagia (deliberate ingestion of soil) by has not been documented how soil contamination affects young children.1,2 Contamination of drinking water, a direct ingestion pathway, Received: April 3, 2017 has been studied extensively, and water treatment has been Revised: June 23, 2017 shown to improve microbiological water quality as measured by Accepted: July 7, 2017 fecal indicator bacteria (FIB) and reduce self-reported Published: July 7, 2017 © 2017 American Chemical Society 8725 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article subsequent contamination of ambient and drinking waters, were collected, presence of a handwashing station with soap hands and food. and water (tubewell, pond, or container with water) within 10 Sanitation interventions are a primary barrier to disease m of the latrine, and presence and number of latrines in the transmission and should block enteric pathogens both by compound and within 10 m of tubewells and ponds. They stopping feces from spreading into the environment as well as differentiated improved latrines based on Joint Monitoring eliminating fly breeding sites. However, recent sanitation trials Programme (JMP) categories26 and observed whether the have shown limited health impact. Two trials in India found no latrine drained into a septic tank, pit, or the environment effect of sanitation improvements on child diarrhea, parasite (pond, ditch, etc.). When collecting soil samples, field staff infections, and growth,15,16 while a trial in Mali demonstrated observed whether the sampled area was visibly wet and in the improved child growth but no diarrhea reduction.17 These trials sun or shade. also found no reduction in FIB measured in source and stored Sample Collection. Field workers collected samples from water, and on hands and fomites.15−17 A systematic review the compound, including tubewell water, drinking water stored identified no overall reductions in environmental contami- in the home, pond water, child hand rinses, complementary nation in response to sanitation improvements.18 Possible foods given to young children, flies caught in the food explanations include low latrine uptake and continuing open preparation area, and courtyard soil from young children’s defecation; Clasen et al. (2014) and Patil et al. (2014) reported outdoor play area. Samples were collected in sterile Whirlpak that <50% of households in intervention villages had a bags (Nasco Modesto, Salida, CA). To collect source water functional or improved latrine, respectively, and Patil et al. (tubewell) samples, field staff removed fabric or other materials found that >70% of adults in intervention villages reported daily attached to the tubewell mouth and flushed the tubewell by open defecation.15,16 pumping five times before collecting 250 mL of water. To Another potential explanation for the failure of sanitation collect stored water, field workers asked the respondent to improvements to reduce domestic fecal contamination and provide a glass of water from their primary drinking water diarrhea is residual contamination from animal feces. Sanitation storage container as if giving it to their children <5 years and programs focus on isolating human feces from the environ- pour 250 mL from the glass into a Whirlpak. Pond samples ment, typically with no measures to reduce exposure to animal were collected by dipping a Whirlpak into the pond and feces. Many households in low-income countries keep livestock collecting 250 mL of water from the area where the household in close proximity to living quarters.19 Microbial source tracking reported most commonly accessing the pond. To sample child studies in rural India and Bangladesh suggest that fecal hands, field workers asked the respondent to place both hands contamination from animals is more prevalent than human of the youngest child <5 years, one at a time, into a Whirlpak contamination in the domestic environment, including source prefilled with 250 mL of distilled water. Each hand was and stored drinking water, hands, and soil.20−22 Courtyard soil, massaged from outside the bag for 15 s, followed by 15 s of household floors, and child hands have been shown to contain shaking, and the rinsewater was preserved in the Whirlpak.27 animal fecal molecular markers.20,23 Presence of animal feces in To collect soil samples, the respondent was asked to identify the compound has been associated with visible dirtiness of the outdoor area where the youngest child <5 years had most caregivers’ and children’s hands and faces.24 There is also recently spent time. Field workers marked a 30 × 30 cm2 area increasing evidence that exposure to domestic animals is with a sterile stencil, and scraped the top layer of soil within the associated with increased diarrhea.19 However, the contribution stencil into a Whirlpak using a sterile scoop to collect of animal feces to fecal contamination along different approximately 50 g of soil. To sample complementary food, transmission pathways in settings with on-site sanitation has field workers identified stored food to be served to children <5 not been assessed. years and asked the respondent to provide a small amount of The objectives of our study were to (1) characterize levels of food in the same manner they feed their children. Food was fecal contamination along multiple environmental transmission scooped to fill a 50 mL sterile plastic tube using a sterile spoon. pathways (source, stored and ambient waters, child hands, Finally, field workers identified a suitable location in the food complementary food, courtyard soil, and flies caught in the preparation area (away from the stove and smoke, under a roof compound) in rural Bangladeshi households, (2) determine or protected from rain if possible) and hung three horizontal how the presence of domestic animals, household sanitary 1.5-foot strips of nonbaited sticky fly tape. The tape was left in infrastructure and ambient climate conditions affect contami- place for 3−6 h to capture flies. Field workers removed one fly nation levels, and (3) assess how different environmental from the center of the strip with the most flies using sterile pathways affect each other. ■ tweezers and placed it into a Whirlpak. Clean gloves were worn to collect pond, hand rinse, soil, and food samples. MATERIALS AND METHODS Sample Processing. Samples were preserved on ice and Data Collection. Our study was nested within a processed on the same day, typically within 12 h of collection. randomized controlled trial in rural central Bangladesh Tubewell and stored water samples were analyzed without (WASH Benefits).25 We randomly enrolled households from dilution. Pond samples were diluted 1:100 and hand rinses 1:2 the trial’s control arm between July 2013 and March 2014. with distilled water. Food and soil were homogenized with During household visits, field workers conducted spot check distilled water using a sterile blending bag (BagFilter P, 400 observations on the presence of human and animal feces in the mL, Interscience, Saint Nom, France) and a laboratory-scale courtyard; human vs specific animal (cow, goat/sheep, chicken) food processor (BagMixer C, Interscience, Saint Nom, France) feces were distinguished based on their visual characteristics. for 1 min at a specified mixing speed. A 10 g aliquot of food was Field workers also administered a structured questionnaire on homogenized with 100 mL of distilled water and then diluted animal husbandry. Additionally, they observed water, sanitation, 1:10. A 20 g aliquot of soil was homogenized with 200 mL of and hygiene indicators, including the cover status of the storage distilled water and then diluted 1:104. An additional 5 g food containers from which the drinking water and food samples and soil aliquot was oven-dried overnight to determine the 8726 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article moisture content and dry weight. Flies were homogenized with Table 1. Characteristics of Enrolled Households (N = 608) a pestle from outside the Whirlpak and mixed with 100 mL of household characteristics % distilled water; this slurry was diluted 1:100. One field blank per sample collector per week, one household water, sanitation, hygiene conditions laboratory blank per laboratory assistant per day, 10% field latrine in compound 97 duplicates (two samples from one household), and 5% improved latrine in compound (JMP definitiona) 68 laboratory replicates (two aliquots from the same sample) latrine flushes to environment 29 were processed for quality control. Field workers collected two household owns child potty 17 types of field blanks (1) by asking the respondent to pour human feces observed in courtyard 4 distilled water from a sterile bottle into a Whirlpak and (2) by stored water covered 17 water present in latrine 47 opening and shaking a prefilled Whirlpak in the field as if soap present in latrine 7 collecting a hand rinse. Samples were analyzed using IDEXX food container covered 85 Quantitray with Colilert-18 media (IDEXX Laboratories, flies captured in food preparation area 32 Maine, U.S.A.) and incubated at 44.5 °C for 18 h to enumerate presence of domestic animals and animal feces E. coli with the most probable number (MPN) method. The compound has animals 94 Quantitray-2000 system with a wide detection range of 1−2419 chickens 91 MPN per tray was selected to accommodate variability within cows 69 sample types. goats/sheep 39 Statistical Methods. We tabulated the presence/absence, animals roam free in compound 56 log10-transformed counts, and geometric means of E. coli; we animal feces observed in courtyard 89 substituted the value of 0.5 MPN for samples below and 2420 chicken feces 87 MPN for samples above the detection limit to calculate the cow feces 30 logarithm. We assessed the association between log10-trans- goat/sheep feces 19 formed E. coli counts and ambient climate factors (e.g., season, a sunlight and visible moisture in soil sampling area, measured JMP: Joint Monitoring Programme. soil moisture content), presence of animals, observed human/ animal feces, and household sanitary infrastructure. Season was Animals and Animal Feces. Almost all compounds (94%) defined as wet vs dry as Bangladesh receives >80% of its rain had domestic animals and the most common animal was during the monsoon season from June through October and is chickens, while 89% of compounds had observed animal feces typically dry otherwise.28 We also assessed the relationships in the courtyard and chicken feces were the most common type between different transmission pathways by separately estimat- of feces observed. Whether or not a household had animals was ing the association of E. coli levels in different sample types not associated with socioeconomic proxies; however, house- (e.g., log10 increase in E. coli on hands for every log10 increase in holds that owned land were more likely to have >1 cow or >10 E. coli in soil). We used generalized linear models with robust chickens (p < 0.05). Compounds were more likely to have standard errors to account for the clustered design of the animal feces in the courtyard if they had unfinished (e.g., WASH Benefits trial. We assessed whether housing materials, bamboo, mud) walls or no electricity (p < 0.05). reported income, land ownership, presence of electricity, and Fecal Contamination. We tested 3254 samples and female education (≥1 year of formal schooling) as socio- detected E. coli in every sample type, including 25% of source economic proxies were associated with the presence of animals water, 77% of stored water, 43% of child hands, 58% of and animal feces using chi-square tests; all models controlled complementary foods, 50% of flies, 97% of ponds, and 95% of for these potential confounders. soil (Table 2). Geometric mean E. coli was <10 MPN per ■ reporting unit in drinking water, food and on hands. The RESULTS AND DISCUSSION geometric mean E. coli for flies was 663 MPN per fly. Ponds and soil had extremely high contamination; geometric mean E. Household Characteristics. Of the 699 households coli was >5000 MPN per 100 mL for ponds and >120 000 randomly selected from the control arm of the parent trial, MPN per dry gram for soil. Across all samples types, 5% of we successfully enrolled 608 (87%) households with 13% lost samples exceeded the detection limit. to follow-up (7% stillbirth, miscarriage, abortion, or death of Ambient Climate Conditions Vs Fecal Contamination. children in the target age range, 5% relocation, and 1% refusal). During the rainy season, E. coli was detected significantly more Among 608 enrolled households, 97% had a latrine and 68% frequently and at higher concentrations along all pathways had an improved latrine as per the JMP definition;26 29% of compared to the dry season (all p < 0.05 except for log10 E. coli latrines drained into the environment (Table 1). The presence in soil) (Figure 1). Soil E. coli counts were not affected by and number of latrines were positively associated with all whether the soil was visibly wet at the time of sampling. proxies of higher socioeconomic status (finished walls, However, soil samples with above-median moisture content electricity access, above-median reported income, land owner- (median = 7%, range = 0−34%) had 0.70 log10 MPN higher E. ship, ≥1 years of female education, all p-values <0.05) while the coli per dry gram (p < 0.005). Soil from areas sunlit at the time presence of an improved latrine was not associated with any of of collection had 0.48 log10 MPN fewer E. coli per dry gram these proxies. Human feces were observed in 4% of than soil from shaded areas (p < 0.005). compounds. Half (47%) of households had water in the latrine Presence of Animals and Animal Feces Vs Fecal area while 7% had soap. Fewer than 20% of drinking water Contamination. Animal presence was associated with higher storage containers were covered in contrast to 85% of food levels of fecal contamination along multiple pathways; soil storage containers. At least one fly was caught in the food contamination in particular was independently associated with preparation area in 32% of households. the presence of individual animal species (chickens, goats/ 8727 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article Table 2. E. coli Detection among Environmental Pathways type of sample N unit lower detection limit (MPNa) upper detection limit (MPNa) geometric mean (MPNa) % positive soil 591 1 dry gram 1000−1515 b 2.4 × 10 to 3.7 × 10 6 6b 125 530 95 ponds 277c 100 mL 100 241 900 5918 97 tubewells 563 100 mL 1 2419 1 25 flies 193d 1 fly 100 241 900 663 50 child hands 584 2 hands 5 12 095 7 43 stored water 497 100 mL 1 2419 9 77 food 549 1 dry gram 1−8e 2426−20 158e 2 58 a MPN: Most probable number. bCorresponds to lower limit of 1000 MPN and upper limit of 2 419 000 MPN per wet gram given soil moisture content range of 0−34%. cApproximately half of households reported accessing a pond (typically to wash dishes and clothes). dA fly was captured in one-third of households. eCorresponds to lower limit of 1 MPN and upper limit of 2419 MPN per wet gram given food moisture content range of 3−88%. Figure 1. E. coli detection during wet season (Jun−Oct) vs dry season (Nov−May). The y-axis shows the percentage of E. coli positive samples. Geometric mean E. coli counts are displayed beneath the bars. sheep, cows) as well as the presence of any animal in the the presence of animal feces was associated with lower E. coli compound (Figure 2). Compounds with animals had 0.54 log10 levels in tubewells and not associated with E. coli levels on flies. MPN higher E. coli in soil, 0.40 log10 MPN higher E. coli in Because human feces were observed very infrequently (4% of stored water, and 0.61 log10 MPN higher E. coli in food (all p < households), we did not have sufficient statistical power to 0.05). This was primarily driven by the presence of chickens; assess associations with this variable. compounds with chickens had 0.70 log10 MPN higher E. coli in Sanitary Infrastructure vs Fecal Contamination. The soil, 0.49 log10 MPN higher E. coli in stored water, and 0.40 presence of a latrine was associated with significantly lower E. log10 MPN higher E. coli in food (all p < 0.05). Compounds coli in soil and ponds, while the presence of an improved latrine where animals roamed freely had 0.22 log10 MPN higher E. coli was associated with reduced contamination of ponds, and a in soil and 0.27 log10 MPN higher E. coli in ponds (all p < 0.05) higher number of latrines in the compound was associated with than compounds with no animals at all or no free-roaming reduced contamination of soil, child hands and stored drinking animals. Food had 0.32 log10 MPN higher E. coli in compounds water (Figure 2). In contrast, ponds had increased E. coli if where ≥1 fly was captured in the food preparation area (p = there was a latrine within 10 m (Δlog10=0.21, 0.02−0.41) or if 0.02). the latrine was observed to drain into the environment (Δlog10 Similarly, the presence of animal feces in the courtyard was = 0.22, 0.00−0.45) or directly into the pond (Δlog10 = 0.30, significantly associated with increased contamination in the 0.13−0.47). The presence, number, improved vs unimproved domestic environment; especially soil E. coli was independently status, proximity or drainage location of latrines in the associated with the presence of feces from individual animal compound was not associated with tubewell water quality or species as well as the presence of any animal feces in the E. coli on flies. compound (Figure 2). Compounds with animal feces had 0.55 Associations between Pathways. Contamination levels log10 MPN higher soil E. coli; the increase was 0.51 log10 for along different environmental pathways were associated with chicken feces, 0.33 log10 for goat/sheep feces and 0.25 log10 for each other (Figure 3). Pond E. coli increased for each log10 E. cow feces (all p < 0.05). Animal feces were associated with coli increase in soil (Δlog10 = 0.13, 0.03−0.23). E. coli on child higher levels of E. coli in ponds and food as well. Surprisingly, hands increased for each log10 E. coli in soil (Δlog10 = 0.07, 8728 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article Figure 2. Increase in log10 E. coli associated with the presence of animals, animal and human feces, and sanitary infrastructure in compound. 0.01−0.12) and ponds (Δlog10 = 0.15, 0.05−0.25). E. coli in 0.22), ponds (Δlog10 = 0.28, 0.08−0.48), hands (Δlog10 = 0.18, stored water increased for each log10 E. coli in soil (Δlog10 = 0.04−0.32), source water (Δlog10 = 0.21, 0.05−0.37), stored 0.15, 0.06−0.24), ponds (Δlog10 = 0.27, 0.09−0.44), hands water (Δlog10 = 0.28, 0.17−0.39), and flies caught in the food (Δlog10 = 0.21, 0.08−0.33), and source water (Δlog10 = 0.39, preparation area (Δlog10 = 0.21, 0.08−0.34). 0.23−0.55). Finally, E. coli in food increased with each log10 E. Discussion. We found ubiquitous fecal contamination along coli in all other pathways, including soil (Δlog10 = 0.12, 0.02− multiple environmentally mediated pathways in rural Banglade- 8729 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article Bangladeshi households than human feces; 4% of compounds had observed human feces in the courtyard vs almost 90% having animal feces. This is not surprising considering that 97% of enrolled households had a latrine. Open defecation is commonly practiced by young children in this setting;43 nonetheless, our infrequent observation of human feces indicates that child feces are removed from the compound’s living area. Indeed, other work in our study area indicated that, among households where child feces are not disposed of in a latrine, 64% reported disposing of them in the bushes surrounding the compound, 18% in open waste heaps and 13% in drains while only 11% left the feces on the ground.44 However, human feces likely contribute to domestic fecal contamination through other routes, such as latrines draining into ponds/canals or pits leaking into the environment. Indeed, ponds with a latrine within 10 m and ponds receiving latrine effluent had higher E. coli levels in our study, consistent with Figure 3. Associations between environmental transmission pathways, measured as increase in log10 E. coli on a pathway associated with each previous evidence from rural Bangladesh.40,45 log10 increase in E. coli along another pathway. Arrows indicate Chickens presented the most prevalent domestic animal associations that are significant at the p < 0.05 level; the lack of an exposure in our study. Roughly 90% of compounds had arrow between two sample types indicates that we did not observe a chickens, followed by cows (69%) and goats/sheep (39%). significant association. Similarly, 87% of courtyards had chicken feces, followed by cow feces (30%) and goat/sheep feces (19%). Chickens typically shi households with on-site sanitation access. We detected E. roam and deposit feces throughout the compound while coli in 25% of tubewells, compared to 77% of stored drinking scavenging for food,46 and because their feces are small and water, supporting prior evidence on tubewell water quality in relatively odorless, they are likely to be left in place, even Bangladesh29−31 and subsequent contamination at the point-of- though some households collect chicken feces to use as use.32,33 We found E. coli on 43% of child hands. Hand fertilizer.47 Cow dung is often collected and used as cooking contamination levels were similar to findings from Tanzania34 fuel and housing material in rural Bangladesh.48 This could and urban Bangladesh23 but much higher than in high-income explain the relatively low prevalence of cow feces; while 69% of countries.35 E. coli was detected in 58% of complementary food, compounds had cows, only 30% had observed cow feces. consistent with previous studies in Bangladesh.6,36,37 Among Due to the infrequency of observed human feces, we had flies captured at the food preparation area, 50% had E. coli, limited power to detect associations between this exposure and which could have been present on the outside or in the gut of E. coli . Animal feces were associated with increased the fly. Soil and ponds had high levels of E. coli, suggesting contamination of soil, ponds and complementary foods. The these are major reservoirs for fecal organisms. Soil E. coli levels association with food contamination might indicate that, when in our study were substantially higher than previously preparing food, caregivers do not wash hands after handling documented in Tanzania and Zimbabwe.12,38 Our method animal feces. Previous work in Bangladesh found that, during may have had higher recovery efficiency since it did not require food preparation, caregivers feed dung cakes to the fire with a settling step like the protocol used in the Tanzania study. Our bare hands and resume food handling or feed children without results could also indicate heavier fecal input into the washing hands.49 However, while dung cakes are moist when environment due to the high population density of Bangladesh handled to form them for subsequent use, they are sun-dried and/or enhanced bacterial growth in soil due to the wet climate before being used as fuel, and desiccation should substantially and high groundwater table in Bangladesh; saturated subsurface reduce pathogen concentrations.50 Surprisingly, the presence of conditions favor the transport, survival and growth of animal (as well as human) feces was associated with lower microorganisms.39 This is consistent with our finding that soil tubewell contamination. Tubewells in rural Bangladesh are with higher moisture content had higher E. coli counts. typically located on the periphery of the compound rather than However, soil in our study was also more contaminated than in the central courtyard area. Animal and child feces are often measured in a similar rural Bangladeshi setting by enumerating disposed of in bushes or open waste heaps on the compound soil homogenates on Petrifilm without a settling step;13 one periphery as well. Observed feces in the courtyard area could reason for this could be our method’s high upper detection indicate that feces have not been disposed of near the tubewell, limit (2 419 000 MPN per wet gram of soil). We also found where they could more easily infiltrate into the well. E. coli on associations between E. coli levels measured in soil, ponds, flies was not associated with animal feces in the courtyard, groundwater, hands, flies, stored water, and food. Previous potentially indicating that flies can acquire fecal contamination evidence supports these findings; however, few studies have from distal sources beyond a given compound. explored associations between several different pathways. Evidence from microbial source tracking supports the Ambient (pond) water quality has been shown to affect contribution of animal feces to domestic fecal contamination groundwater quality.40 Source water quality, in turn, is a known in our study setting. A subset of 500 stored drinking water, determinant of stored water quality.41 A link between child hand and soil samples from our study were analyzed by contamination of stored water and hands has also been quantitative polymerase chain reaction (qPCR) for human, demonstrated.22,34,42 ruminant and avian molecular fecal markers.20 Over 50% of soil Our findings suggest that animal feces contribute more and hands and 22% of stored water contained ruminant substantially to domestic fecal contamination in rural markers while the avian marker was detected in 33% of soil, 8730 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article 16% of hand rinses and 9% of water samples.20 Ruminant and eggs), corralling poultry inside the home overnight was avian markers were more commonly detected in compounds associated with growth faltering; indoor corralling of other that had ruminants and birds, respectively,20 consistent with domestic animals was not associated with adverse growth our finding of higher E. coli levels in compounds with animals. outcomes.46 Similarly, keeping animals in the room where In contrast, human fecal markers were detected in 9% of soil, children sleep was associated with environmental enteric 2% of hand rinse and none of the water samples.20 Others have dysfunction scores and stunting among rural Bangladeshi reported similar findings. A recent study in India detected children; chickens were the most common animal corralled in animal fecal markers in 75% of ponds, 15% of tubewells, 52% of the sleeping area (61%) followed by cows (39%).57 In Peruvian stored water, and 96% of hands in contrast to human markers shantytowns, children living in households with chickens were detected in 8% of ponds, 2% of tubewells, 20% of stored water at increased risk of Campylobacter infections;58 an intervention and 37% of hands.22 A similar study found evidence of animal to corral chickens in an attempt to reduce children’s exposure contamination in 70% of households in rural India compared to to feces deposited by free-ranging chickens substantially human contamination in 35%, based on testing stored drinking increased rather than decreased the risk of Campylobacter- water and hands.21 Ruminant fecal markers have also been related diarrhea compared to letting the chickens free-range.59 detected on child hands and household floors in urban This could have been due to exposure to concentrated rather Bangladesh.23 than dispersed fecal matter from chickens. In contrast, cow The high prevalence of animals and animal feces in our study exposure was not associated with child diarrhea or growth in and their associations with fecal contamination in the domestic rural India.60 environment suggest that animals can be a source of fecal Limitations. One limitation of our study is that E. coli is an pathogen exposure. A previous study in Bangladesh found 8.5 imperfect proxy for fecal contamination. It has been suggested log10 MPN E. coli and 7.8 log10 MPN Enterococcus per gram of that tropical soils and waters can harbor naturally present E. chicken feces23 and 6.8 log10 MPN E. coli and 3.8 log10 MPN coli;61,62 these are phenotypically identical to E. coli from fecal Enterococcus per gram of cow feces.23 Animal feces also carry sources and can only be distinguished by genotypic pathogens that infect humans, such as pathogenic E. coli, comparison.63,64 While soil collected from compounds with Salmonella and Campylobacter.50 A study in Ecuador found that animals and observed animal feces consistently had higher 76% of chickens were positive for Campylobacter;51 Campylo- levels of E. coli in our study, the level of contamination in bacter can persist in chicken feces for days after deposition.38,52 compounds without animal feces was still high (4.7 log10 Animal feces pose variable levels of human health risk, MPN). This could indicate that soil accumulates fecal indicator depending on the prevalence of human-infective pathogen contamination beyond the immediate contribution of feces strains in the host species.53−55 A study in rural India found observed at the time of sampling; however, it could also point similar odds of diarrhea associated with animal and human fecal to the presence of naturally present E. coli. Soilborne E. coli can markers in the domestic environment.21 Identical strains of persist and multiply outside animal hosts, especially in warm Campylobacter were isolated from the feces of children and and moist tropical conditions; when incubated at 30−37 °C in chickens in Ecuador, suggesting zoonotic transmission.51 the laboratory, naturally present E. coli can grow in soil to Pathogens can be transmitted from animal feces to human concentrations of ∼5 log10 per gram (similar to the soil E. coli hosts through direct and indirect routes. Previous studies have levels in our study).65 However, testing of a subset of our soil observed children ingesting chicken feces.38,52 Structured samples with biochemical assays, phylogrouping and PCR observations of 148 children in our study demonstrated that detection of genes associated with enteric vs environmental roughly 20% of young children touched animal feces but direct origin showed no differences between E. coli isolates in soil vs ingestion was rare (<3%).56 However, up to 35% of children those from fecal samples collected from cattle, chickens, and placed soil in their mouth or put their hands in their mouth humans in the study area.66 Additionally, qPCR testing of our without handwashing after touching soil.56 Compounds with soil samples for microbial source tracking markers revealed high animals had higher levels of soil contamination in our study, as prevalence of ruminant and avian fecal molecular markers, well as higher contamination of stored water and food. Taken providing evidence for contamination of fecal origin from together, these findings suggest that animal feces are a source of animal sources, while human fecal markers were rare.20 This fecal exposure for children in this setting. Environmental evidence suggests that, while E. coli can signal fecal pathways, including highly contaminated soil, potentially contamination, its presence should not be interpreted as mediate transmission by direct and indirect ingestion. evidence of strictly human fecal contamination when animals Our findings are consistent with an emerging body of are present. literature that exposure to domestic animals, especially E. coli is also imperfectly correlated with the presence of fecal chickens, is associated with increased risk of enteric infection pathogens.50 The associations we observed between animal and adverse child growth. A meta-analysis found associations feces and E. coli therefore do not provide standalone evidence between diarrheal infections and domestic animal exposure, for pathogen transmission from animal feces to the domestic with an almost 3-fold increase in Campylobacter infections environment.67 Multiplex PCR testing of a subset of E. coli- associated with poultry exposure.19 The presence of animal positive food and fly samples from our study found pathogenic fecal markers in the household environment was associated E. coli genes in 14% of E. coli-positive food and 2% of E. coli- with an over 4-fold increase in the odds of child diarrhea in positive flies.68 A previous study in rural Bangladesh found that rural India; the magnitude of the effect was similar to that among tubewells with 1−10 MPN/100 mL E. coli (similar to observed for the presence of human markers.21 The presence of our tubewell E. coli levels), pathogenic E. coli was detected by animal feces was associated with lower height-for-age in qPCR in 21%, rotavirus in 57%, Shigella in 7% and Vibrio children in Ethiopia and Bangladesh.24 In rural Ethiopia, cholerae in 7% of wells.69 Another study in a similar Bangladeshi while poultry ownership was associated with improved child setting found that, while 97% of soil samples were positive for growth (presumably by providing nutrition-rich foods such as E. coli, only 14% contained pathogenic E. coli detected by 8731 DOI: 10.1021/acs.est.7b01710 Environ. Sci. Technol. 2017, 51, 8725−8734 Environmental Science & Technology Article multiplex PCR.13 However, despite its limitations as an indicator organism, E. coli is used globally to monitor ■ AUTHOR INFORMATION Corresponding Author microbiological contamination.70−72 A systematic review has *Phone: (510) 225 8828; e-mail: aercumen@berkeley.edu demonstrated that E. coli in drinking water is associated with (A.E.). diarrhea, supporting its use as an indicator for diarrhea-causing ORCID pathogens.73 Ayse Ercumen: 0000-0001-6002-1514 Another limitation is that we collected all environmental Author Contributions samples simultaneously; we therefore cannot ascertain the ○ Co-primary author with equal contribution. causal direction of observed associations. For example, we can only hypothesize that soil contamination preceded the Notes The authors declare no competing financial interest. ■ associated contamination of hands, stored water and food, and not vice versa. However, while we cannot directly compare ACKNOWLEDGMENTS levels of contamination along the different pathways due to different reporting units and detection limits, the environmental This research was financially supported [in part] by Grant media we would expect to be more proximal to fecal sources OPPGD759 from the Bill & Melinda Gates Foundation to the (e.g., soil, ponds, flies) were more heavily contaminated than University of California, Berkeley, and by a grant from the those further down the transmission pathway (e.g., hands, World Bank to the International Centre for Diarrhoeal Disease Research, Bangladesh. ■ stored water, food). Similarly, all molecular fecal markers had higher prevalence in soil than on hands or in stored water,20 REFERENCES although it is difficult to compare PCR results across sample (1) Eisenberg, J. N. S.; Scott, J. C.; Porco, T. Integrating disease types because of differential recovery efficiency. 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