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Green stormwater infrastructure systems, such as biofilters, provide many water quality and other environmental benefits, but their ability to remove human pathogens and antibiotic resistance genes (ARGs) from stormwater runoff is not well documented. In this study, a field scale biofilter in Southern California (USA) was simultaneously evaluated for the breakthrough of a conservative tracer (bromide), conventional fecal indicators, bacterial and viral human-associated fecal source markers (HF183, crAssphage, and PMMoV), ARGs, and bacterial and viral pathogens. When challenged with a 50:50 mixture of untreated sewage and stormwater (to mimic highly contaminated storm flow) the biofilter significantly removed (p < 0.05) 14 of 17 microbial markers and ARGsin descending order of concentration reduction: ermB (2.5 log(base 10) reduction) > Salmonella (2.3) > adenovirus (1.9) > coliphage (1.5) > crAssphage (1.2) > E. coli (1.0) ∼ 16S rRNA genes (1.0) ∼ fecal coliform (1.0) ∼ intl1 (1.0) > Enterococcus (0.9) ∼ MRSA (0.9) ∼ sul1 (0.9) > PMMoV (0.7) > Entero1A (0.5). No significant removal was observed for GenBac3, Campylobacter, and HF183. From the bromide data, we infer that 0.5 log-units of attenuation can be attributed to the dilution of incoming stormwater with water stored in the biofilter; removal above this threshold is presumably associated with non-conservative processes, such as physicochemical filtration, die-off, and predation. Our study documents high variability (>100-fold) in the removal of different microbial contaminants and ARGs by a field-scale stormwater biofilter operated under transient flow and raises further questions about the utility of human-associated fecal source markers as surrogates for pathogen removal., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In sub-Saharan Africa, many families travel to collect water and store it in their homes for daily use, presenting an opportunity for the introduction of fecal contamination. One stored and one source water sample were each collected from 45 households in rural Kenya. All 90 samples were analyzed for fecal indicator bacteria ( E. coli and enterococci) and species-specific contamination using molecular microbial source tracking assays. Human (HF183), avian (GFD), and ruminant (BacR) contamination were detected in 52, two, and four samples, respectively. Stored water samples had elevated enterococci concentrations ( p < 0.01, Wilcoxon matched pairs test) and more frequent BacR detection (89% versus 27%, p < 0.01, McNemar's exact test) relative to source water samples. fsQCA (fuzzy set qualitative comparative analysis) was conducted on the subset of households with no source water BacR contamination to highlight combinations of factors associated with the introduction of BacR contamination to stored water supplies. Three combinations were identified: (i) ruminants in the compound, safe water extraction methods, and long storage time, (ii) ruminants, unsafe water extraction methods, and no soap at the household handwashing station, and (iii) long storage time and no soap. This suggests that multiple pathways contribute to the transmission of ruminant fecal contamination in this context, which would have been missed if data were analyzed using standard regression techniques.

Stormwater biofilters are used to attenuate the flow and volume of runoff and reduce pollutant loading to aquatic systems. However, the capacity of biofilters to remove microbial contaminants remains inadequate. While biochar has demonstrated promise as an amendment to improve microbial removal in laboratory-scale biofilters, it is uncertain if the results are generalizable to the field. To assess biochar performance in a simulated field setting, sand and biochar-amended sand biofilters were periodically dosed with natural stormwater over a 61-week conditioning phase. Impact of media saturation was assessed by maintaining biofilters with and without a saturated zone. Biochar-amended biofilters demonstrated improved Escherichia coli removal over sand biofilters during the first 31 weeks of conditioning though media type did not impact E. coli removal during the last 30 weeks of conditioning. Presence of a saturated zone was not a significant factor influencing E. coli removal across the entire conditioning phase. Following conditioning, biofilters underwent challenge tests using stormwater spiked with wastewater to assess their capacity to remove wastewater-derived E. coli, enterococci, and male-specific (F+) coliphage. In challenge tests, biochar-amended biofilters demonstrated enhanced removal of all fecal indicators relative to sand biofilters. Additionally, saturated biofilters demonstrated greater removal of fecal indicators than unsaturated biofilters for both media types. Discrepant conclusions from the conditioning phase and challenge tests may be due to variable influent chemistry, dissimilar transport of E. coli indigenous to stormwater and those indigenous to wastewater, and differences in E. coli removal mechanisms between tests. Mobilization tests conducted following challenge tests showed minimal (<2.5%) observable mobilization of fecal indicators, regardless of media type and presence of a saturated zone. While our results emphasize the challenge of translating biochar's performance from the laboratory to the field, findings of this study inform biofilter design to remove microbial contaminants from urban stormwater., Competing Interests: The authors have declared that no competing interests exist.

Fecal indicator organisms are measured to indicate the presence of fecal pollution, yet the association between indicators and pathogens varies by context. The goal of this study was to empirically evaluate the relationships between indicator Escherichia coli , microbial source tracking markers, select enteric pathogen genes, and potential sources of enteric pathogens in 600 rural Bangladeshi households. We measured indicators and pathogen genes in stored drinking water, soil, and on mother and child hands. Additionally, survey and observational data on sanitation and domestic hygiene practices were collected. Log 10 concentrations of indicator E. coli were positively associated with the prevalence of pathogenic E. coli genes in all sample types. Given the current need to rely on indicators to assess fecal contamination in the field, it is significant that in this study context indicator E. coli concentrations, measured by IDEXX Colilert-18, provided quantitative information on the presence of pathogenic E. coli in different sample types. There were no significant associations between the human fecal marker (HumM2) and human-specific pathogens in any environmental sample type. There was an increase in the prevalence of Giardia lamblia genes, any E. coli virulence gene, and the specific E. coli virulence genes stx1/2 with every log 10 increase in the concentration of the animal fecal marker (BacCow) on mothers' hands. Thus, domestic animals were important contributors to enteric pathogens in these households.

Exposure to fecal contamination continues to be a major public health concern for low-income households in sub-Saharan Africa. Drinking water and hands are known transmission routes for pathogens in household environments. In an effort to identify explanatory variables of water and hand contamination, a variety of analytical approaches have been employed that model variation in E. coli contamination as a function of behaviors and household characteristics. Using data collected from 1217 households in Bagamoyo, Tanzania, this investigation compares the explanatory variables identified in the three different modeling methods to explain hand and water contamination: ordinary least squares regression, logistic regression, and classification tree. Although the modeling approaches varied, there were some similarities in the results, with certain explanatory variables being consistently identified as being related to hand and water contamination (e.g., water source type for the water models and activity prior to sampling for the hand models). At the same time, there were also marked differences across the models. In sum, these results suggest there are benefits to using multiple analysis methods to assess relationships in complex systems. The models were also characterized by low explanatory power, suggesting that variation in hand and water contamination is difficult to capture when analyzing one-time water and hand rinse samples. For improved model performance, future studies could explore modeling of repeat measures of water quality and hand contamination.

The decay of sewage-sourced enterococci, Escherichia coli, three human-associated microbial source tracking (MST) markers, Salmonella, Campylobacter, and norovirus GII was measured in situ in coastal, marine waters. Experiments examined the effects of sunlight intensity and season on decay. Seawater was seeded with untreated sewage, placed into permeable dialysis bags, and deployed in the coastal ocean near the water surface, and at 18 cm, and 99 cm depths, to vary solar intensity, during winter and summer seasons. Microbial decay was modeled using a log-linear or shoulder log-linear decay model. Pathogen levels were too low in sewage to obtain kinetic parameters. Human-associated MST markers all decayed with approximately the same rate constant (k ∼ 1.5 d -1 ) in all experimental treatments, suggesting markers could be detectable up to ∼6 days after a raw sewage spill. E. coli and enterococci (culturable and molecular marker) k significantly varied with season and depth; enterococci decayed faster at shallow depths and during the summer, while E. coli decayed faster at shallow depths and during the winter. Rate constants for MST markers and culturable FIB diverged except at the deepest depth in the water column potentially complicating the use of MST marker concentrations to allocate sources of FIB contamination., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The presence of microbial contaminants in urban stormwater is a significant concern for public health; however, their removal by traditional stormwater biofilters has been reported as inconsistent and inadequate. Recent work has explored the use of biochar to improve performance of stormwater biofilters under simplified conditions that do not consider potential effects of biofilm development on filter media. The present study investigates the role of biofilm on microbial contaminant removal performance of stormwater biofilters. Pseudomonas aeruginosa biofilms were formed in laboratory-scale sand and biochar-modified sand packed columns, which were then challenged with Escherichia coli laden synthetic stormwater containing natural organic matter. Results suggests that the presence of biofilm influences the removal of E. coli. However, the nature of the influence depends on the specific surface area and the relative hydrophobicity of filter media. The distribution of attached bacteria within the columns indicates that removal by filter media varies along the length of the column: the inlet was the primary removal zone regardless of experimental conditions. Findings from this research inform the design of field-scale biofilters for better and consistent performance in removing microbial contaminants from urban stormwater., Competing Interests: The authors have declared that no competing interests exist.

Fecal indicator bacteria (FIB) are leading causes of impaired surface waters. Innovative and environmentally appropriate best management practices are needed to reduce FIB concentrations and associated risk. This study examines the ability of the native freshwater mussel Anodonta californiensis and an invasive freshwater clam Corbicula fluminea to reduce concentrations of the FIB Escherichia coli in natural waters. Laboratory batch experiments were used to show bivalve species-specific E. coli removal capabilities and to develop a relationship between bivalve size and clearance rates. A field survey within an impaired coastal river containing both species of bivalves in an agricultural- and grazing-dominated area of the central coast of California showed a significant inverse correlation between E. coli concentration and bivalve density. An in situ field spiking and sampling study showed filtration by freshwater bivalves resulting in 1-1.5 log 10 reduction of E. coli over 24 h, and calculated clearance rates ranged from 1.2 to 7.4 L hr -1 bivalve -1 . Results of this study show the importance of freshwater bivalves for improving water quality through the removal of E. coli. While both native and invasive bivalves can reduce E. coli levels, the use of native bivalves through integration into best management practices is recommended as a way to improve water quality and protect and encourage re-establishment of native bivalve species that are in decline.

The decay of sewage-sourced Escherichia coli and enterococci was measured at multiple depths in a freshwater marsh, a brackish water lagoon, and a marine site, all located in California. The marine site had very clear water, while the waters from the marsh and lagoon contained colored dissolved organic matter that not only blocked light but also produced reactive oxygen species. First order decay rate constants of both enterococci and E. coli were between 1 and 2 d(-1) under low light conditions and as high as 6 d(-1) under high light conditions. First order decay rate constants were well correlated to the daily average UVB light intensity corrected for light screening incorporating water absorbance and depth, suggesting endogenous photoinactivation is a major pathway for bacterial decay. Additional laboratory experiments demonstrated the presence of colored dissolved organic matter in marsh water enhanced photoinactivation of a laboratory strain of Enterococcus faecalis, but depressed photoinactivation of sewage-sourced enterococci and E. coli after correcting for UVB light screening, suggesting that although the exogenous indirect photoinactivation mechanism may be active against Ent. faecalis, it is not for the sewage-source organisms. A simple linear regression model based on UVB light intensity appears to be a useful tool for predicting inactivation rate constants in natural waters of any depth and absorbance.

In Dhaka, Bangladesh, the sensitivity and specificity of three human, three ruminant, and one avian source-associated QPCR microbial source tracking assays were evaluated using fecal samples collected on site. Ruminant-associated assays performed well, whereas the avian and human assays exhibited unacceptable cross-reactions with feces from other hosts. Subsequently, child hand rinses (n = 44) and floor sponge samples (n = 44) from low-income-households in Dhaka were assayed for fecal indicator bacteria (enterococci, Bacteroidales, and Escherichia coli) and a ruminant-associated bacterial target (BacR). Mean enterococci concentrations were of 100 most probable number (MPN)/2 hands and 1000 MPN/225 cm(2) floor. Mean concentrations of Bacteroidales were 10(6) copies/2 hands and 10(5) copies/225 cm(2) floor. E. coli were detected in a quarter of hand rinse and floor samples. BacR was detected in 18% of hand rinse and 27% of floor samples. Results suggest that effective household fecal management should account not only for human sources of contamination but also for animal sources. The poor performance of the human-associated assays in the study area calls into the question the feasibility of developing a human-associated marker in urban slum environments, where domestic animals are exposed to human feces that have been disposed in pits and open drains.

To improve bacterial removal, a traditional stormwater biofilter can be augmented with biochar, but it is unknown whether bacterial removal remains consistent as the biochar weathers during intermittent exposure to stormwater under dry-wet and freeze-thaw cycles. To examine the effect of weathering on bacterial removal capacity of biochar, we subjected biochar-augmented sand filters (or simplified biofilters) to multiple freeze-thaw or dry-wet cycles for a month and then compared their bacterial removal capacity with the removal capacity of unweathered biofilters. To isolate the effect of physical and chemical weathering processes from that of biological processes, the biofilters were operated without any developed biofilm. Biochar particles were mobilized during intermittent infiltration of stormwater, but the mobilization depended on temperature and antecedent conditions. During stormwater infiltration without intermediate drying, exposure to natural organic matter (NOM) in the stormwater decreased the bacterial removal capacity of biochar, partly due to exhaustion of attachment sites by NOM adsorption. In contrast, exposure to the same amount of NOM during stormwater infiltration with intermediate drying resulted in higher bacterial removal. This result suggests that dry-wet cycles may enhance recovery of the previously exhausted attachment sites, possibly due to diffusion of NOM from biochar surfaces into intraparticle pores during intermediate drying periods. Overall, these results indicate that physical weathering has net positive effect on bacterial removal by biochar-augmented biofilters., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

High levels of fecal indicator bacteria, such as Escherichia coli, can be indicative of poor water quality. The use of shellfish to reduce eutrophication has been proposed, but application of bivalves to reduce bacterial levels has not been extensively reported. Removal of E. coli by the native freshwater mussel Anodonta californiensis was studied using laboratory batch systems and field-based flow-through systems. Batch systems were utilized to determine the fate and inactivation of E. coli after uptake by the mussel. Batch experiments demonstrated that uptake patterns followed first order kinetics and E. coli was inactivated with less than 5% of the initial colonies recoverable in fecal matter or tissue. Flow-through systems located at an urban impaired lake in San Francisco, CA were utilized to determine uptake kinetics under environmentally relevant conditions. The bivalves maintained a 1-log removal of E. coli for the duration of exposure. The calculated uptake rates can be used in conjunction with hydrologic models to determine the number of bivalves needed to maintain removal of E. coli in different freshwater systems. The outcomes of this study support the use of native freshwater bivalves to achieve the co-benefits of rehabilitating a freshwater ecosystem and improving water quality via reduction of E. coli in contaminated freshwater systems.

Bioretention systems and biofilters are used in low impact development to passively treat urban stormwater. However, these engineered natural systems are not efficient at removing fecal indicator bacteria, the contaminants responsible for a majority of surface water impairments. The present study investigates the efficacy of biochar-augmented model sand biofilters for Escherichia coli removal under a variety of stormwater bacterial concentrations and infiltration rates. Additionally, we test the role of biochar particle size and "presence of compost on model" biofilter performance. Our results show that E. coli removal in a biochar-augmented sand biofilter is ∼ 96% and is not greatly affected by increases in stormwater infiltration rates and influent bacterial concentrations, particularly within the ranges expected in field. Removal of fine (<125 μm) biochar particles from the biochar-sand biofilter decreased the removal capacity from 95% to 62%, indicating biochar size is important. Addition of compost to biochar-sand biofilters not only lowered E. coli removal capacity but also increased the mobilization of deposited bacteria during intermittent infiltration. This result is attributed to exhaustion of attachment sites on biochar by the dissolved organic carbon leached from compost. Overall, our study indicates that biochar has potential to remove bacteria from stormwater under a wide range of field conditions, but for biochar to be effective, the size should be small and biochar should be applied without compost. Although the results aid in the optimization of biofilter design, further studies are needed to examine biochar potential in the field over an entire rainy season.

Biofilters, designed to facilitate the infiltration of stormwater into soil, are generally ineffective in removing bacteria from stormwater, thereby causing pollution of groundwater and receiving surface waters. The bacterial removal capacity of biofilters has been shown to be lower in the presence of natural organic matter (NOM) and during intermittent infiltration of stormwater. To improve the removal of fecal indicator bacteria (Escherichia coli) under these conditions, we amended sand with 5% (by weight) biochar, a carbonaceous geomedia produced by pyrolysis of biomass, and investigated the removal and remobilization of E. coli. Three types of biochar were used to evaluate the role of biochar properties on the removal. Compared to sand, biochar not only retained up to 3 orders of magnitude more E. coli, but also prevented their mobilization during successive intermittent flows. In the presence of NOM, the removal capacity of biochar was lower, but remained higher than sand alone. The improved retention with the biochar amendment is attributed to an increase in the attachment of E. coli at the primary minimum and to an increase in the water-holding capacity of biochar-amended sand, which renders driving forces such as moving air-water interfaces less effective in detaching bacteria from grain surfaces. Biochars with lower volatile matter and polarity appear to be more effective in removing bacteria from stormwater. Overall, our results suggest that a biochar amendment to biofilter media has the potential to effectively remove bacteria from stormwater., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bioinfiltration systems facilitate the infiltration of urban stormwater into soil and reduce high flow events and flooding. Stormwater carries a myriad of pollutants including fecal indicator bacteria (FIB). Significant knowledge gaps exist about the ability of bioinfiltration systems to remove and retain FIB. The present study investigates the ability of model, simplified bioinfiltration systems containing quartz sand and iron oxide-coated quartz sand (IOCS) to remove two FIB (Enterococcus faecalis and Escherichia coli) suspended in synthetic stormwater with and without natural organic matter (NOM) as well as the potential for accumulated FIB to be remobilized during intermittent flow. The experiments were conducted in two phases: (1) the saturated columns packed with either sand or IOCS were contaminated by injecting stormwater with bacteria followed by injection of sterile stormwater and (2) the contaminated columns were subjected to intermittent infiltration of sterile stormwater preceded by a pause during which columns were either kept saturated or drained by gravity. During intermittent flow, fewer bacteria were released from the saturated column compared to the column drained by gravity: 12% of attached E. coli and 3% of attached Ent. faecalis were mobilized from the drained sand column compared to 3% of attached E. coli and 2% attached Ent. faecalis mobilized from the saturated sand column. Dry and wet cycles introduce moving air-water interfaces that can scour bacteria from grain surfaces. During intermittent flows, less than 0.2% of attached bacteria were mobilized from IOCS, which bound both bacteria irreversibly in the absence of NOM. Addition of NOM, however, increased bacterial mobilization from IOCS: 50% of attached E. coli and 8% of attached Ent. faecalis were released from IOCS columns during draining and rewetting. Results indicate that using geomedia such as IOCS that promote irreversible attachment of bacteria, and maintaining saturated condition, could minimize the mobilization of previous attached bacteria from bioinfiltration systems, although NOM may significantly decrease these benefits.

Consistently high levels of bacterial indicators of fecal pollution rank Cowell Beach as the most polluted beach in California. High levels of fecal indicator bacteria (FIB), E. coli and enterococci, are measured throughout the summer, resulting in beach advisories with social and economic consequences. The source of FIB, however, is unknown. Speculations have been made that the wrack accumulating on the beach is a major source of FIB to the surf zone. The present study uses spatial and temporal sampling coupled with process-modeling to investigate potential FIB sources and the relative contributions of those sources. Temporal sampling showed consistently high FIB concentrations in the surf zone, sand, and wrack at Cowell Beach, and ruled out the storm drain, the river, the harbor, and the adjacent wharf as the sources of the high concentrations observed in the surf zone. Spatial sampling confirmed that the source of FIB to the beach is terrestrial rather than marine. Modeling results showed two dominant FIB sources to the surf zone: sand for enterococci and groundwater for E. coli. FIB from wrack represented a minor contribution to bacterial levels in the water. Molecular source tracking methods indicate the FIB at the beach is of human and bird origin. The microbial source tracking (MST) approach presented here provides a framework for future efforts.

Algae on freshwater beaches can serve as reservoirs for fecal indicator bacteria (FIB). Wrack (especially kelp) at marine beaches might sustain FIB as well. This study examines the relationship between beach wrack, FIB, and surrounding water and sediment at marine beaches along the California coast. Surveys of southern and central California beaches were conducted to observe environmental wrack-associated FIB concentrations. FIB concentrations normalized to dry weight were the highest in stranded dry wrack, followed by stranded wet and suspended 'surf' wrack. Laboratory microcosms were conducted to examine the effect of wrack on FIB persistence in seawater and sediment. Indigenous enterococci and Escherichia coli incubated in a seawater microcosm containing wrack showed increased persistence relative to those incubated in a microcosm without wrack. FIB concentrations in microcosms containing wrack-covered sand were significantly higher than those in uncovered sand after several days. These findings implicate beach wrack as an important FIB reservoir. The presence of wrack may increase water and sediment FIB levels, altering the relationship between FIB levels and actual health risk while possibly leading to beach closures. Further work will need to investigate the possibility of FIB growth on wrack and the potential for pathogen presence., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Low- and middle-income countries (LMICs) bear the largest mortality burden of antibiotic-resistant infections. Small-scale animal production and free-roaming domestic animals are common in many LMICs, yet data on zoonotic exchange of gut bacteria and antibiotic resistance genes (ARGs) in low-income communities are sparse. Differences between rural and urban communities with regard to population density, antibiotic use, and cohabitation with animals likely influence the frequency of transmission of gut bacterial communities and ARGs between humans and animals. Here, we determined the similarity in gut microbiomes, using 16S rRNA gene amplicon sequencing, and resistomes, using long-read metagenomics, between humans, chickens, and goats in a rural community compared to an urban community in Bangladesh. Gut microbiomes were more similar between humans and chickens in the rural (where cohabitation is more common) than the urban community, but there was no difference for humans and goats in the rural versus the urban community. Human and goat resistomes were more similar in the urban community, and ARG abundance was higher in urban animals than rural animals. We identified substantial overlap of ARG alleles in humans and animals in both settings. Humans and chickens had more overlapping ARG alleles than humans and goats. All fecal hosts from the urban community and rural humans carried ARGs on chromosomal contigs classified as potentially pathogenic bacteria, including Escherichia coli, Campylobacter jejuni, Clostridioides difficile, and Klebsiella pneumoniae. These findings provide insight into the breadth of ARGs circulating within human and animal populations in a rural compared to urban community in Bangladesh. IMPORTANCE While the development of antibiotic resistance in animal gut microbiomes and subsequent transmission to humans has been demonstrated in intensive farming environments and high-income countries, evidence of zoonotic exchange of antibiotic resistance in LMIC communities is lacking. This research provides genomic evidence of overlap of antibiotic resistance genes between humans and animals, especially in urban communities, and highlights chickens as important reservoirs of antibiotic resistance. Chicken and human gut microbiomes were more similar in rural Bangladesh, where cohabitation is more common. Incorporation of long-read metagenomics enabled characterization of bacterial hosts of resistance genes, which has not been possible in previous culture-independent studies using only short-read sequencing. These findings highlight the importance of developing strategies for combatting antibiotic resistance that account for chickens being reservoirs of ARGs in community environments, especially in urban areas.

Fresh produce items can become contaminated with enteric pathogens along the supply chain at the preharvest (e.g., irrigation water, soil, fertilizer) or postharvest (e.g., vendor handling or consumer handling) stages. This study assesses the concentrations of fecal indicator bacteria Escherichia coli, enterococci (ENT), and Bacteriodales on surfaces of carrots, eggplants, red amaranth leaves, and tomatoes obtained from both a wholesale market (recently harvested) and neighborhood retail markets in Dhaka, Bangladesh. We detected E. coli in 100% of carrot and red amaranth rinses, 92% of eggplant rinses, and 46% of tomato rinses. Using a molecular microbial source tracking assay, we found that 32% of produce samples were positive for ruminant fecal contamination. Fecal indicator bacteria were more likely to be detected on produce collected in retail markets compared with that in the wholesale market; retail market produce were 1.25 times more likely to have E. coli detected (P = 0.03) and 1.24 times more likely to have ENT detected (P = 0.03) as compared with wholesale market produce. Bacteriodales was detected in higher concentrations in retail market produce samples compared with wholesale market produce samples (0.40 log10 gene copies per 100 cm2 higher, P = 0.03). Our results suggest that ruminant and general fecal contamination of produce in markets in Dhaka is common, and suggest that unsanitary conditions in markets are an important source of produce fecal contamination postharvest.

BackgroundDiarrhea is one of the leading causes of mortality in young children. Diarrheal pathogens are transmitted via the fecal-oral route, and for children the majority of this transmission is thought to occur within the home. However, very few studies have documented enteric pathogens within households of low-income countries.Methods and findingsThe presence of molecular markers for three enteric viruses (enterovirus, adenovirus, and rotavirus), seven Escherichia coli virulence genes (ECVG), and human-specific Bacteroidales was assessed in hand rinses and household stored drinking water in Bagamoyo, Tanzania. Using a matched case-control study design, we examined the relationship between contamination of hands and water with these markers and child diarrhea. We found that the presence of ECVG in household stored water was associated with a significant decrease in the odds of a child within the home having diarrhea (OR = 0.51; 95% confidence interval 0.27-0.93). We also evaluated water management and hygiene behaviors. Recent hand contact with water or food was positively associated with detection of enteric pathogen markers on hands, as was relatively lower volumes of water reportedly used for daily hand washing. Enteropathogen markers in stored drinking water were more likely found among households in which the markers were also detected on hands, as well as in households with unimproved water supply and sanitation infrastructure.ConclusionsThe prevalence of enteric pathogen genes and the human-specific Bacteroidales fecal marker in stored water and on hands suggests extensive environmental contamination within homes both with and without reported child diarrhea. Better stored water quality among households with diarrhea indicates caregivers with sick children may be more likely to ensure safe drinking water in the home. Interventions to increase the quantity of water available for hand washing, and to improve food hygiene, may reduce exposure to enteric pathogens in the domestic environment.

Handwashing education and promotion are well established as effective strategies to reduce diarrhea and respiratory illness in countries around the world. However, access to reliable water supplies has been identified as an important barrier to regular handwashing in low-income countries. Alcohol-based hand sanitizer (ABHS) is an effective hand hygiene method that does not require water, but its use is not currently recommended when hands are visibly soiled. This study evaluated the efficacy of ABHS on volunteers' hands artificially contaminated with Escherichia coil in the presence of dirt (soil from Tanzania) and cooking oil. ABHS reduced levels of E. coil by a mean of 2.33 log colony forming units (CFU) per clean hand, 2.32 log CFU per dirt-covered hand, and 2.13 log CFU per oil-coated hand NO significant difference in efficacy was detected between hands that were clean versus dirty or oily ABHS may be an appropriate hand hygiene method for hands that are moderately soiled, and an attractive option for field settings in which access to water and soap is limited. [ABSTRACT FROM AUTHOR]

To characterize mechanisms of hand contamination with faecal indicator bacteria and to assess the presence of selected pathogens on mothers' hands in Tanzania. A household observational study combined with repeated microbiological hand rinse sampling was conducted among 119 mothers in Dar es Salaam, Tanzania. All hand rinse samples were analysed for enterococci and Escherichia coli, and selected samples were analysed for genetic markers of Bacteroidales, enterovirus and pathogenic E. coli. Using the toilet, cleaning up a child's faeces, sweeping, cleaning dishes, preparing food and bathing were all found to increase faecal indicator bacterial levels on hands. Geometric mean increases in colony forming units per two hands ranged from 50 (cleaning dishes) to 6310 (food preparation). Multivariate modelling of hand faecal indicator bacteria as a function of activities recently performed shows that food handling, exiting the household premises and longer time since last handwashing with soap are positively associated with bacterial levels on hands, while bathing is negatively associated. Genetic markers of Bacteroidales, enterovirus and pathogenic E. coli were each detected on a subset of mothers' hands. Escherichia coli and enterococci on hands can be significantly increased by various household activities, including those involving the use of soap and water. Thus, faecal indicator bacteria should be considered highly variable when used as indicators of handwashing behaviour. This work corroborates hands as important vectors of disease among Tanzanian mothers and highlights the difficulty of good personal hygiene in an environment characterized by the lack of networked sanitation and water supply services. [ABSTRACT FROM AUTHOR]

In Papua New Guinea, a significant portion of morbidity and mortality is attributed to water-borne diseases. To reduce incidence of disease, communities and non-governmental organizations have installed rain catchments to provide drinking water of improved quality. However, little work has been done to determine whether these rain catchments provide drinking water of better quality than traditional drinking water sources, and if morbidity is decreased in villages with rain catchments. The specific aim of this study was to evaluate the quality of water produced by rain catchments in comparison with traditional drinking water sources in rural villages in the East Sepik Province. Fifty-four water sources in 22 villages were evaluated for enterococci and Escherichia coli densities as well as 14 health-relevant metals. In addition, we examined how the prevalence of diarrhoeal illness in villages relates to the type of primary drinking water source. The majority of tested metals were below World Health Organization safety limits. Catchment water sources had lower enterococci and E. coli than other water sources. Individuals in villages using Sepik River water as their primary water source had significantly higher incidence of diarrhoea than those primarily using other water sources (streams, dug wells and catchments). [ABSTRACT FROM AUTHOR]

Sunlight influences microbial water quality of surface waters. Previous studies have investigated photoinactivation mechanisms and cellular photostress responses of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, but further work is needed to characterize photostress responses of bacterial pathogens. Here we investigate the photoinactivation of Staphylococcus aureus (strain Newman), a pigmented, waterborne pathogen of emerging concern. We measured photodecay using standard culture-based assays and cellular membrane integrity and investigated photostress response by measuring the relative number of mRNA transcripts of select oxidative stress, DNA repair, and metabolism genes. Photoinactivation experiments were performed in both oxic and anoxic systems to further investigate the role of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. S. aureus lost culturability much faster in oxic systems than in anoxic systems, indicating an important role for oxygen in photodecay mechanisms. S. aureus cell membranes were damaged by sunlight exposure in anoxic systems but not in oxic systems, as measured by cell membrane permeability to propidium iodide. After sunlight exposure, S. aureus increased expression of a gene coding for methionine sulfoxide reductase after 12 h of sunlight exposure in the oxic system and after 6 h of sunlight exposure in the anoxic system, suggesting that methionine sulfoxide reductase is an important enzyme for defense against both oxygen-dependent and oxygen-independent photostresses. This research highlights the importance of oxygen in bacterial photoinactivation in environmentally relevant systems and the complexity of the bacterial photostress response with respect to cell structure and transcriptional regulation. IMPORTANCE Staphylococcus aureus is a pathogenic bacterium that causes gastrointestinal, respiratory, and skin infections. In severe cases, S. aureus infection can lead to life-threatening diseases, including pneumonia and sepsis. Cases of communityacquired S. aureus infection have been increasing in recent years, pointing to the importance of considering S. aureus transmission pathways outside the hospital environment. Associations have been observed between recreational water contact and staphylococcal skin infections, suggesting that recreational waters may be an important environmental transmission pathway for S. aureus. However, prediction of human health risk in recreational waters is hindered by incomplete knowledge of pathogen sources, fate, and transport in this environment. This study is an in-depth investigation of the inactivation of a representative strain of S. aureus by sunlight exposure, one of the most important factors controlling the fate of microbial contaminants in clear waters, which will improve our ability to predict water quality changes and human health risk in recreational waters. [ABSTRACT FROM AUTHOR]

A pilot-scale open-water unit process wetland was monitored for one year and found to be effective in enhancing sunlight inactivation of fecal indicator bacteria (FIB). The removal of Escherichia coli and enterococci in the open-water wetland receiving non-disinfected secondary municipal wastewater reached 3 logs and 2 logs in summer time, respectively. Pigmented enterococci were shown to be significantly more resistant to sunlight inactivation than non-pigmented enterococci. A model was developed to predict the inactivation of E. coli , and pigmented and non-pigmented enterococci that accounts for endogenous and exogenous sunlight inactivation mechanisms and dark processes. Endogenous inactivation rates were modeled using the sum of UVA and UVB irradiance. Exogenous inactivation was only significant for enterococci, and was modeled as a function of steady-state singlet oxygen concentration. The rate constants were determined from lab experiments and an empirical correction factor was used to account for differences between lab and field conditions. The model was used to predict removal rate constants for FIB in the pilot-scale wetland; considering the variability of the monitoring data, there was general agreement between the modeled values and those determined from measurements. Using the model, we estimate that open-water wetlands at 40° latitude with practical sizes can achieve 3-log (99.9%) removal of E. coli and non-pigmented enterococci throughout the year [5.5 ha and 7.0 ha per million gallons of wastewater effluent per day (MGD), respectively]. Differences in sunlight inactivation rates observed between pigmented and non-pigmented enterococci, as well as between lab-cultured and indigenous wastewater bacteria highlight the challenges of using FIB as model organisms for actual pathogens in natural sunlit environments. [ABSTRACT FROM AUTHOR]

Abstract: Fecal pollution enters coastal waters through multiple routes, many of which originate from land-based activities. Runoff from pervious and impervious land surfaces transports pollutants from land to sea and can cause impairment of coastal ocean waters. To understand how land use practices and water characteristics influence concentrations of fecal indicator bacteria (FIB) and pathogens in natural waters, fourteen coastal streams, rivers, and tidal lagoons, surrounded by variable land use and animal densities, were sampled every six weeks over two years (2008 & 2009). Fecal indicator bacteria (FIB; Escherichia coli and Enterococci) and Salmonella concentrations, the occurrence of Bacteroidales human, ruminant, and pig-specific fecal markers, E. coli O157:H7, and Shiga toxin (stx) genes present in E. coli, were measured. In addition, environmental and climatic variables (e.g., temperature, salinity, rainfall), as well as human and livestock population densities and land cover were quantified. Concentrations of FIB and Salmonella were correlated with each other, but the occurrence of host-specific Bacteroidales markers did not correlate with FIB or pathogens. FIB and Salmonella concentrations, as well as the occurrence of E. coli harboring stx genes, were positively associated with the fraction of the surrounding subwatershed that was urban, while the occurrence of E. coli O157:H7 was positively associated with the agricultural fraction. FIB and Salmonella concentrations were negatively correlated to salinity and temperature, and positively correlated to rainfall. Areal loading rates of FIB, Salmonella and E. coli O157:H7 to the coastal ocean were calculated for stream and river sites and varied with land cover, salinity, temperature, and rainfall. Results suggest that FIB and pathogen concentrations are influenced, in part, by their flux from the land, which is exacerbated during rainfall; once waterborne, bacterial persistence is affected by water temperature and salinity. [Copyright &y& Elsevier]

Abstract: The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers. [Copyright &y& Elsevier]

Diarrheal disease is a leading cause of under-five childhood mortality worldwide, with at least half of these deaths occurring in sub-Saharan Africa. Transmission of diarrheal pathogens occurs through several exposure routes including drinking water and hands, but the relative importance of each route is not well understood. Using molecular methods, this study examines the relative importance of different exposure routes by measuring enteric bacteria (pathogenic Escherichia coli) and viruses (rotavirus, enterovirus, adenovirus) in hand rinses, stored water, and source waters in Bagamoyo, Tanzania. Viruses were most frequently found on hands, suggesting that hands are important vectors for viral illness. The occurrence of E. coli virulence genes (ECVG) was equivalent across all sample types, indicating that both water and hands are important for bacterial pathogen transmission. Fecal indicator bacteria and turbidity were good predictors of ECVG, whereas turbidity and human-specific Bacteroidales were good predictors of viruses. ECVG were more likely found in unimproved water sources, but both ECVG and viral genes were detected in improved water sources. ECVG were more likely found in stored water of households with unimproved sanitation facilities. The results provide insights into the distribution of pathogens in Tanzanian households and offer evidence that hand-washing and improved water management practices could alleviate viral and bacterial diarrhea. [ABSTRACT FROM AUTHOR]

Little is known about the extent or pattern of environmental fecal contamination among households using low-cost, on-site sanitation facilities, or what role environmental contamination plays in the transmission of diarrheal disease. A microbial survey of fecal contamination and selected diarrheal pathogens in soil (n = 200), surface (n = 120), and produce samples (n = 24) was conducted in peri-urban Bagamoyo, Tanzania, among 20 households using private pit latrines. All samples were analyzed for E. coli and enterococci. A subset was analyzed for enterovirus, rotavirus, norovirus GI, norovirus GII, diarrheagenic E. coli, and general and human-specific Bacteroidales fecal markers using molecular methods. Soil collected from the house floor had significantly higher concentrations of E. coli and enterococci than soil collected from the latrine floor. There was no significant difference in fecal indicator bacteria levels between households using pit latrines with a concrete slab (improved sanitation) versus those without a slab. These findings imply that the presence of a concrete slab does not affect the level of fecal contamination in the household environment in this setting. Human Bacteroidales, pathogenic E. coli, enterovirus, and rotavirus genes were detected in soil samples, suggesting that soil should be given more attention as a transmission pathway of diarrheal illness in low-income countries. [ABSTRACT FROM AUTHOR]

Almost half of the world's population relies on non-networked water supply services, which necessitates in-home water storage. It has been suggested that dirty hands play a role in microbial contamination of drinking water during collection, transport, and storage. However, little work has been done to evaluate quantitatively the association between hand contamination and stored water quality within households. This study measured levels of E. coli, fecal streptococci, and occurrence of the general Bacteroidales fecal DNA marker in source water, in stored water, and on hands in 334 households among communities in Dar es Salaam, Tanzania, where residents use non-networked water sources. Levels of fecal contamination on hands of mothers and children were positively correlated to fecal contamination in stored drinking water within households. Household characteristics associated with hand contamination included mother's educational attainment, use of an improved toilet, an infant in the household, and dissatisfaction with the quantity of water available for hygiene. In addition, fecal contamination on hands was associated with the prevalence of gastrointestinal and respiratory symptoms within a household. The results suggest that reducing fecal contamination on hands should be investigated as a strategy for improving stored drinking water quality and health among households using non-networked water supplies. [ABSTRACT FROM AUTHOR]

Fecal indicator bacteria (FIB) are nearly ubiquitous in California (CA) beach sands. Sands were collected from 55 beaches along the CA coast. Ninety-one percent of the beaches had detectable enterococci (ENT) while 62% had detectable E. coli (EC) in their sands. The presence of a putative bacterial source (such as a river), the degree of wave shelter, and surrounding land use explained a significant (p < 0.05) fraction of the variation in both ENT and EC densities between beaches. Sand characteristics including moisture content, organic carbon, and percent fines, significantly (p < 0.05) influenced only EC densities in beach sand. We assayed 34 of 163 sand samples for salmonellae, but did not detect this bacterial pathogen. The potential for FIB to be transported from the sand to sea was investigated at a single wave-sheltered beach with high densities of ENT in beach sand: Lovers Point, CA (LP). We collected samples of exposed and submerged sands as well as water over a 24 h period in order to compare the disappearance or appearance of ENT in sand and the water column. Exposed sands had significantly higher densities of ENT than submerged sands with the highest densities located near the high tide line. Water column ENT densities began low, increased sharply during the first flood tide and slowly decreased over the remainder of the study. During the first flood tide, the number of ENT that entered the water column was nearly equivalent to the number of ENT lost from exposed sands when they were submerged by seawater. The decrease in nearshore ENT concentrations after the initial influx can be explained by ENT die-off and dilution with clean ocean water. While some ENT in the water and sand at LP might be of human origin because they were positive for the esp gene, others lacked the esp gene and were therefore equivocal with respect to their origin. Follow-up sampling at LP revealed the presence of the human specific Bacteroides marker in water and sand. [ABSTRACT FROM AUTHOR]