Correction: Effects of chronic exposure to arsenic on the fecal carriage of antibiotic-resistant Escherichia coli among people in rural Bangladesh.

[This corrects the article DOI: 10.1371/journal.ppat.1010952.].

Sanitary sewage overflows, boil water advisories, and emergency room and urgent care visits for gastrointestinal illness: a case-crossover study in South Carolina, USA, 2013-2017.

BACKGROUND: Sanitary sewage overflows (SSOs) release raw sewage, which may contaminate the drinking water supply. Boil water advisories (BWAs) are issued during low or negative pressure events, alerting customers to potential contamination in the drinking water distribution system. OBJECTIVE: We evaluated the associations between SSOs and BWAs and diagnoses of gastrointestinal (GI) illness in Columbia, South Carolina, and neighboring communities, 2013-2017. METHODS: A symmetric bi-directional case-crossover study design was used to assess the role of SSOs and BWAs on Emergency Room and Urgent Care visits with a primary diagnosis of GI illness. Cases were considered exposed if an SSO or BWA occurred 0-4 days, 5-9 days, or 10-14 days prior to the diagnosis, within the same residential zip code. Effect modification was explored via stratification on participant-level factors (e.g., sex, race, age) and season (January-March versus April-December). RESULTS: There were 830 SSOs, 423 BWAs, and 25,969 cases of GI illness. Highest numbers of SSOs, BWAs and GI cases were observed in a zip code where >80% of residents identified as Black or African-American. SSOs were associated with a 13% increase in the odds of a diagnosis for GI illness during the 0-4 day hazard period, compared to control periods (Odds Ratio: 1.13, 95% Confidence Interval: 1.09, 1.18), while no associations were observed during the other hazard periods. BWAs were not associated with increased or decreased odds of GI illness during all three hazard periods. However, in stratified analyses BWAs issued between January-March were associated with higher odds of GI illness, compared to advisories issued between April-December, in all three hazard periods. SIGNIFICANCE: SSOs (all months) and BWAs (January-March) were associated with increased odds of a diagnosis of GI illness. Future research should examine sewage contamination of the drinking water distribution system, and mechanisms of sewage intrusion from SSOs. IMPACT: Sewage contains pathogens, which cause gastrointestinal (GI) illness. In Columbia, South Carolina, USA, between 2013-2017, there were 830 sanitary sewage overflows (SSOs). There were also 423 boil water advisories, which were issued during negative pressure events. Using case-crossover design, SSOs (all months) and boil water advisories (January-March) were associated with increased odds of Emergency Room and Urgent Care diagnoses of GI illness, potentially due to contamination of the drinking water distribution system. Lastly, we identified a community where >80% of residents identified as Black or African-American, which experienced a disproportionate burden of sewage exposure, compared to the rest of Columbia.

Effects of chronic exposure to arsenic on the fecal carriage of antibiotic-resistant Escherichia coli among people in rural Bangladesh.

Antibiotic resistance is a leading cause of hospitalization and death worldwide. Heavy metals such as arsenic have been shown to drive co-selection of antibiotic resistance, suggesting arsenic-contaminated drinking water is a risk factor for antibiotic resistance carriage. This study aimed to determine the prevalence and abundance of antibiotic-resistant Escherichia coli (AR-Ec) among people and drinking water in high (Hajiganj, >100 µg/L) and low arsenic-contaminated (Matlab, <20 µg/L) areas in Bangladesh. Drinking water and stool from mothers and their children (<1 year) were collected from 50 households per area. AR-Ec was detected via selective culture plating and isolates were tested for antibiotic resistance, arsenic resistance, and diarrheagenic genes by PCR. Whole-genome sequencing (WGS) analysis was done for 30 E. coli isolates from 10 households. Prevalence of AR-Ec was significantly higher in water in Hajiganj (48%) compared to water in Matlab (22%, p <0.05) and among children in Hajiganj (94%) compared to children in Matlab (76%, p <0.05), but not among mothers. A significantly higher proportion of E. coli isolates from Hajiganj were multidrug-resistant (83%) compared to isolates from Matlab (71%, p <0.05). Co-resistance to arsenic and multiple antibiotics (MAR index >0.2) was observed in a higher proportion of water (78%) and child stool (100%) isolates in Hajiganj than in water (57%) and children (89%) in Matlab (p <0.05). The odds of arsenic-resistant bacteria being resistant to third-generation cephalosporin antibiotics were higher compared to arsenic-sensitive bacteria (odds ratios, OR 1.2-7.0, p <0.01). WGS-based phylogenetic analysis of E. coli isolates did not reveal any clustering based on arsenic exposure and no significant difference in resistome was found among the isolates between the two areas. The positive association detected between arsenic exposure and antibiotic resistance carriage among children in arsenic-affected areas in Bangladesh is an important public health concern that warrants redoubling efforts to reduce arsenic exposure.

Resistome characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment utilities in Oregon.

Infections resistant to broad spectrum antibiotics due to the emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is of global concern. This study characterizes the resistome (i.e., entire ecology of resistance determinants) of 11 ESBL-producing Escherichia coli isolates collected from eight wastewater treatment utilities across Oregon. Whole genome sequencing was performed to identify the most abundant antibiotic resistance genes including ESBL-associated genes, virulence factors, as well as their sequence types. Moreover, the phenotypes of antibiotic resistance were characterized. ESBL-associated genes (i.e., blaCMY, blaCTX, blaSHV, blaTEM) were found in all but one of the isolates with five isolates carrying two of these genes (four with blaCTX and blaTEM; one with blaCMY and blaTEM). The ampC gene and virulence factors were present in all the E. coli isolates. Across all the isolates, 31 different antibiotic resistance genes were identified. Additionally, all E. coli isolates harbored phenotypic resistance to beta-lactams (penicillins and cephalosporins), while 8 of the 11 isolates carried multidrug resistance phenotypes (resistance to three or more classes of antibiotics). Findings highlight the risks associated with the presence of ESBL-producing E. coli isolates in wastewater systems that have the potential to enter the environment and may pose direct or indirect risks to human health.

Impact of pH and removed filtrate on E. coli regrowth and microbial community during storage of electro-dewatered biosolids.

Residual biosolids can be land applied if they meet microbiological requirements at the time of application. Electro-dewatering technology is shown to reduce biosolids bacterial counts to detection limits with little potential for bacterial regrowth during incubations. Here, we investigated the impacts on Escherichia coli regrowth and microbial communities of biosolids pH, removed nutrients via the filtrate, and inhibitory compounds produced in electro-dewatered biosolids. Findings suggest pH as the primary mechanism impacting E. coli regrowth in electro-dewatered biosolids. Propidium monoazide treatments were effective at removing DNA from dead cells, based on the removal of obligate anaerobes observed after anaerobic incubation. Analyses of high throughput sequenced data showed lower alpha-diversities associated with electro-dewatering treatment and incubation time. Moreover, biosolids pH and incubation period were the main factors contributing to the variations in microbial community compositions after incubation. Results highlight the role of electro-dewatered biosolids' low pH on inhibiting the regrowth of culturable bacteria as well as reducing the microbial community variance.

Impact of biosolids amendment and wastewater effluent irrigation on enteric antibiotic-resistant bacteria - a greenhouse study.

Reuse of wastewater effluent and biosolids in agriculture is essential to sustainable water and nutrient resource management practices. Wastewater and biosolids, however, are reportedly the recipients, reservoirs, and sources of antibiotic-resistant enteric pathogens. While decay rates of fecal bacterial indicators in soil are frequently studied, very few studies have reported on the persistence of the antibiotic-resistant sub-populations. Little is known about how multi-drug resistance phenotypes of enteric bacteria in agricultural soil change over time. In this study, germinated carrot seeds were planted in soil that received biosolids amendment and/or wastewater effluent irrigation in a greenhouse setting. We quantified total and antibiotic-resistant fecal bacterial indicators (Escherichia coli and enterococci) weekly in soil and total E. coli at harvest (day 77) on carrots. Antibiotic susceptibility of 121 E. coli and 110 enterococci collected isolates were determined. E. coli or enterococci were not recovered from the soil without biosolids amendment regardless of the irrigation water source. After biosolids amendment, soil E. coli and enterococci concentrations increased more than 3 log10 CFU/g-TS within the first week, declined slowly over time, but stayed above the detection limit (0.39 CFU/g-TS) over the entirety of the study. No statistical difference was found between effluent wastewater or water irrigation in soil total and antibiotic-resistant E. coli and enterococci concentrations or carrots E. coli levels. Soil antibiotic-resistant E. coli and enterococci decayed significantly faster than total E. coli and enterococci. Moreover, the prevalence of multi-drug resistant (resistance to three or more antibiotics) E. coli declined significantly over time, while almost all collected enterococci isolates showed multi-drug resistance phenotypes. At harvest, E. coli were present on carrots; the majority of which were resistant to ampicillin. The survival of antibiotic-resistant enteric bacteria in soil and on harvested carrots indicates there are transmission risks associated with biosolids amendment use in root crops.

Antibiotic resistance in fecal sludge and soil in Ho Chi Minh City, Vietnam.

This study investigated the prevalence of antibiotic-resistant bacteria and genes in fecal sludge and soil in Ho Chi Minh City, Vietnam, and identified the factors contributing to the survival of antibiotic-resistant bacteria in soil. Sludge and soil samples (n = 24 and 55, respectively) were collected from residential septic systems and environmental reservoirs (i.e., canals, rivers, and parks) in twelve districts of Ho Chi Minh City and tested against a library of 12 antibiotic-resistant genes and 1 integron gene. The susceptibility of isolated Escherichia coli from sludge and soil (n = 104 and 129, respectively) was tested against nine antibiotics. Over 60% of sludge and soil samples harbored sul1, ere(A), intI1, cmIA, and tet(A) genes. The three most common phenotypic resistances found in E. coli isolated from sludge and soil were to ampicillin, tetracycline, and sulfamethoxazole/trimethoprim. In a temporal microcosm study of antibiotic-susceptible and multi-drug-resistant E. coli inoculated in soil, temperature (21.4 vs. 30 °C), resistance phenotype, and soil background microbial community were associated with E. coli decay rates over 73 days. This is the first study that provides insights into the high prevalence of antibiotic resistance in septic systems and environmental reservoirs in Ho Chi Minh City, Vietnam. Findings highlight that the fecal sludge and soil environments in Vietnam are likely reservoirs for dissemination of and human exposure to antibiotic resistance.

Fecal Colonization With Multidrug-Resistant E. coli Among Healthy Infants in Rural Bangladesh.

Third generation cephalosporins (3GC) are one of the main choices for treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Due to their overuse, an increasing trend of resistance to 3GC has been observed in developing countries. Here, we describe fecal colonization of 3GC-resistant (3GCr) Escherichia coli in healthy infants (1-12 months old) living in rural areas of Bangladesh. We found that stool samples of 82% of infants (n = 100) were positive for 3GCr E. coli with a mean ± standard deviation of 6.21 ± 1.32 log10 CFU/g wet weight of stool. 3GCr E. coli encompasses an average one third (33%) of the total E. coli of stool. Almost 77% (n = 63) of these 3GCr E. coli were MDR (or resistant to ≥3 classes of antibiotics). Around 90% (n = 74) of 3GCr E. coli were extended spectrum beta-lactamase (ESBL)-producing in which bla CTX-M-group-1 was the predominant (96%, n = 71) ESBL-gene followed by bla TEM (41%, n = 30) and bla OXA-1 (11%, n = 8). A significant proportion (26.5%, n = 22) of 3GCr E. coli was pathogenic, comprising two types, enteroaggregative (EAEC, n = 19) and enteropathogenic (EPEC, n = 3). Colonization of 3GCr E. coli in infant guts was not associated with demographic characteristics such as age, sex, mode of delivery, maternal and infant antibiotic use, disease morbidity, and feeding practices. The high rate of colonization of 3GCr E. coli in infants' guts is a serious public health concern which needs immediate attention and warrants further studies to explore the cause.

Safely Managed Hygiene: A Risk-Based Assessment of Handwashing Water Quality.

Sustainable Development Goal (SDG) Indicator 6.2.1 requires household handwashing facilities to have soap and water, but there are no guidelines for handwashing water quality. In contrast, drinking water quality guidelines are defined: water must be "free from contamination" to be defined as "safely managed" (SDG Indicator 6.1.1). We modeled the hypothesized mechanism of infection due to contaminated handwashing water to inform risk-based guidelines for microbial quality of handwashing water. We defined two scenarios that should not occur: (1) if handwashing caused fecal contamination, indicated using Escherichia coli, on a person's hands to increase rather than decrease and (2) if hand-to-mouth contacts following handwashing caused an infection risk greater than an acceptable threshold. We found water containing <1000 E. coli colony-forming units (CFU) per 100 mL removes E. coli from hands with>99.9% probability. However, for the annual probability of infection to be <1:1000, handwashing water must contain <2 × 10-6 focus-forming units of rotavirus, <1 × 10-4 CFU of Vibrio cholerae, and <9 × 10-6 Cryptosporidium oocysts per 100 mL. Our model suggests that handwashing with nonpotable water will generally reduce fecal contamination on hands but may be unable to lower the annual probability of infection risks from hand-to-mouth contacts below 1:1000.

Risk Factors for Detection, Survival, and Growth of Antibiotic-Resistant and Pathogenic Escherichia coli in Household Soils in Rural Bangladesh.

Soils in household environments in low- and middle-income countries may play an important role in the persistence, proliferation, and transmission of Escherichia coli Our goal was to investigate the risk factors for detection, survival, and growth of E. coli in soils collected from household plots. E. coli was enumerated in soil and fecal samples from humans, chickens, and cattle from 52 households in rural Bangladesh. Associations between E. coli concentrations in soil, household-level risk factors, and soil physicochemical characteristics were investigated. Susceptibility to 16 antibiotics and the presence of intestinal pathotypes were evaluated for 175 E. coli isolates. The growth and survival of E. coli in microcosms using soil collected from the households were also assessed. E. coli was isolated from 44.2% of the soil samples, with an average of 1.95 log10 CFU/g dry soil. Soil moisture and clay content were associated with E. coli concentrations in soil, whereas no household-level risk factor was significantly correlated. Antibiotic resistance and pathogenicity were common among E. coli isolates, with 42.3% resistant to at least one antibiotic, 12.6% multidrug resistant (≥3 classes), and 10% potentially pathogenic. Soil microcosms demonstrate growth and/or survival of E. coli, including an enteropathogenic extended-spectrum beta-lactamase (ESBL)-producing isolate, in some, but not all, of the household soils tested. In rural Bangladesh, defined soil physicochemical characteristics appear more influential for E. coli detection in soils than household-level risk factors. Soils may act as reservoirs in the transmission of antibiotic-resistant and potentially pathogenic E. coli and therefore may impact the effectiveness of water, sanitation, and hygiene interventions.IMPORTANCE Soil may represent a direct source or act as an intermediary for the transmission of antibiotic-resistant and pathogenic Escherichia coli strains, particularly in low-income and rural settings. Thus, determining risk factors associated with detection, growth, and long-term survival of E. coli in soil environments is important for public health. Here, we demonstrate that household soils in rural Bangladesh are reservoirs for antibiotic-resistant and potentially pathogenic E. coli strains and can support E. coli growth and survival, and defined soil physicochemical characteristics are drivers of E. coli survival in this environment. In contrast, we found no evidence that household-level factors, including water, sanitation, and hygiene indicators, were associated with E. coli contamination of household soils.

Escherichia coli Contamination across Multiple Environmental Compartments (Soil, Hands, Drinking Water, and Handwashing Water) in Urban Harare: Correlations and Risk Factors.

Escherichia coli pathotypes (i.e., enteropathogenic and enterotoxigenic) have been identified among the pathogens most responsible for moderate-to-severe diarrhea in low- and middle-income countries (LMICs). Pathogenic E. coli are transmitted from infected human or animal feces to new susceptible hosts via environmental reservoirs such as hands, water, and soil. Commensal E. coli, which includes nonpathogenic E. coli strains, are widely used as fecal bacteria indicator, with their presence associated with increased likelihood of enteric pathogens and/or diarrheal disease. In this study, we investigated E. coli contamination in environmental reservoirs within households (N = 142) in high-population density communities of Harare, Zimbabwe. We further assessed the interconnectedness of the environmental compartments by investigating associations between, and household-level risk factors for, E. coli contamination. From the data we collected, the source and risk factors for E. coli contamination are not readily apparent. One notable exception is the presence of running tap water on the household plot, which is associated with significantly less E. coli contamination of drinking water, handwashing water, and hands after handwashing. In addition, E. coli levels on hands after washing are significantly associated with handwashing water contamination, hand contamination before washing, and diarrhea incidence. Finally, we observed that animal ownership increases E. coli contamination in soil, and E. coli in soil are correlated with contamination on hands before washing. This study highlights the complexity of E. coli contamination in household environments within LMICs. More, larger, studies are needed to better identify sources and exposure pathways of E. coli-and enteric pathogens generally-to identify effective interventions.

Impact of Joule Heating and pH on Biosolids Electro-Dewatering.

Electro-dewatering (ED) is a novel technology to reduce the overall costs of residual biosolids processing, transport, and disposal. In this study, we investigated Joule heating and pH as parameters controlling the dewaterability limit, dewatering rate, and energy efficiency. Temperature-controlled electrodes revealed that Joule heating enhances water removal by increasing evaporation and electro-osmotic flow. High temperatures increased the dewatering rate, but had little impact on the dewaterability limit and energy efficiency. Analysis of horizontal layers after 15-min ED suggests electro-osmotic flow reversal, as evidenced by a shifting of the point of minimum moisture content from the anode toward the cathode. This flow reversal was also confirmed by the pH at the anode being below the isoelectric point, as ascertained by pH titration. The important role of pH on ED was further studied by adding acid/base solutions to biosolids prior to ED. An acidic pH reduced the biosolids charge while simultaneously increasing the dewatering efficiency. Thus, process optimization depends on trade-offs between speed and efficiency, according to physicochemical properties of the biosolids microstructure.

Bacterial pathogen indicators regrowth and reduced sulphur compounds' emissions during storage of electro-dewatered biosolids.

Electro-dewatering (ED) increases biosolids dryness from 10-15 to 30-50%, which helps wastewater treatment facilities control disposal costs. Previous work showed that high temperatures due to Joule heating during ED inactivate total coliforms to meet USEPA Class A biosolids requirements. This allows biosolids land application if the requirements are still met after the storage period between production and application. In this study, we examined bacterial regrowth and odour emissions during the storage of ED biosolids. No regrowth of total coliforms was observed in ED biosolids over 7d under aerobic or anaerobic incubations. To mimic on-site contamination during storage or transport, ED samples were seeded with untreated sludge. Total coliform counts decreased to detection limits after 4d in inoculated samples. Olfactometric analysis of ED biosolids odours showed that odour concentrations were lower compared to the untreated and heat-treated control biosolids. Furthermore, under anaerobic conditions, odorous reduced sulphur compounds (methanethiol, dimethyl sulphide and dimethyl disulphide) were produced by untreated and heat-treated biosolids, but were not detected in the headspaces above ED samples. The data demonstrate that ED provides advantages not only as a dewatering technique, but also for producing biosolids with lower microbial counts and odour levels.

Inactivation mechanisms of bacterial pathogen indicators during electro-dewatering of activated sludge biosolids.

Electro-dewatering is an energy-efficient technology in which an electric field can increase the dryness of biosolids from secondary wastewater treatment from 15% w/w to 30-50% w/w. Here, we address bacterial pathogen indicators inactivation (total coliforms, Escherichia coli and aerobic endospores) during electro-dewatering, investigating the roles of electrochemically generated oxidants, extreme pH, and high temperature (from Joule heating). Our results demonstrate that temperature is the primary factor affecting total coliforms and E. coli inactivation. First, several electro-dewatering cycles were used to increase sludge temperature to about 100 °C after 6 min, during which time the average pH decreased from 7 to 3.6 after 10 min. Total coliforms and E. coli MPNs reached their detection limits after 6 min (with 4-5 logs of inactivation for total coliforms and 3-4 logs for E. coli). In contrast, aerobic endospores were not inactivated under these conditions; rather, their germination appeared to be stimulated by 6-8 min of electro-dewatering. Second, the dewatering cake was separated into four horizontal layers. After 8 min of electro-dewatering, the pH in the top layers decreased to 3, whereas the pH in the bottom layers increased to 8. Inactivation of total coliforms and E. coli in the sludge cake was similar in all layers, increasing with time, suggesting that oxidants and extreme pH are secondary inactivation factors. Finally, electrodes were cooled to maintain a temperature less than 34 °C. Although pH decreased significantly after 12 min of electro-dewatering, there was no significant bacterial pathogen indicator inactivation at low temperature.