Erin M. Symonds

Pepper Mild Mottle Virus as an Indicator of Fecal Pollution

ABSTRACT Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.

Eukaryotic Viruses in Wastewater Samples from the United States

ABSTRACT Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments.

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem.

A case study of enteric virus removal and insights into the associated risk of water reuse for two wastewater treatment pond systems in Bolivia.

Wastewater treatment ponds (WTP) are one of the most widespread treatment technologies in the world; however, the mechanisms and extent of enteric virus removal in these systems are poorly understood. Two WTP systems in Bolivia, with similar overall hydraulic retention times but different first stages of treatment, were analyzed for enteric virus removal. One system consisted of a facultative pond followed by two maturation ponds (three-pond system) and the other consisted of an upflow anaerobic sludge blanket (UASB) reactor followed by two maturation (polishing) ponds (UASB-pond system). Quantitative polymerase chain reaction with reverse transcription (RT-qPCR) was used to measure concentrations of norovirus, rotavirus, and pepper mild mottle virus, while cell culture methods were used to measure concentrations of culturable enteroviruses (EV). Limited virus removal was observed with RT-qPCR in either system; however, the three-pond system removed culturable EV with greater efficiency than the UASB-pond system. The majority of viruses were not associated with particles and only a small proportion was associated with particles larger than 180 μm; thus, it is unlikely that sedimentation is a major mechanism of virus removal. High concentrations of viruses were associated with particles between 0.45 and 180 μm in the UASB reactor effluent, but not in the facultative pond effluent. The association of viruses with this size class of particles may explain why only minimal virus removal was observed in the UASB-pond system. Quantitative microbial risk assessment of the treated effluent for reuse for restricted irrigation indicated that the three-pond system effluent requires an additional 1- to 2-log10 reduction of viruses to achieve the WHO health target of <10(-4) disability-adjusted life years (DALYs) lost per person per year; however, the UASB-pond system effluent may require an additional 2.5- to 4.5-log10 reduction of viruses.

Reduction of nutrients, microbes, and personal care products in domestic wastewater by a benchtop electrocoagulation unit

To preserve environmental and human health, improved treatment processes are needed to reduce nutrients, microbes, and emerging chemical contaminants from domestic wastewater prior to discharge into the environment. Electrocoagulation (EC) treatment is increasingly used to treat industrial wastewater; however, this technology has not yet been thoroughly assessed for its potential to reduce concentrations of nutrients, a variety of microbial surrogates, and personal care products found in domestic wastewater. This investigations objective was to determine the efficiency of a benchtop EC unit with aluminum sacrificial electrodes to reduce concentrations of the aforementioned biological and chemical pollutants from raw and tertiary-treated domestic wastewater. EC treatment resulted in significant reductions (p < 0.05, α = 0.05) in phosphate, all microbial surrogates, and several personal care products from raw and tertiary-treated domestic wastewater. When wastewater was augmented with microbial surrogates representing bacterial, viral, and protozoan pathogens to measure the extent of reduction, EC treatment resulted in up to 7-log10 reduction of microbial surrogates. Future pilot and full-scale investigations are needed to optimize EC treatment for the following: reducing nitrogen species, personal care products, and energy consumption; elucidating the mechanisms behind microbial reductions; and performing life cycle analyses to determine the appropriateness of implementation.

Managing Microbial Risks from Indirect Wastewater Reuse for Irrigation in Urbanizing Watersheds.

Limited supply of clean water in urbanizing watersheds creates challenges for safely sustaining irrigated agriculture and global food security. On-farm interventions, such as riverbank filtration (RBF), are used in developing countries to treat irrigation water from rivers with extensive fecal contamination. Using a Bayesian approach incorporating ethnographic data and pathogen measurements, quantitative microbial risk assessment (QMRA) methods were employed to assess the impact of RBF on consumer health burdens for Giardia, Cryptosporidium, rotavirus, norovirus, and adenovirus infections resulting from indirect wastewater reuse, with lettuce irrigation in Bolivia as a model system. Concentrations of the microbial source tracking markers pepper mild mottle virus and HF183 Bacteroides were respectively 2.9 and 5.5 log10 units lower in RBF-treated water than in the river water. Consumption of lettuce irrigated with river water caused an estimated median health burden that represents 37% of Bolivias overall diarrheal disease burden, but RBF resulted in an estimated health burden that is only 1.1% of this overall diarrheal disease burden. Variability and uncertainty associated with environmental and cultural factors affecting exposure correlated more with QMRA-predicted health outcomes than factors related to disease vulnerability. Policies governing simple on-farm interventions like RBF can be intermediary solutions for communities in urbanizing watersheds that currently lack wastewater treatment.

Environmental Factors Correlated with Culturable Enterococci Concentrations in Tropical Recreational Waters: A Case Study in Escambron Beach, San Juan, Puerto Rico

Enterococci concentration variability at Escambron Beach, San Juan, Puerto Rico, was examined in the context of environmental conditions observed during 2005–2015. Satellite-derived sea surface temperature (SST), turbidity, direct normal irradiance, and dew point were combined with local precipitation, winds, and mean sea level (MSL) observations in a stepwise multiple regression analyses (Akaike Information Criteria model selection). Precipitation, MSL, irradiance, SST, and turbidity explained 20% of the variation in observed enterococci concentrations based upon these analyses. Changes in these parameters preceded increases in enterococci concentrations by 24 h up to 11 days, particularly during positive anomalies of turbidity, SST, and 480–960 mm of accumulated (4 days) precipitation, which relates to bacterial ecology. Weaker, yet still significant, increases in enterococci concentrations were also observed during positive dew point anomalies. Enterococci concentrations decreased with elevated irradiance and MSL anomalies. Unsafe enterococci concentrations per US EPA recreational water quality guidelines occurred when 4-day cumulative precipitation ranged 481–960 mm; irradiance < 667 W·m−2; daily average turbidity anomaly >0.005 sr−1; SST anomaly >0.8 °C; and 3-day average MSL anomaly <−18.8 cm. This case study shows that satellite-derived environmental data can be used to inform future water quality studies and protect human health.

Pepper mild mottle virus: A plant pathogen with a greater purpose in (waste)water treatment development and public health management

An enteric virus surrogate and reliable domestic wastewater tracer is needed to manage microbial quality of food and water as (waste)water reuse becomes more prevalent in response to population growth, urbanization, and climate change. Pepper mild mottle virus (PMMoV), a plant pathogen found at high concentrations in domestic wastewater, is a promising surrogate for enteric viruses that has been incorporated into over 29 water- and food-related microbial quality and technology investigations around the world. This review consolidates the available literature from across disciplines to provide guidance on the utility of PMMoV as either an enteric virus surrogate and/or domestic wastewater marker in various situations. Synthesis of the available research supports PMMoV as a useful enteric virus process indicator since its high concentrations in source water allow for identifying the extent of virus log-reductions in field, pilot, and full-scale (waste)water treatment systems. PMMoV reduction levels during many forms of wastewater treatment were less than or equal to the reduction of other viruses, suggesting this virus can serve as an enteric virus surrogate when evaluating new treatment technologies. PMMoV excels as an index virus for enteric viruses in environmental waters exposed to untreated domestic wastewater because it was detected more frequently and in higher concentrations than other human viruses in groundwater (72.2%) and surface waters (freshwater, 94.5% and coastal, 72.2%), with pathogen co-detection rates as high as 72.3%. Additionally, PMMoV is an important microbial source tracking marker, most appropriately associated with untreated domestic wastewater, where its pooled-specificity is 90% and pooled-sensitivity is 100%, as opposed to human feces where its pooled-sensitivity is only 11.3%. A limited number of studies have also suggested that PMMoV may be a useful index virus for enteric viruses in monitoring the microbial quality of fresh produce and shellfish, but further research is needed on these topics. Finally, future work is needed to fill in knowledge gaps regarding PMMoVs global specificity and sensitivity.

Predicting culturable enterococci exceedances at escambron beach, San Juan, Puerto Rico using satellite remote sensing and artificial neural networks

Predicting recreational water quality is key to protecting public health from exposure to wastewater-associated pathogens. It is not feasible to monitor recreational waters for all pathogens; therefore, monitoring programs use fecal indicator bacteria (FIB), such as enterococci, to identify wastewater pollution. Artificial neural networks (ANNs) were used to predict when culturable enterococci concentrations exceeded the U.S. Environmental Protection Agency (U.S. EPA) Recreational Water Quality Criteria (RWQC) at Escambron Beach, San Juan, Puerto Rico. Ten years of culturable enterococci data were analyzed together with satellite-derived sea surface temperature (SST), direct normal irradiance (DNI), turbidity, and dew point, along with local observations of precipitation and mean sea level (MSL). The factors identified as the most relevant for enterococci exceedance predictions based on the U.S. EPA RWQC were DNI, turbidity, cumulative 48 h precipitation, MSL, and SST; they predicted culturable enterococci exceedances with an accuracy of 75% and power greater than 60% based on the Receiving Operating Characteristic curve and F-Measure metrics. Results show the applicability of satellite-derived data and ANNs to predict recreational water quality at Escambron Beach. Future work should incorporate local sanitary survey data to predict risky recreational water conditions and protect human health.