Characterizing Spatial Information Loss for Wastewater Surveillance Using crAssphage: Effect of Decay, Temperature, and Population Mobility.

Populations contribute information about their health status to wastewater. Characterizing how that information degrades in transit to wastewater sampling locations (e.g., wastewater treatment plants and pumping stations) is critical to interpret wastewater responses. In this work, we statistically estimate the loss of information about fecal contributions to wastewater from spatially distributed populations at the census block group resolution. This was accomplished with a hydrologically and hydraulically influenced spatial statistical approach applied to crAssphage (Carjivirus communis) load measured from the influent of four wastewater treatment plants in Hamilton County, Ohio. We find that we would expect to observe a 90% loss of information about fecal contributions from a given census block group over a travel time of 10.3 h. This work demonstrates that a challenge to interpreting wastewater responses (e.g., during wastewater surveillance) is distinguishing between a distal but large cluster of contributions and a near but small contribution. This work demonstrates new modeling approaches to improve measurement interpretation depending on sewer network and wastewater characteristics (e.g., geospatial layout, temperature variability, population distribution, and mobility). This modeling can be integrated into standard wastewater surveillance methods and help to optimize sewer sampling locations to ensure that different populations (e.g., vulnerable and susceptible) are appropriately represented.

Characterization of roof runoff microbial quality in four U.S. cities with varying climate and land use characteristics.

Roof runoff has the potential to serve as an important local water source in regions with growing populations and limited water supply. Given the scarcity of guidance regulating the use of roof runoff, a need exists to characterize the microbial quality of roof runoff. The objective of this 2-year research effort was to examine roof runoff microbial quality in four U.S. cities: Fort Collins, CO; Tucson, AZ; Baltimore, MD; and Miami, FL. Seven participants, i.e., homeowners and schools, were recruited in each city to collect roof runoff samples across 13 precipitation events. Sample collection was done as part of a citizen science approach. The presence and concentrations of indicator organisms and potentially human-infectious pathogens in roof runoff were determined using culture methods and digital droplet polymerase chain reaction (ddPCR), respectively. The analyzed pathogens included Salmonella spp., Campylobacter spp., Giardia duodenalis, and Cryptosporidium parvum. Several factors were evaluated to study their influence on the presence of potentially human-infectious pathogens including the physicochemical characteristics (total suspended solids, volatile suspended solids, total dissolved solids, chemical oxygen demand, and turbidity) of roof runoff, concentrations of indicator organisms, presence/absence of trees, storm properties (rainfall depth and antecedent dry period), percent of impervious cover surrounding each sampling location, seasonality, and geographical location. E. coli and enterococci were detected in 73.4% and 96.2% of the analyzed samples, respectively. Concentrations of both E. coli and enterococci ranged from <0 log10 to >3.38 log10 MPN/100 mL. Salmonella spp. invA, Campylobacter spp. ceuE, and G. duodenalis ß - giardin gene targets were detected in 8.9%, 2.5%, and 5.1% of the analyzed samples, respectively. Campylobacter spp. mapA and C. parvum 18S rRNA gene targets were not detected in any of the analyzed samples. The detection of Salmonella spp. invA was influenced by the geographical location of the sampling site (Chi-square p-value < 0.001) as well as the number of antecedent dry days prior to a rain event (p-value = 0.002, negative correlation). The antecedent dry period was negatively correlated with the occurrence of Campylobacter spp. ceuE as well (p-value = 0.07). On the other hand, the presence of G. duodenalis ß-giardin in roof runoff was positively correlated with rainfall depth (p-value = 0.05). While physicochemical parameters and impervious area were not found to be correlated with the presence/absence of potentially human-infectious pathogens, significant correlations were found between meteorological parameters and the presence/absence of potentially human-infectious pathogens. Additionally, a weak, yet significant positive correlation, was found only between the concentrations of E. coli and those of Giardia duodenalis ß-giardin. This dataset represents the largest-scale study to date of enteric pathogens in U.S. roof runoff collections and will inform treatment targets for different non-potable end uses for roof runoff. However, the dataset is limited by the low percent detection of bacterial and protozoan pathogens, an issue that is likely to persist challenging the characterization of roof runoff microbial quality given sampling limitations related to the volume and number of samples.

Geospatial Patterns of Antimicrobial Resistance Genes in the US EPA National Rivers and Streams Assessment Survey.

Antimicrobial resistance (AR) is a serious global problem due to the overuse of antimicrobials in human, animal, and agriculture sectors. There is intense research to control the dissemination of AR, but little is known regarding the environmental drivers influencing its spread. Although AR genes (ARGs) are detected in many different environments, the risk associated with the spread of these genes to microbial pathogens is unknown. Recreational microbial exposure risks are likely to be greater in water bodies receiving discharge from human and animal waste in comparison to less disturbed aquatic environments. Given this scenario, research practitioners are encouraged to consider an ecological context to assess the effect of environmental ARGs on public health. Here, we use a stratified, probabilistic survey of nearly 2000 sites to determine national patterns of the anthropogenic indicator class I integron Integrase gene (intI1) and several ARGs in 1.2 million kilometers of United States (US) rivers and streams. Gene concentrations were greater in eastern than in western regions and in rivers and streams in poor condition. These first of their kind findings on the national distribution of intI1 and ARGs provide new information to aid risk assessment and implement mitigation strategies to protect public health.

Monoassociation with bacterial isolates reveals the role of colonization, community complexity and abundance on locomotor behavior in larval zebrafish.

BACKGROUND: Across taxa, animals with depleted intestinal microbiomes show disrupted behavioral phenotypes. Axenic (i.e., microbe-free) mice, zebrafish, and fruit flies exhibit increased locomotor behavior, or hyperactivity. The mechanism through which bacteria interact with host cells to trigger normal neurobehavioral development in larval zebrafish is not well understood. Here, we monoassociated zebrafish with either one of six different zebrafish-associated bacteria, mixtures of these host-associates, or with an environmental bacterial isolate. RESULTS: As predicted, the axenic cohort was hyperactive. Monoassociation with three different host-associated bacterial species, as well as with the mixtures, resulted in control-like locomotor behavior. Monoassociation with one host-associate and the environmental isolate resulted in the hyperactive phenotype characteristic of axenic larvae, while monoassociation with two other host-associated bacteria partially blocked this phenotype. Furthermore, we found an inverse relationship between the total concentration of bacteria per larvae and locomotor behavior. Lastly, in the axenic and associated cohorts, but not in the larvae with complex communities, we detected unexpected bacteria, some of which may be present as facultative predators. CONCLUSIONS: These data support a growing body of evidence that individual species of bacteria can have different effects on host behavior, potentially related to their success at intestinal colonization. Specific to the zebrafish model, our results suggest that differences in the composition of microbes in fish facilities could affect the results of behavioral assays within pharmacological and toxicological studies.

Droplet digital PCR quantification of norovirus and adenovirus in decentralized wastewater and graywater collections: Implications for onsite reuse.

Risk-based treatment of onsite wastewaters for decentralized reuse requires information on the occurrence and density of pathogens in source waters, which differ from municipal wastewater due to scaling and dilution effects in addition to variable source contributions. In this first quantitative report of viral enteric pathogens in onsite-collected graywater and wastewater, untreated graywater (n = 50 samples) and combined wastewater (i.e., including blackwater; n = 28) from three decentralized collection systems were analyzed for two norovirus genogroups (GI/GII) and human adenoviruses using droplet digital polymerase chain reaction (ddPCR). Compared to traditional quantitative PCR (qPCR), which had insufficient sensitivity to quantify viruses in graywater, ddPCR allowed quantification of norovirus GII and adenovirus in 4% and 14% of graywater samples, respectively (none quantifiable for norovirus GI). Norovirus GII was routinely quantifiable in combined wastewater by either PCR method (96% of samples), with well-correlated results between the analyses (R2 = 0.96) indicating a density range of 5.2-7.9 log10 genome copies/L. These concentrations are greater than typically reported in centralized municipal wastewater, yet agree well with an epidemiology-based model previously used to develop pathogen log-reduction targets (LRTs) for decentralized non-potable water systems. Results emphasize the unique quality of onsite wastewaters, supporting the previous LRTs and further quantitative microbial risk assessment (QMRA) of decentralized water reuse.

Triclosan-Selected Host-Associated Microbiota Perform Xenobiotic Biotransformations in Larval Zebrafish.

Microbiota regulate important physiologic processes during early host development. They also biotransform xenobiotics and serve as key intermediaries for chemical exposure. Antimicrobial agents in the environment may disrupt these complex interactions and alter key metabolic functions provided by host-associated microbiota. To examine the role of microbiota in xenobiotic metabolism, we exposed zebrafish larvae to the antimicrobial agent triclosan. Conventionally colonized (CC), microbe-free axenic (AX), or axenic colonized on day 1 (AC1) zebrafish were exposed to 0.16-0.30 µM triclosan or vehicle on days 1, 6, 7, 8, and 9 days post fertilization (dpf). After 6 and 10 dpf, host-associated microbial community structure and putative function were assessed by 16S rRNA gene sequencing. At 10 dpf, triclosan exposure selected for bacterial taxa, including Rheinheimera. Triclosan-selected microbes were predicted to be enriched in pathways related to mechanisms of antibiotic resistance, sulfonation, oxidative stress, and drug metabolism. Furthermore, at 10 dpf, colonized zebrafish contained 2.5-3 times more triclosan relative to AX larvae. Nontargeted chemical analysis revealed that, relative to AX larvae, both cohorts of colonized larvae showed elevations in 23 chemical features, including parent triclosan and putative triclosan sulfate. Taken together, these data suggest that triclosan exposure selects for microbes that harbor the capacity to biotransform triclosan into chemical metabolites with unknown toxicity profiles. More broadly, these data support the concept that microbiota modify the toxicokinetics of xenobiotic exposure.

Microbiota alter metabolism and mediate neurodevelopmental toxicity of 17ß-estradiol.

Estrogenic chemicals are widespread environmental contaminants associated with diverse health and ecological effects. During early vertebrate development, estrogen receptor signaling is critical for many different physiologic responses, including nervous system function. Recently, host-associated microbiota have been shown to influence neurodevelopment. Here, we hypothesized that microbiota may biotransform exogenous 17-ßestradiol (E2) and modify E2 effects on swimming behavior. Colonized zebrafish were continuously exposed to non-teratogenic E2 concentrations from 1 to 10 days post-fertilization (dpf). Changes in microbial composition and predicted metagenomic function were evaluated. Locomotor activity was assessed in colonized and axenic (microbe-free) zebrafish exposed to E2 using a standard light/dark behavioral assay. Zebrafish tissue was collected for chemistry analyses. While E2 exposure did not alter microbial composition or putative function, colonized E2-exposed larvae showed reduced locomotor activity in the light, in contrast to axenic E2-exposed larvae, which exhibited normal behavior. Measured E2 concentrations were significantly higher in axenic relative to colonized zebrafish. Integrated peak area for putative sulfonated and glucuronidated E2 metabolites showed a similar trend. These data demonstrate that E2 locomotor effects in the light phase are dependent on the presence of microbiota and suggest that microbiota influence chemical E2 toxicokinetics. More broadly, this work supports the concept that microbial colonization status may influence chemical toxicity.

Host Developmental Toxicity of BPA and BPA Alternatives Is Inversely Related to Microbiota Disruption in Zebrafish.

Host-associated microbiota can biotransform xenobiotics, mediate health effects of chemical exposure, and play important roles in early development. Bisphenol A (BPA) is a widespread environmental chemical that has been associated with adverse endocrine and neurodevelopmental effects, some of which may be mediated by microbiota. Growing public concern over the safety of BPA has resulted in its replacement with structurally similar alternatives. In this study, we evaluated whether BPA and BPA alternatives alter microbiota and modulate secondary adverse behavioral effects in zebrafish. Zebrafish were developmentally exposed to BPA, Bisphenol AF (BPAF), Bisphenol B (BPB), Bisphenol F (BPF), or Bisphenol S (BPS). At 10 days post fertilization (dpf), toxicity assessments were completed and 16S rRNA gene sequencing was performed to evaluate potential chemical-dependent shifts in microbial community structure and predicted function. A standard light/dark behavioral assay was used to assess locomotor activity. Based on developmental toxicity assessments at 10 dpf, a range of potencies was observed: BPAF > BPB > BPF ∼ BPA > BPS. Analysis of 16S rRNA gene sequencing data showed significant concentration-dependent disruption of microbial community structure and enrichment of putative microbial functions with exposure to BPS, BPA, or BPF, but not BPB or BPAF. Interestingly, microbial disruption was inversely related to host developmental toxicity and estrogenicity. Exposure to BP analogs did not cause behavioral effects at 10 dpf. Our findings indicate that some BP analogs disrupt host microbiota early in life and demonstrate novel chemical-microbiota interactions that may add important context to current hazard identification strategies.