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.

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.

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.