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  1. Abstract Background There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. Results The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75–90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40–73 to 31–68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35–13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4–2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating verticalmore »gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. Conclusions Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention.« less
    Free, publicly-accessible full text available December 1, 2023
  2. Free, publicly-accessible full text available July 14, 2023
  3. We conducted a critical review to establish what is known about the sources, characteristics, and dissemination of ARGs in the atmosphere. We identified 52 papers that reported direct measurements of bacterial ARGs in air samples and met other inclusion criteria. The settings of the studies fell into the following categories: urban, rural, hospital, industrial, wastewater treatment plants (WWTPs), composting and landfill sites, and indoor environments. Certain genes were commonly studied and generally abundant: sul1 , intI1 , β-lactam ARGs, and tetracycline ARGs. Abundances of total ARGs varied by season and setting, with air in urban areas having higher ARG abundance than rural areas during the summer and vice versa during the winter. There was greater consistency in the types and abundances of ARGs throughout the seasons in urban areas. Human activity within indoor environments was also linked to increased ARG content (abundance, diversity, and concentration) in the air. Several studies found that human exposure to ARGs through inhalation was comparable to exposure through drinking water or ingesting soil. Detection of ARGs in air is a developing field, and differences in sampling and analysis methods reflect the many possible approaches to studying ARGs in air and make direct comparisons between studiesmore »difficult. Methodologies need to be standardized to facilitate identification of the dominant ARGs in the air, determine their major sources, and quantify the role of atmospheric transport in dissemination of ARGs in the environment. With such knowledge we can develop better policies and guidelines to limit the spread of antimicrobial resistance.« less
    Free, publicly-accessible full text available June 22, 2023
  4. Antibiotic resistance is a continually rising threat to global health. A primary driver of the evolution of new strains of resistant pathogens is the horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). However, identifying and quantifying ARGs subject to HGT remains a significant challenge. Here, we introduce HT-ARGfinder (horizontally transferred ARG finder), a pipeline that detects and enumerates horizontally transferred ARGs in metagenomic data while also estimating the directionality of transfer. To demonstrate the pipeline, we applied it to an array of publicly-available wastewater metagenomes, including hospital sewage. We compare the horizontally transferred ARGs detected across various sample types and estimate their directionality of transfer among donors and recipients. This study introduces a comprehensive tool to track mobile ARGs in wastewater and other aquatic environments.
    Free, publicly-accessible full text available June 3, 2023
  5. Free, publicly-accessible full text available May 19, 2023
  6. To evaluate the use of wastewater-based surveillance and epidemiology to monitor and predict SARS-CoV-2 virus trends, over the 2020–2021 academic year we collected wastewater samples twice weekly from 17 manholes across Virginia Tech’s main campus. We used data from external door swipe card readers and student isolation/quarantine status to estimate building-specific occupancy and COVID-19 case counts at a daily resolution. After analyzing 673 wastewater samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR), we reanalyzed 329 samples from isolation and nonisolation dormitories and the campus sewage outflow using reverse transcription digital droplet polymerase chain reaction (RT-ddPCR). Population-adjusted viral copy means from isolation dormitory wastewater were 48% and 66% higher than unadjusted viral copy means for N and E genes (1846/100 mL to 2733/100 mL/100 people and 2312/100 mL to 3828/100 mL/100 people, respectively; n = 46). Prespecified analyses with random-effects Poisson regression and dormitory/cluster-robust standard errors showed that the detection of N and E genes were associated with increases of 85% and 99% in the likelihood of COVID-19 cases 8 days later (incident–rate ratio (IRR) = 1.845, p = 0.013 and IRR = 1.994, p = 0.007, respectively; n = 215), and one-log increases in swipe card normalized viral copiesmore »(copies/100 mL/100 people) for N and E were associated with increases of 21% and 27% in the likelihood of observing COVID-19 cases 8 days following sample collection (IRR = 1.206, p < 0.001, n = 211 for N; IRR = 1.265, p < 0.001, n = 211 for E). One-log increases in swipe normalized copies were also associated with 40% and 43% increases in the likelihood of observing COVID-19 cases 5 days after sample collection (IRR = 1.403, p = 0.002, n = 212 for N; IRR = 1.426, p < 0.001, n = 212 for E). Our findings highlight the use of building-specific occupancy data and add to the evidence for the potential of wastewater-based epidemiology to predict COVID-19 trends at subsewershed scales.« less
    Free, publicly-accessible full text available May 13, 2023
  7. Free, publicly-accessible full text available March 1, 2023
  8. Antibiotic resistance genes (ARGs) are commonly detected in the atmosphere, but questions remain regarding their sources and relative contributions, bacterial hosts, and corresponding human health risks. Here, we conducted a qPCR- and metagenomics-based investigation of inhalable fine particulate matter (PM2.5) at a large wastewater treatment plant (WWTP) and in the ambient air of Hong Kong, together with an in-depth analysis of published data of other potential sources in the area. PM2.5 was observed with increasing enrichment of total ARGs along the coastal–urban–WWTP gradient and clinically relevant ARGs commonly identified in urban and WWTP sites, illustrating anthropogenic impacts on the atmospheric accumulation of ARGs. With certain kinds of putative antibiotic-resistant pathogens detected in urban and WWTP PM2.5, a comparable proportion of ARGs that co-occurred with MGEs was found between the atmosphere and WWTP matrices. Despite similar emission rates of bacteria and ARGs within each WWTP matrix, about 11–13% of the bacteria and >57% of the relevant ARGs in urban and WWTP PM2.5 were attributable to WWTPs. Our study highlights the importance of WWTPs in disseminating bacteria and ARGs to the ambient air from a quantitative perspective and, thus, the need to control potential sources of inhalation exposure to protect the healthmore »of urban populations.« less
    Free, publicly-accessible full text available January 1, 2023
  9. Free, publicly-accessible full text available November 15, 2022
  10. Free, publicly-accessible full text available November 1, 2022