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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 1, 2023
  3. 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
  4. Free, publicly-accessible full text available November 1, 2022