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  1. Abstract

    Wastewater surveillance has proven to be an effective tool to monitor the transmission and emergence of infectious agents at a community scale. Workflows for wastewater surveillance generally rely on concentration steps to increase the probability of detection of low-abundance targets, but preconcentration can substantially increase the time and cost of analyses while also introducing additional loss of target during processing. To address some of these issues, we conducted a longitudinal study implementing a simplified workflow for SARS-CoV-2 detection from wastewater, using a direct column-based extraction approach. Composite influent wastewater samples were collected weekly for 1 year between June 2020 and June 2021 in Athens-Clarke County, Georgia, USA. Bypassing any concentration step, low volumes (280 µl) of influent wastewater were extracted using a commercial kit, and immediately analyzed by RT-qPCR for the SARS-CoV-2 N1 and N2 gene targets. SARS-CoV-2 viral RNA was detected in 76% (193/254) of influent samples, and the recovery of the surrogate bovine coronavirus was 42% (IQR: 28%, 59%). N1 and N2 assay positivity, viral concentration, and flow-adjusted daily viral load correlated significantly with per-capita case reports of COVID-19 at the county-level (ρ = 0.69–0.82). To compensate for the method’s high limit of detection (approximately 106–107 copies l−1 in wastewater), we extracted multiple small-volume replicates of each wastewater sample. With this approach, we detected as few as five cases of COVID-19 per 100 000 individuals. These results indicate that a direct-extraction-based workflow for SARS-CoV-2 wastewater surveillance can provide informative and actionable results.

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  2. Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has demonstrated useful correlation with both coronavirus disease 2019 (COVID-19) cases and clinical testing positivity at the community level. Wastewater surveillance on college campuses has also demonstrated promising predictive capacity for the presence and absence of COVID-19 cases. However, to date, such monitoring has most frequently relied upon composite samplers and reverse transcription quantitative PCR (RT-qPCR) techniques, which limits the accessibility and scalability of wastewater surveillance, particularly in low-resource settings. In this study, we trialed the use of tampons as passive swabs for sample collection and reverse transcription loop-mediated isothermal amplification (RT-LAMP), which does not require sophisticated thermal cycling equipment, to detect SARS-CoV-2 RNA in wastewater. Results for the workflow were available within three hours of sample collection. The RT-LAMP assay is approximately 20 times less analytically sensitive than RT-droplet digital PCR. Nonetheless, during a building-level wastewater surveillance campaign concurrent with independent weekly clinical testing of all students, the method demonstrated a three-day positive predictive value (PPV) of 75% (excluding convalescent cases) and same-day negative predictive value (NPV) of 80% for incident COVID-19 cases. These predictive values are comparable to that reported by wastewater monitoring using RT-qPCR. These observations suggest that even with lower analytical sensitivity the tampon swab and RT-LAMP workflow offers a cost-effective and rapid approach that could be leveraged for scalable building-level wastewater surveillance for COVID-19 potentially even in low-resource settings. 
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  3. Reptile-associated human salmonellosis cases have increased recently in the United States. It is not uncommon to find healthy chelonians shedding Salmonella enterica . The rate and frequency of bacterial shedding are not fully understood, and most studies have focused on captive vs. free-living chelonians and often in relation to an outbreak. Their ecology and significance as sentinels are important to understanding Salmonella transmission. In 2012–2013, Salmonella prevalence was determined for free-living aquatic turtles in man-made ponds in Clarke and Oconee Counties, in northern Georgia (USA) and the correlation between species, basking ecology, demographics (age/sex), season, or landcover with prevalence was assessed. The genetic relatedness between turtle and archived, human isolates, as well as, other archived animal and water isolates reported from this study area was examined. Salmonella was isolated from 45 of 194 turtles (23.2%, range 14–100%) across six species. Prevalence was higher in juveniles (36%) than adults (20%), higher in females (33%) than males (18%), and higher in bottom-dwelling species (31%; common and loggerhead musk turtles, common snapping turtles) than basking species (15%; sliders, painted turtles). Salmonella prevalence decreased as forest cover, canopy cover, and distance from roads increased. Prevalence was also higher in low-density, residential areas that have 20–49% impervious surface. A total of 9 different serovars of two subspecies were isolated including 3 S. enterica subsp. arizonae and 44 S. enterica subsp. enterica (two turtles had two serotypes isolated from each). Among the S. enterica serovars, Montevideo ( n = 13) and Rubislaw ( n = 11) were predominant. Salmonella serovars Muenchen, Newport, Mississippi, Inverness, Brazil, and Paratyphi B. var L(+) tartrate positive (Java) were also isolated. Importantly, 85% of the turtle isolates matched pulsed-field gel electrophoresis patterns of human isolates, including those reported from Georgia. Collectively, these results suggest that turtles accumulate Salmonella present in water bodies, and they may be effective sentinels of environmental contamination. Ultimately, the Salmonella prevalence rates in wild aquatic turtles, especially those strains shared with humans, highlight a significant public health concern. 
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  4. Abstract

    Deposition of aerosolized desert dust can affect marine microbial community structure and function through pulsed addition of limiting micro‐ and macronutrients. However, few studies have captured responses to dust deposition in situ following trans‐oceanic transport. We conducted a 26‐d time series evaluating biogeochemical and microbial community response to Saharan dust deposition in surface waters in the subtropical western Atlantic (Florida Keys National Marine Sanctuary, U.S.A.). Following periods of elevated atmospheric dust concentrations, particulate and dissolved iron concentrations increased in surface waters. Autotrophic picoeukaryote abundance increased rapidly, followed by increases in the abundance of heterotrophic bacteria andSynechococcus. Concomitant to cell count changes, we observed successional shifts in bacterial community composition. The relative abundances ofProchlorococcusandPelagibacterdeclined with dust arrival, while relative abundance of heterotrophic bacteria increased, beginning with Vibrionales and followed sequentially by Chrysophyceae, Rhodobacteriaceae, and Flavobacteriaceae. Finally, a peak inSynechococcuscyanobacteria was observed. These results provide new insight into microbial community succession in response to Saharan dust deposition, their association with temporal dynamics in surface water dissolved and particulate iron concentrations, and a potential role for bioprocessing of dust particles in shaping marine microbial responses to deposition events.

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