Examining the stability of viral RNA and DNA in wastewater: Effects of storage time, temperature, and freeze-thaw cycles.

Wastewater-based epidemiology (WBE) has been demonstrably successful as a relatively unbiased tool for monitoring levels of SARS-CoV-2 virus circulating in communities during the COVID-19 pandemic. Accumulated biobanks of wastewater samples allow retrospective exploration of spatial and temporal trends for public health indicators such as chemicals, viruses, antimicrobial resistance genes, and the possible emergence of novel human or zoonotic pathogens. We investigated virus resilience to time, temperature, and freeze-thaw cycles, plus the optimal storage conditions to maintain the stability of genetic material (RNA/DNA) of viral +ssRNA (Envelope - E, Nucleocapsid - N and Spike protein - S genes of SARS-CoV-2), dsRNA (Phi6 phage) and circular dsDNA (crAssphage) in wastewater. Samples consisted of (i) processed and extracted wastewater samples, (ii) processed and extracted distilled water samples, and (iii) raw, unprocessed wastewater samples. Samples were stored at -80 °C, -20 °C, 4 °C, or 20 °C for 10 days, going through up to 10 freeze-thaw cycles (once per day). Sample stability was measured using reverse transcription quantitative PCR, quantitative PCR, automated electrophoresis, and short-read whole genome sequencing. Exploring different areas of the SARS-CoV-2 genome demonstrated that the S gene in processed and extracted samples showed greater sensitivity to freeze-thaw cycles than the E or N genes. Investigating surrogate and normalisation viruses showed that Phi6 remains a stable comparison for SARS-CoV-2 in a laboratory setting and crAssphage was relatively resilient to temperature variation. Recovery of SARS-CoV-2 in raw unprocessed samples was significantly greater when stored at 4 °C, which was supported by the sequencing data for all viruses - both time and freeze-thaw cycles negatively impacted sequencing metrics. Historical extracts stored at -80 °C that were re-quantified 12, 14 and 16 months after original quantification showed no major changes. This study highlights the importance of the fast processing and extraction of wastewater samples, following which viruses are relatively robust to storage at a range of temperatures.

National-scale antimicrobial resistance surveillance in wastewater: A comparative analysis of HT qPCR and metagenomic approaches.

Wastewater serves as an important reservoir of antimicrobial resistance (AMR), and its surveillance can provide insights into population-level trends in AMR to inform public health policy. This study compared two common high-throughput screening approaches, namely (i) high-throughput quantitative PCR (HT qPCR), targeting 73 antimicrobial resistance genes, and (ii) metagenomic sequencing. Weekly composite samples of wastewater influent were taken from 47 wastewater treatment plants (WWTPs) across Wales, as part of a national AMR surveillance programme, alongside 4 weeks of daily wastewater effluent samples from a large municipal hospital. Metagenomic analysis provided more comprehensive resistome coverage, detecting 545 genes compared to the targeted 73 genes by HT qPCR. It further provided contextual information critical to risk assessment (i.e. potential bacterial hosts). In contrast, HT qPCR exhibited higher sensitivity, quantifying all targeted genes including those of clinical relevance present at low abundance. When limited to the HT qPCR target genes, both methods were able to reflect the spatiotemporal dynamics of the complete metagenomic resistome, distinguishing that of the hospital and the WWTPs. Both approaches revealed correlations between resistome compositional shifts and environmental variables like ammonium wastewater concentration, though differed in their interpretation of some potential influencing factors. Overall, metagenomics provides more comprehensive resistome profiling, while qPCR permits sensitive quantification of genes significant to clinical resistance. We highlight the importance of selecting appropriate methodologies aligned to surveillance aims to guide the development of effective wastewater-based AMR monitoring programmes.

Leaf litter processing and exoenzyme production on leaves in streams of different pH.

We examined microbial colonization, exoenzyme activity, and processing of leaves of yellow poplar (Liriodendron tulipifera), red maple (Acer rubrum), and white oak (Quercus alba) in three streams on the Allegheny Plateau of West Virginia, United States. Leaf packs were placed in streams that varied in their underlying bedrock geology, and therefore in their sensitivity to the high level of acidic precipitation that occurs in this region. The mean pH of the streams was 4.3 in the South Fork of Red Run (SFR), 6.2 in Wilson Hollow Run (WHR), and 7.7 in the North Fork of Hickman Slide Run (HSR). Through time, the patterns of microbial biomass and exoenzyme activity were generally similar among leaf species, but the magnitude of microbial biomass and exoenzyme activity differed among leaf species. Pectinase activity was greatest in HSR, the most alkaline stream, whereas the activity of exocellulase and xylanase was greatest in WHR and SFR, the intermediate and acidic streams. This variation in the activity of different exoenzymes was consistent with published pH optima for these exoenzymes. Variation in processing rates, both among leaf species and among streams, seems to be related to the level of microbial exoenzyme activity on the leaf detritus.

Pneumoretroperitoneum in two patients with Clostridium perfringens necrotizing pancreatitis.

Pancreatic gas gangrene is an uncommon and often fatal complication of acute pancreatitis, due to the sporulating anaerobe Clostridium perfringens. C. perfringens is a normal constituent of colonic flora, but infects the pancreas by either transmural spread from the colon or via the biliary tree. Only three reported cases in the world literature describe acute pancreatitis with pneumoretroperitoneum and clostridial infection. Two separate cases, at the same institution, of acute pancreatitis complicated by C. perfringens were analyzed. The records of patients were reviewed for admission history, laboratory and radiology results, intensive care support, surgical intervention, and outcome. Retroperitoneal air was visualized early in the clinical course of both patients by computed tomography. Early surgical debridement, drainage, parental antibiotics, and reexploration resulted in an uncomplicated recovery. Early computed tomography in patients with suspected necrotizing pancreatitis contributes to early intervention and may advantageously enhance survival.

Everglades restoration and water quality challenges in south Florida.

This paper provides background information and a brief overview of water quality issues for the rest of the papers in this volume that are concerned with Everglades restoration. The Everglades of Florida have been diminished over 50% of their former extent. The Everglades are no longer a free-flowing wetland ecosystem, but are now subject to a complicated system of water management that is regulated primarily for flood control and consumptive use. Attempts to restore a more natural hydropattern to the remaining undeveloped Everglades are made more difficult by the natural extremes in rainfall, flat landscape, highly porous geology, and inaccessibility of the remaining natural areas. The Comprehensive Everglades Restoration Plan (CERP) seeks ecosystem restoration by adding water storage capacity, reducing groundwater seepage, improving regulatory delivery and timing of water to avoid environmental damage, and where feasible, improving the quality of water to be used for Everglades restoration. Water quality issues that currently exist for south Florida include eutrophication (especially phosphorus), mercury, and contaminants from agricultural production and the urban environment. Lands once in agricultural production that will be converted back to wetlands or will become reservoirs may contribute to the water quality concerns. Stormwater runoff from managed lands that will be used for restoration purposes will also present water quality challenges. The state continues to seek water quality improvement with a number of pollution reduction programs, and CERP attempts to improve water quality without sacrificing even more natural areas; however providing water quality sufficient for use in recovery of remaining Everglades wetlands and estuaries will remain a daunting challenge.