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Prophage Activation as an Overlooked Mechanism Underlying the Biocidal Effect of Free Nitrous Acid in Sewers.

Biogenic hydrogen sulfide produced in sewer systems causes odor nuisance and concrete corrosion, necessitating effective biocidal control. Free nitrous acid (FNA) has emerged as a promising biocide, but its unclear mechanisms complicate dosage optimization and risk assessment. Here, using Desulfovibrio vulgaris as a model lysogenic bacterium, we demonstrate that low-dose FNA (0.2-4.0 mg N/L) induces bacterial inactivation via prophage activation-associated lysis in addition to chemical oxidation. Reactive nitrogen species (RNS) scavenging tests revealed that RNS-mediated oxidative stress was closely associated with prophage activation. Activated phages further infected new hosts, reducing the viability of freshly cultured D. vulgaris cells by 25.7% and increasing total phage production 15.2-fold. The phenomenon was further validated in real sewage biofilms, where increased phage production and decreased bacterial viability were observed at a low FNA dose of 0.2 mg N/L, below the dose required for cell destruction by chemical oxidation. Furthermore, metagenomic analysis of 896 sewage samples worldwide revealed that 71.9% of recovered sewage-derived microbial genomes harbor prophages, indicating a widespread genomic basis for prophage activation-mediated bacterial inactivation. Overall, this study expands current understanding of the biocidal mechanisms of FNA and contributes to the development of environmentally sustainable biocidal strategies.

Nitrous Acid

Modelling peak microbial pollution events caused by combined sewer overflows in a source-to-sea system.

Predicting peak microbial pollution events in downstream coastal bathing waters caused by combined sewer overflows (CSOs) is essential for protecting public health. In urban areas, wastewater effluents, CSOs, and surface runoff can contribute to elevated microorganism loads to downstream waters. These pressures are likely to be intensified by growing population density and more frequent heavy rainfalls due to climate change. This study developed a process-based model to simulate Escherichia coli (E. coli) emissions, transport, and fate from the initial sources to coastal beaches. A three-year retrospective simulation (2017-2019) shows that E. coli concentrations in CSO discharges varied widely across the catchment (4.6 - 7.3 (log10 CFU 100 ml-1)). 99th percentile E. coli concentrations (4.0 (log10 CFU 100 ml-1)) at the inland water outlet were dominated by local CSO emissions, whereas 90th percentile E. coli concentrations (3.6 (log10 CFU 100 ml-1)) reflected cumulative upstream contributions from both CSO and effluent emissions. With the simulation accuracy of 89%, the model reliably reproduced the E. coli dynamics on the downstream beach and showed strong performance in representing peak concentrations based on Complementary Cumulative Distribution Function (CCDF) analysis. The process-based model enables quantitative tracking of source contributions and identification of pollution hotspots, providing support for mitigation measures. The study lays down a source-to-sea modelling framework for representing pollution transport across the aquatic continuum and provides a transferable tool for microbial pollution forecasting and climate adaptation planning.

Climate projection

Inclusion of Physical-Chemical Water Quality Measurements Can Improve Associations between SARS-CoV-2 RNA Levels in Wastewater and COVID-19 Cases within Smaller Sewersheds.

Measurements of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater can be used to understand the prevalence of COVID-19 cases within a community. Environmental conditions inclusive of physical-chemical water quality characteristics are known to impact wastewater SARS-CoV-2 signals, but they are rarely measured within the sewer infrastructure in areas upstream of wastewater treatment plants (WWTPs). The objectives of this study were to report on measurements of environmental parameters [flow and physical-chemical water quality (water temperature, pH, specific conductivity, dissolved oxygen, and turbidity)] upstream of a WWTP and to evaluate whether the inclusion of these environmental parameters improves correlations between SARS-CoV-2 RNA levels in wastewater, and COVID-19 prevalence in the sewershed community. Measurements of environmental parameters and SARS-CoV-2 RNA in wastewater spanned different time scales (minutes, hours and weeks) and population scales (building, campus, community). For short time scales, water quality parameters did not improve correlations between SARS-CoV-2 in wastewater and COVID-19 prevalence due to high variability of water quality and flows within the sewer system. When averaging data over weekly time scales, regressions showed that inclusion of pH improved correlations between RNA and COVID-19 prevalence. At the cluster scale, for the entire data set, the root mean square error decreased from 6.9 cases per week to 6.5 cases per week. At the community scale benefits were observed only for the delta wave with a decrease in root mean square error from 539 cases per week to 430 cases per week. The inclusion of pH improved correlations between wastewater SARS-CoV-2 and COVID-19 prevalence more frequently when evaluating the cluster sewershed scale (populations of a few thousand) in comparison to the community scale (populations of several 100,000). Given the simplicity of measuring pH and other physical-chemical water quality parameters, their inclusion should be considered as part of wastewater-based epidemiology programs.

COVID-19

Cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes.

To prevent vaccine-associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype-2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus-free and cold chain-free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low-cost, cold chain/poliovirus-free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non-toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site-specific integration of CTB-VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9-15-fold in chloroplasts. GM1-ganglioside receptor-binding ELISA confirmed pentamer assembly of CTB-VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1-VLPs of 22.3&#xa0;nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB-VP1co, formulated with plant-derived oral adjuvants, enhanced VP1-specific IgG1, VP1-IgA titres and neutralization (80%-100% seropositivity of Sabin-1, 2, 3). In contrast, IPV single dose resulted in <50% VP1-IgG1 and negligible VP1-IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB-VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low-cost solution to eradicate polio.

Animals

Dynamics and virulence of Enterobacteriaceae reservoirs harboring blaCTX-M group 1 in community wastewater.

UNLABELLED: Extended-spectrum beta-lactamase (ESBL)-producing bacteria are ubiquitous and can cause serious infections. Here, we examined untreated community wastewater influent as a reservoir for blaCTX-M group 1 organisms and their virulence potential. Raw influent samples (n = 268) were collected from four wastewater treatment plants (WWTPs) representing dense urban populations. We observed that blaCTX-M group 1 levels were high at all WWTPs and only ~1-2 log10 lower and not correlated to common human-specific microbiome fecal markers, Lachno3 and HF183, indicating a lack of connection to human fecal inputs. Concentrations of blaCTX-M group 1 genes and markers for presumptive host organisms Escherichia coli and Klebsiella pneumoniae were influenced by travel time and season. Amplicon sequencing revealed high diversity of blaCTX-M group 1-9 genes, with 63% belonging to group 1. Selective culture and 16S rRNA gene sequencing showed blaCTX-M group 1 isolates were 26% E. coli, 26% K. pneumoniae, 40% other Enterobacteriaceae, and 8% Aeromonas. Overall, E. coli averaged 3.6E7 cells/L, with 3% of all E. coli found to contain blaCTX-M group 1. Whole-genome sequencing of blaCTX-M group 1 E. coli from wastewater revealed resistance and virulence gene profiles similar to clinical isolates and distinct from other wastewater ESBL-resistant and non-resistant E. coli. Interpretation of wastewater data needs to consider both the existence of environmental reservoirs that contain potentially pathogenic organisms and the strong influence the dynamics of the conveyance system can have on final concentrations measured at the WWTP. IMPORTANCE: The CTX-M enzyme family is highly abundant in nosocomial, community, and environmental settings and is leading to treatment of infections with carbapenem antibiotics, a last-line therapeutic option. The progressive increase of the clinically relevant blaCTX-M group 1 resistance genes in the human population warrants investigation, particularly to understand the establishment and dynamics of environmental reservoirs. This study utilized molecular and culture methods to gain insight into the possible origin, abundance, and dynamics of blaCTX-M group 1 genes in untreated wastewater influent samples. We found extremely high levels of these genes, with Escherichia coli as a major host organism that closely resembled clinical strains, suggesting they are seeded and propagate in sewer pipe systems. The significance of our research is in developing approaches to monitor antimicrobial resistance reservoirs in community wastewater, which could shed light on global burdens and potential transmission cycles and indicate increasing inputs of clinically relevant strains originating from human populations.

E. coli