Monitoring SARS-CoV-2 in sewage: toward sentinels with analytical accuracy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effect of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested by electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

Activation of innate immunity by neutral polysaccharides from broccoli buds

Edible substances that stimulate the innate immune system are good candidates for functional foods to improve human health. We have previously reported that acidic polysaccharides from broccoli extract exhibit immunostimulatory effects, but neutral polysaccharides have been overlooked. In the present study, we found that neutral polysaccharides have significantly stronger (higher specific activity) immunostimulatory activity than acidic polysaccharides. The hot water extract of broccoli showed the immunostimulatory activity in the silkworm muscle contraction assay, suggesting that it stimulates innate immunity via paralytic peptide pathway. The activity was concentrated in the buds, but not in the stems and stalk. The active substance was recovered in the flow-through fraction of diethylaminoethyl-cellulose column chromatography with neutral polysaccharides. The specific activity of the fraction was significantly higher than that of the acidic polysaccharides from broccoli reported previously. These results suggest that the neutral polysaccharide present in broccoli buds stimulates innate immunity and can be semi-purified by one-step chromatography.

Prospective application of diethylaminoethyl cellulose (DEAE-cellulose) with a high adsorption capacity toward the detoxification of 2,4-dichlorophenoxyacetic acid (2,4-D) from water

Dextoxification of 2,4-D was achieved using DEAE-cellulose.

A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

Antibiotic resistant genes (ARGs) and mobile genetic elements (MGEs) can be found in the free-floating extracellular DNA (eDNA) fraction of microbial systems. These xenogenic components can generate bacterial cells resistant to one or more antibiotics by natural transformation. Because of low concentration in wastewater, the obtaining of a high quality and a high yield of eDNA extract is challenging. We developed a method using chromatography to isolate eDNA without causing cell lysis (often unchecked) from complex wastewater matrices. The chromatographic step involved a diethylaminoethyl-cellulose-monolithic column to capture the eDNA found in cell-free filtered wastewater samples (e.g. influent wastewater, activated sludge and treated effluent wastewaster). Free-floating eDNA yields from 1 L of influent, activated sludge and treated effluent water reached 12.5 ± 1.9 μg, 12.3 ± 1 μg and 5.6 ± 2.9 μg of raw eDNA and 9.0 ± 0.7 μg, 5.6 ± 0.46 μg and 2.6 ± 1.3 μg of purified eDNA, respectively. In order to check the suitability of free-floating eDNA extracts for molecular analysis, qPCR and metagenomics were performed. eDNA extracts from treated effluent water were analyzed by qPCR to quantify a selected panel of ARGs and MGEs. Microbiome, resistome, and mobilome profiles from activated sludge free-floating eDNA were measured by metagenomic sequencing. Between iDNA and eDNA fractions, qPCR showed differences of 0.94, 1.11, 1.92 and 1.32 log10 gene copies mL−1 for sulfonamides resistant genes (sul1 and sul2), β-lactamase resistance gene blaCTXM, and the class 1 integron-integrase (intI1) MGE, respectively. These differences highlighted the crucial need for an isolation method to discern both iDNA and eDNA to understand ARGs persistence and quantity in complex cultures. The eDNA yields obtained from 1 L of activated sludge (3.6 g of total suspended solids L−1) samples were substantially higher than the amount of DNA template needed for high-throughput sequencing (>1 μg) in service facilities. Subsystems classification showed that the eDNA metagenome was mainly composed by MGEs (65.1%). The 35.9% rest related to traditional functional genetic signatures. It was the first time the resistome from the eDNA fraction was analyzed showing lower number of primary aligned reads when compared to the iDNA and a predominance of aminoglycosides and β-lactamams. Metagenome results showed that eDNA can not be discarded as a pool of ARGs and MGEs for horizontal gene transfer. This novel isolation method was powerful to elucidate the molecular compositions of free-floating eDNA fractions in complex environmental samples such as wastewater environments at different microbial densities. Data obtained using this extraction method will foster xenogenic and microbial risk assessments across urban and natural water systems. This will support water authorities in the delineation of measures to adopt at wastewater treatment plants to remove them and safeguard environmental and public health.Graphical abstractPicture created with BioRender