Coprostanol as a Population Biomarker for SARS-CoV-2 Wastewater Surveillance Studies

Wastewater surveillance is a cost-effective tool for monitoring SARS-CoV-2 transmission in a community. However, challenges remain with regard to interpretating such studies, not least in how to compare SARS-CoV-2 levels between different-sized wastewater treatment plants. Viral faecal indicators, including crAssphage and pepper mild mottle virus, have been proposed as population biomarkers to normalise SARS-CoV-2 levels in wastewater. However, as these indicators exhibit variability between individuals and may not be excreted by everyone, their utility as population biomarkers may be limited. Coprostanol, meanwhile, is a bacterial metabolite of cholesterol which is excreted by all individuals. In this study, composite influent samples were collected from a large- and medium-sized wastewater treatment plant in Dublin, Ireland and SARS-CoV-2 N1, crAssphage, pepper mild mottle virus, HF183 and coprostanol levels were determined. SARS-CoV-2 N1 RNA was detected and quantified in all samples from both treatment plants. Regardless of treatment plant size, coprostanol levels exhibited the lowest variation in composite influent samples, while crAssphage exhibited the greatest variation. Moreover, the strongest correlations were observed between SARS-CoV-2 levels and national and Dublin COVID-19 cases when levels were normalised to coprostanol. This work demonstrates the usefulness of coprostanol as a population biomarker for wastewater surveillance studies.

Flood and rainfall mobilisation of E. coli and faecal source tracking markers from decomposing cowpats

<p>The intensification of dairy farming on the agricultural landscape in New Zealand has raised concerns about pollution sources from dairy faecal runoff into waterways. The transport of faecal pollution from farms into waterways is facilitated by overland flow, which can result from rain and flood events, poorly designed irrigation practices and the washing down of milking sheds.</p><p>An important step for mitigation of pollution is the identification of the source(s) of faecal contamination. When elevated levels of faecal indicator bacteria (FIB) such as <em>Escherichia coli </em>are identified in a waterway, faecal source tracking (FST) tools such as microbial source tracking (MST) using quantitative polymerase chain reaction (qPCR), and faecal steroids (for example, cholesterol) provide information about the sources of faecal contamination. The understanding of the fate (degradation/persistence) and transport of these FST markers in the environment is recognised as an important requirement for the interpretation of water quality monitoring in aquatic environments.</p><p>This study investigated the effects of faecal decomposition on bovine faecal indicators (<em>E. coli </em>and FST markers: bovine-associated qPCR markers and ten faecal steroids) by monitoring the effect of flood and rainfall events on simulated cowpats over a five and a half month period under field conditions. Two separate spring/summer trials were conducted to evaluate: Trial 1) the mobilisation under simulated flood conditions of the faecal indicators from irrigated versus non-irrigated cowpats, Trial 2) the mobilisation of faecal indicators from non-irrigated cowpat flood runoff versus runoff after simulated rainfall onto non-irrigated cowpats.</p><p>The microbial community changes within the decomposing cowpat (as illustrated by amplicon-based metagenomic analysis) were expected to impact on the survival/persistence of the bacterial targets of the MST markers, and also alter the ratio between faecal sterols and their biodegradation products, the stanols. It was hypothesised, therefore, that there would be:</p><ul><li>Changes over time in the concentration of<em> E. coli </em>and the bovine-associated MST markers mobilised into the cowpat runoff</li> <li>Alterations in the FST ratio signature of the ten measured faecal steroids, resulting in a change from a bovine faecal steroid signature in fresh cowpat runoff to other animal faecal signatures in the runoff from decomposing cowpats</li> <li>A difference in the mobilisation decline rates of all FST and microbial markers within a treatment regime and between treatments.</li> </ul><p>Linear regression analysis was undertaken to establish mobilisation decline rates for each of the analytes in the mobilisable phase from the cowpat runoff treatments, with calculation of the time taken in days for reduction in 90% of the concentration (T<sub>90</sub>), and statistical comparison of the regression coefficients (slopes) of all analytes. The results will include a discussion of the impacts of the study’s observations on the interpretation of faecal indicator assessments for water quality monitoring in waterways influenced by sources of faecal contamination.</p>

Removal performance of faecal indicators by natural and silver-modified zeolites under dynamic batch experiments

<p>The use of natural zeolites (NZs) in waste-water treatment plants is one of the oldest and most promising applications.  Modified natural zeolites (MZs) have shown improved ion exchange and adsorption capacities and have been extensively applied for the removal of pollutants (metal(loid) ions, ammonia etc) from aqueous solutions. However, MZs application in biological pollutants such as indicator organisms or pathogens has not been extensively explored. This study examines the antimicrobial effect of both natural Greek zeolite (NZ), with clinoptilolite content up to 85% (OLYMPOS SA), and modified Greek zeolite by incorporation with silver ions (Ag-MNZ) on the survival of two selected bacteria. The chosen organisms, <em>Escherichia coli </em>and <em>Enterococcus faecalis</em>, constitute indicators of fecal contamination in both soils and water. Scanning electron microscopy and energy dispersive X-ray detection (SEM-EDX) were used for the surface morphology and elemental composition of the NZ and Ag-MNZ samples, respectively. A series of dynamic batch experiments were conducted at constant room temperature (22°C) in order to examine the inactivation of the above bacteria by NZ and Ag-MNZ.  It was found that the Ag-MNZ resulted in much higher reduction of the bacterial numbers when compared to the NZ and control (absence of zeolites). Moreover, the reduction in the bacterial numbers was affected by NZ particle size with higher reduction observed for coarse (1-3 mm) than fine (0-1) NZ. Finally, the<em> E. faecalis</em> was found to be more resistant than <em>E.coli </em>to Ag-MNZ.</p>

Tryptophan-like and humic-like fluorophores are extracellular in groundwater: implications as real-time faecal indicators

Fluorescent natural organic matter at tryptophan-like (TLF) and humic-like fluorescence (HLF) peaks is associated with the presence and enumeration of faecal indicator bacteria in groundwater. We hypothesise, however, that it is predominantly extracellular material that fluoresces at these wavelengths, not bacterial cells. We quantified total (unfiltered) and extracellular (filtered at < 0.22 µm) TLF and HLF in 140 groundwater sources across a range of urban population densities in Kenya, Malawi, Senegal, and Uganda. Where changes in fluorescence occurred following filtration they were correlated with potential controlling variables. A significant reduction in TLF following filtration (ΔTLF) was observed across the entire dataset, although the majority of the signal remained and thus considered extracellular (median 96.9%). ΔTLF was only significant in more urbanised study areas where TLF was greatest. Beneath Dakar, Senegal, ΔTLF was significantly correlated to total bacterial cells (ρs 0.51). No significant change in HLF following filtration across all data indicates these fluorophores are extracellular. Our results suggest that TLF and HLF are more mobile than faecal indicator bacteria and larger pathogens in groundwater, as the predominantly extracellular fluorophores are less prone to straining. Consequently, TLF/HLF are more precautionary indicators of microbial risks than faecal indicator bacteria in groundwater-derived drinking water.

Microbiological characterisation and impact of suspended solids on pathogen removal from wastewaters in dairy processing factories

In this Research Communication we investigate the microbiological profile of 12 dairy wastewater streams from three contrasting Irish dairy processing factories to determine whether faecal indicators/pathogens were present and in turn, whether disinfection may be required for potential water reuse within the factory. Subsequently, the impact of suspended solids on the inactivation efficiency of Escherichia coli via two means of ultravoilet (UV) disinfection; flow-through pulsed UV (PUV) and continuous low pressure UV (LPUV) disinfection was analysed. Faecal indicators total coliforms and E. coli were detected in 10 out of the 12 samples collected at the dairy processing factories while pathogenic bacteria Listeria monocytogenes was detected in all samples collected at 2 out of the 3 factories. Salmonella spp. was undetected in all samples. The results also indicated that organic dairy wastewater solids had an impact on the performance efficiency of the PUV system and, to a lesser extent, the LPUV system. The findings indicate that the targeting of key pathogens would be required to enable wastewater reuse (and indeed effluent discharges if regulation continues to become more stringent) and that LPUV may offer a more robust disinfection method as it appears to be less susceptible to the presence of suspended solids.