Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS^® miniMAG^®, or PowerViral^® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral^® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

Novel foods represent sustainable alternatives to traditional farming and conventional foodstuffs. The house cricket (Acheta domesticus) is considered as one of the most promising reared insects due to their attractive nutritional profile and lower feed conversion ratio compared to other animals. However, putative health hazards associated with consumption of crickets have previously not been investigated. The present study assesses the risks of A. domesticus reared in closed systems controlled by the implementation of hazard analysis and critical control points and good farming practices. Due to the novelty of the topic, data scarcity has been a limiting factor, hence comparative evidence from closely related species belonging to the order Orthoptera (e.g. grasshoppers, locusts, and other cricket species) have been included. The present risk profile identified as main hazards: (1) high total counts of aerobic bacteria; (2) presence of spore-forming bacteria post thermal processing; (3) accumulation of cadmium and other heavy metals; and (4) a possible increase of allergenic reactions due to exposure to insects and insect derived products. Important data gaps regarding edible crickets and their safety as novel foods have been highlighted in the future perspective section, representing aims for future research. Identified data gaps include: (1) farming conditions of the insects being studied; (2) data on the impact of thermal processing of the products prior to consumption; (3) fungal communities and mycotoxins-producing fungi in reared crickets; and (4) heavy metals not fully assessed (chromium, aluminium and arsenic) and other chemical hazards produced during processing (i.e. heterocyclic aromatic amines, acrylamide). The present risk profile explores food safety risks related to consumption of A. domesticus, thereby constituting an example of chemical and microbial hazards risk profiling on edible insects, covering rearing to consumption. Graphical abstract - Risk profile of edible crickets.

This was a detailed investigation of the seasonal occurrence, dynamics, removal and resistance of human-associated genetic Bacteroidetes faecal markers (GeBaM) compared with ISO-based standard faecal indicator bacteria (SFIB), human-specific viral faecal markers and one human-associated Bacteroidetes phage in raw and treated wastewater of municipal and domestic origin. Characteristics of the selected activated sludge wastewater treatment plants (WWTPs) from Austria and Germany were studied in detail (WWTPs, n = 13, connected populations from 3 to 49000 individuals), supported by volume-proportional automated 24-h sampling and chemical water quality analysis. GeBaM were consistently detected in high concentrations in raw (median log10 8.6 marker equivalents (ME) 100 ml(-1)) and biologically treated wastewater samples (median log10 6.2-6.5 ME 100 ml(-1)), irrespective of plant size, type and time of the season (n = 53-65). GeBaM, Escherichia coli, and enterococci concentrations revealed the same range of statistical variability for raw (multiplicative standard deviations s* = 2.3-3.0) and treated wastewater (s* = 3.7-4.5), with increased variability after treatment. Clostridium perfringens spores revealed the lowest variability for raw wastewater (s* = 1.5). In raw wastewater correlations among microbiological parameters were only detectable between GeBaM, C. perfringens and JC polyomaviruses. Statistical associations amongst microbial parameters increased during wastewater treatment. Two plants with advanced treatment were also investigated, revealing a minimum log10 5.0 (10th percentile) reduction of GeBaM in the activated sludge membrane bioreactor, but no reduction of the genetic markers during UV irradiation (254 nm). This study highlights the potential of human-associated GeBaM to complement wastewater impact monitoring based on the determination of SFIB. In addition, human-specific JC polyomaviruses and adenoviruses seem to be a valuable support if highly specific markers are needed.