Viral Metagenomics to Assess the Microbial Quality of Reused Wastewater

Abstract

Reused wastewater has been suggested as a stable freshwater source that answers the increased demand linked to the global population growth. Raw wastewater harbors a diverse population of virus and bacteria, including pathogens, of which several can cause disease in humans even at low concentrations. Among these the enteric viruses, including norovirus (NoV), are one of the most frequent causes of food and waterborne diseases worldwide. Moreover, the transmission of pathogens to human via food or water is often due to the reuse of insufficiently cleaned wastewater in food production. To ensure food safety it is therefore important to assure that the reclaimed wastewater is free from viral pathogens before it is used during the production of ready-to-eat food products. Traditionally the microbial water quality has been accessed by use of fecal indicator bacteria. However, a number of studies agree in a lack of correlation between levels of fecal indicator bacteria and the presence of e.g. human pathogenic enteric viruses. To decrease the risk of foodborne outbreaks due to reused wastewater it is thus of great importance to investigate and acknowledge the whole viral content within the water to enable the use of proper treatment measures. To address this question, metagenomics sequencing could be a promising tool. The aim of this PhD thesis was to investigate if viral metagenomics sequencing can been used to assess the microbial quality of water. Further goals were to investigate the change in viral community throughout the wastewater treatment procedures and if possible determinate the treatment needed before reuse of wastewater. Initially the effect of different combinations of viral concentration and extraction methods on the detected viral metagenome in wastewater samples was investigated. The results showed that the particular selected method had a strong effect on the detected viral metagenome (Hellmér I). However, none of the 16 tested method combinations appeared to be superior in all tested parameters. Each method and method combination showed strengths and weaknesses. Based on the results obtained and two specific criteria, favoring the possibility to concentrate viruses from large volumes of water and the detection of genomes from RNA viruses, the monolithic adsorption filtration (MAF) combined with the extraction kit Nucleospin RNA XS were chosen in subsequent studies. Following this an investigation of the usefulness of viral metagenomcis to assess the micobiological water quality was performed. In Hellmér II the viral population was investigated before and after different treatment steps in one conventional urban wastewater treatment plant (WWTP) and in one highly advanced hospital WWTP. In addition, quantitate PCR analysis was applied to investigate the levels of the broadly proposed human viral indicators adenovirus (HAdV) and JC polyomavirus (JCPyV) as well as the pathogens, NoV, Salmonella, and Campylobacter. Results showed that analysis of the total viral community structure could differentiate microbial contaminated water from clean water without looking at specific pathogens. We observed a change in the viral community structure in effluent from the membrane bioreactor (MBR) at the hospital WWTP. The similarity of the viral composition in the MBR effluent to the community composition of sterile molecular grade water indicated a good microbial quality, although NoV and Salmonella were both detected in one of nine MBR effluent samples. At the hospital WWTP, the MBR effluent is further treated by ozone, granular activated carbon, and UV irradiation before being discharged. No microorganisms were detected in these samples. In the raw wastewater at the urban WWTP a seasonal pattern of the levls of NoV and HAdV was observed with a decrease in concentration during the summer months. This result contradicts previous observations of a stable concentration of HAdV throughout the year in wastewater in other countries, and may therefore question the usefulness of HAdV as a fecal indicator in waters. During the PhD project we received drinking water suspected to be the cause of a NoV outbreak (Hellmér III). The received volume of water enabled a small comparison between viral concentration methods on naturally NoV contaminated drinking water. NoV was detected with RT-qPCR and characterized with Sanger sequencing in the water sample concentrated with MAF. It was however not possible to further verify this finding of NoV using viral metagenomcis, although the bioinformatic analysis did detect the two viral process controls initially spiked in the water sample. Failure to detect NoV could be due to method dependent biases observed in Hellmér I or that the initial concentration of NoV in the water sample may have been below the limit of detection. In conclusion this PhD thesis has extended the knowledge of viral metagenomics for microbial water quality assessment and outbreak analysis. The effect of laboratory methods to prepare water samples for viral metagenomics sequencing has been investigated. This has increased our understanding of method dependent biases and stress the importance of carefully choosing the laboratory method best suited for the project in question. The PhD study demonstrated for the first time successful application of MAF to detect NoV in contaminated water implicated in a disease outbreak. The three studies within this PhD thesis also highlight the bioinformatic challenges in analyzing viral metagenomics data. Finally the presented work showed that viral metagenomcis could distinguish between the microbial water qualities throughout different stages of wastewater treatment. In combination with existing control measures metagenomics could be a powerful tool to analyze the effectiveness of the process in a wastewater treatment plant. Future studies are needed to validate these results before the method can be used in water management.