Interaction of enteric viruses with aquatic biofilms in the urban water cycle
Aquatic biofilms are ubiquitous in natural and technical systems of the urban water cycle, where they can act as reservoirs for hygienically relevant microorganisms. Numerous studies have investigated the retention or multiplication of bacteria in different types of biofilms. However, little is known about the interaction of viruses with aquatic biofilms. The aim of this study was to determine the potential of aquatic biofilms in the urban water cycle to retain infectious viruses, including human enteric viruses, animal viruses and bacteriophages. Two field studies were conducted to determine the occurrence of viruses in biofilms from natural and technical systems in the urban water cycle. Moreover, laboratory experiments were performed to study the accumulation of selected viruses in drinking water biofilms and to elucidate the relevance of biological interactions between viruses and biofilms. In a field study involving the urban River Ruhr in Germany, the distribution of human enteric viruses, bacterial and viral indicator organisms between water, biofilms and sediments was assessed. First, different protocols for the isolation of viruses from biofilms and sediments were compared in recovery experiments, a method based on ultrasonication in elution buffer in combination with a subsequent concentration step was most efficient to isolate human adenovirus (HAdV) and coliphage φX174. During this field study, concentrations of bacterial and viral indicator organisms (E. coli and somatic coliphages) were elevated in biofilms and sediments compared to the bulk water by 3-4 log-units, using cultural methods. A period of enhanced rainfall led to a significant increase of E. coli and somatic coliphage concentrations in water, whereas concentrations in biofilms and sediments remained relatively constant. Water, biofilms and sediments were also analyzed for the occurrence of selected enteric viruses including human adenovirus (HAdV), enterovirus (EV), group A rotavirus (RoV) and norovirus genogroup GII (NoV GII). Although the detection frequency for enteric viruses in water was higher, HAdV and EV were detected in biofilms and sediments, using quantitative real-time PCR (qPCR). In a second field study, water and biofilms from drinking water systems for animals from piglet breeding farms in Germany were analyzed for the occurrence of somatic coliphages and selected animal pathogenic viruses. This study revealed a high prevalence of somatic coliphages in biofilms compared to the bulk water, also porcine adenoviruses (PAdV) were frequently detected in water and biofilms, using qPCR. Besides, hepatitis E viruses (HEV) were found in water and biofilms during this field study, however these results are doubtful because the detection via qPCR was shown to be unspecific. Further research is needed to verify the occurrence of HEV in drinking water systems for animals. In laboratory experiments, artificial drinking water biofilms were grown on ethylene propylene diene monomer (EPDM) coupons and incubated with three viruses (human adenovirus (HAdV), murine norovirus (MNV) and coliphage φX174) under different hydraulic and physicochemical conditions in order to simulate various situations in a drinking water distribution system. Viruses were quantified in water and biofilms at different time points using qPCR and cultural methods (viral cell culture, plaque assay) in order to determine the distribution between water and biofilms. This work showed that the concentration of all three viruses in water decreased significantly over time, while viruses were enriched in drinking water biofilms by a factor of up to 3.5 log units compared to the water phase within 24 h of incubation. Increased accumulation of viruses in biofilms was observed under stagnant conditions compared to flow conditions. Using cultural methods for virus quantification, the highest accumulation was found for HAdV in artificially hardened water (containing 4 mM Ca2+) under stagnant conditions, indicating a potential influence of the water hardness on virus retention in biofilms. Moreover, the murine macrophage cell line RAW 264.7 showed cytopathic effects when exposed to drinking water or drinking water biofilms without viruses and thus was not suitable to assess the concentration of infectious MNV in drinking water and drinking water biofilms. In the last part of this work, potential biological interactions of viruses with aquatic biofilms were elucidated. First, it was shown that a propagation of somatic coliphage φX174 in the presence of E. coli as natural host under simulated environmental conditions within 4 h at 20 °C or 36 °C is negligible. Moreover, Acanthamoeba castellanii was found to incorporate human adenoviruses in co-culture within 7 days of incubation, indicating that amoebae could potentially affect the persistence of viruses in aquatic environments. However, further research is needed to determine the relevance of amoebae in the context of aquatic biofilms. Overall, this work showed that aquatic biofilms in natural and technical systems can act as reservoirs for viral indicators as well as relevant human and animal pathogenic viruses. Aquatic biofilms can retain viruses that are present in the bulk water and potentially release them after a certain time, which underlines the significance of aquatic biofilms in water hygiene. Besides, this work suggests that biological interactions within aquatic biofilms could affect the fate of viruses in aquatic ecosystems.