Stationary and portable sequencing approaches for tracking fecal contamination in an urban stormwater system

Urban sewer systems consist of wastewater and stormwater sewers, of which typically onlythe wastewater is processed before being discharged. Occasionally, misconnections ordamages in the network occur, resulting in wastewater entering the stormwater system andbeing discharged without prior processing. Cultivation of faecal indicator bacteria, such asEscherichia coli (E. coli), is the current standard for tracing wastewater contamination. Thismethod is cheap but cannot be employed in the field and is characterised by its limitedspecificity. Here, we compared the E. coli culturing approach with two different DNAsequencing-based methodologies (i.e., 16S rRNA amplicon sequencing on the IlluminaMiSeq platform and shotgun metagenomic sequencing on an Oxford Nanopore MinIOndevice), analysing 73 stormwater samples collected throughout the Stockholm city areas.High correlations were obtained between E. coli culturing counts and frequencies of humangut microbiome sequencing reads (via amplicon sequencing), indicating that E. coli is indeeda good indicator of faecal contamination. In contrast to E.coli culturing, amplicon sequencingcould, however, further distinguish between two different sources of contamination in anarea, where misconnections in the stormwater system were later on detected. Shotgunmetagenomic sequencing on a subset of the samples using the portable Oxford NanoporeMinION real-time sequencing device correlated well with the amplicon sequencing data. Insummary, this study shows that DNA sequencing allows distinguishing differentcontamination sources in stormwater systems and demonstrates the potential of using aportable sequencing device in the field for tracking faecal contamination.