Enhancing Wastewater Surveillance: Optimizing Eukaryotic Enrichment for Improved Pathogen Detection

Background Wastewater surveillance is a valuable tool for detecting viral pathogens in communities, providing early warning signs of outbreaks. Shotgun metagenomics is a powerful approach for pathogen detection in wastewater, but critical pathogens such as viruses are often low in abundance, making detection challenging. Standard sequencing approaches primarily capture bacterial DNA, limiting the recovery of eukaryotic and viral genetic material. Furthermore, a significant gap remains in mapping interactions between viruses and their eukaryotic hosts, which is crucial for understanding transmission pathways and zoonotic risks. To address this, we developed a method for targeted enrichment of eukaryotic cells from wastewater to enhance the detection of viruses and their hosts. Objectives 1. Optimize an enrichment method combining size filtration and enzymatic treatment to increase eukaryotic cell recovery. 2. Quantify enrichment efficiency using shotgun sequencing. 3. Assess applications of this method for improving wastewater-based pathogen surveillance. Methods Wastewater samples underwent two-step filtration, starting with layered cheesecloth filtration to remove large debris, followed by 0.5 µm paper filtration using a vacuum pump to selectively capture eukaryotic cells. A subset of samples was treated with lysozyme to lyse bacterial cells. DNA was extracted and sequenced using shotgun metagenomics, with microbial composition analyzed to assess enrichment efficiency. Results, Implications & Future Directions Our method increased eukaryotic DNA content from 8% to 25%, demonstrating significant enrichment. This approach enhances pathogen detection and has future applications for ligation sequencing, improving the identification of host-virus interactions in wastewater and addressing a key gap in surveillance.