Rapid Identification of Bacterial Composition in Wastewater by Combining Reverse Purification Nucleic Acid Extraction and Nanopore Sequencing

Abstract

Wastewater systems represent a threat for the transmission of pathogens in the case of improper treatment. Systematic surveillance based on wastewater is important as an early warning system, e.g., COVID-19. However, current procedures for microorganism identification are often not applicable at the pointof-need. In this study, a rapid and portable DNA extraction protocol was developed and verified to identify the microbial wastewater composition by nanopore sequencing. The study compared different pretreatment and buffer options combined with the reverse purification principle. Extraction efficacy was assessed using Staphylococcus aureus and Escherichia coli spiked wastewater and crude samples from different collection points at the treatment plant. Results were compared to a spin column-based reference method and a real-time polymerase chain reaction. Lysis buffer combined with bead beating showed the best extraction efficacies using an Oxford nanopore. It demonstrated an adequate sensitivity of down to 10 3 copies. Up to eight different bacterial species could be identified at each collection point by nanopore sequencing. The developed protocol achieved a 3-fold increase of average length, an increase of quality score, and a 15-fold decrease of total reads. The evaluated molecular detection methods are a useful tool for the analysis of bacteria within less than 3.5 h and could contribute to public health monitoring of wastewater.