DNA extraction kit benchmarking for Legionella testing and microbiome mapping in pulp and paper mill wastewater

Sammanfattning

Legionella is a genus of pathogenic bacteria found naturally in freshwater, but that sometimes colonize warm, stagnant water in man-made water systems. Legionella lives a parasitic lifestyle within other cells that they utilize as hosts, and can spread to humans through aerosols, microscopic water particles in the air that can contain bacteria. These are breathed in and travel to the lungs, infecting our alveolar macrophages. This infection can cause pneumonic Legionnaire’s Disease which has a mortality rate of roughly 10% globally. Previous studies have shown that two thirds of Swedens biological wastewater treatment plants (BTPs) which handle pulp and paper mill (PPM) wastewater contain Legionella, which poses an infection risk for workers and nearby residents. When Legionella is detected in the aerobic part of the BTP, part of the plant is sometimes shut down, this in turn leads to only partly treated water being released into the downstream environment. In this study, wastewater from parts of the BTP process known to previously have harbored Legionella were sampled. Extraction of DNA from PPM wastewater samples is challenging and requires well-tuned extraction methods to remove contaminants such as lignin and humic acids that can act as PCR inhibitors. DNA extractions on these samples were performed with different DNA extraction kits, three designed for soil, and one for wastewater. The total DNA yield and purity from the extracts were then compared using fluorometric and spectrophotometric assays with Qubit and NanoDrop, 16S and 18S rDNA PCRs were also performed on the DNA extracts to evaluate their viability for amplicons sequencing. The main findings of the study were the following: For the BTP stages where the water was of a high particulate content, the PowerSoil Pro kit yielded the most DNA. The DNA was also of high purity. For the BTP stages with more aqueous samples, the SwiftX DNA Water kit yielded the highest amount of DNA, but none of the kits tested yielded particularly pure DNA. For all kits except for the NucleoSpin Soil kit, 16S and 18S rDNA PCR was consistently successful independently of the sampling location within the BTP. The DNA extracts were subsequently sent for 16S and 18S rDNA amplicons sequencing to screen microbiome for Legionella, potential Legionella hosts organisms and their free-living bacterial prey.