Benchmarking Protocols for DNA Extraction and Cultivation of Legionella From Biological Treatment Plants From Paper and Pulp Mills

Sammanfattning

Legionella spp. are common environmental bacteria that can cause a form of atypical pneumonia known as Legionnaires’ disease when inhaled. The bacteria enter the macrophages of the lung, and replicate in them. Sometimes Legionella is found in extremely high levels in biological treatment plants (BTP) in connection to paper and pulp mills. This becomes a threat to the workers, and people close to the plant. Chemical oxygen demand, retention time, and temperature are factors known to influence the Legionella levels. The metagenomic factors that influence the Legionella levels are not well studied, and an appropriate DNA extraction method is needed. The goals of the project were to find an appropriate DNA extraction method and to culture Legionella on GVPC-plate to investigate where they can be found within the BTP. Here we show how four different commercially available DNA extraction kits performs on water samples from two different BTPs. The bead beating and spin column-based DNA extraction kit DNeasy® PowerSoil® Pro Kit showed good results and can yield both 16S and 18S PCR product, allowing for amplicon sequencing of both prokaryotes and eukaryotes. The plate cultivation resulted in no identifiable Legionella growth. The difficulties to cultivate the desired bacteria further highlights the importance of non-cultivation-based methods for Legionella surveillance.