Affiliation:
1. Department of Environmental Microbiology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland
2. Department of Environmental Systems Science, Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, Zürich, Switzerland
Abstract
ABSTRACT
Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for
Legionella
monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of
Legionella
enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected
Legionella
in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of
Legionella
, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify
Legionella
, and characterize the associated microbial community.
IMPORTANCE
Monitoring for the presence of the waterborne opportunistic pathogen
Legionella
is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for
Legionella
detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of
Legionella
through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.
Funder
Bundesamt für Lebensmittelsicherheit und Veterinärwesen
Bundesamt für Gesundheit
Bundesamt für Energie
Eawag, Swiss Federal Institute of Aquatic Science and Technology
Publisher
American Society for Microbiology
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