The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies

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.


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Sincerely, Sébastien Faucher Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): no comments Reviewer #2 (Comments for the Author): Dear Editor, in the manuscript titled "The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies" the Authors aimed to quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing.It's a very interesting topic and gives the opportunity to reflect on the importance of the decision of the Legionella detection method.Furthermore, materials and methods and results section are well described.My final opinion is to accept the manuscript with minor revisions.1) At Line 33 of the "Importance" section, you haven't added the entire name of ddPCR but only the acronym.Since this is the first time that you mention this word, you should add the entire name and from the second time you can write only the acronym.2) In the "Introduction" section, you could write some numbers about Legionella infection in your country (if they are available).
3) In the "Materials and methods" section, can you write brand, city and country of every material that you have mentioned for the analysis?(IDEXX's Legiolert liquid culture kit at Line 115, polycarbonate filters at Line 117, etc.).4) You should consult the website of the journal where you want to publish the paper on how the numbers in thousands should be written.Sometimes you put a comma (1,000), sometimes not (1000).All numbers should be written in the same way and it may vary from journal to journal.5) At Line 215 of the "Data analysis" section, the parenthesis hasn't been closed after "(version 1.42.0".6) At Line 268 of the "Results" section, there is a closed parenthesis but there isn't its opening.7) At Line 271-272 of the "Results" section, can you better write the sentence "As expected, there instances when both methods detected L. pneumophila DNA but the cultivation data was negative".8) At Line 290 of the "Results" section, you should write "(a)" in bold, as you did for (b) and (c).9) At Line 316 of the "Results" section, you should write "Segata et al." as "Segata et al.,", as you did for the entire text for the other references.10) At Line 318 of the "Results" section, there is an extra space between the words "5.Based".11) At Line 374 of the "Discussion" section, the closed parenthesis is missing after "(Fig.2".12) At Line 529 of the "Discussion" section, you should write the entire name of CFU (although its meaning is widely known), since this is the first time that you mention it.13) In the "Discussion" section, you could write some examples of studies that have investigated Legionella presence in environmental samples.Don't forget that there are a lot of studies based on culture media methods, such as https://doi.org/10.3390/ijerph20085526. it is a valuable and interesting study that provides a great contribution to the detection of Legionella in water samples.As mentioned by the authors, an efficient DNA extraction is a crucial step for risk assessment analysis but also for understanding the ecology of Legionella within water and biofilm microbial communities.

Remarks 2.Material and methods
Please describe the two methodologies A and B better in the text in order to make it clearer to the reader the link to the Results section where he finds the methods A and B which are not so clearly indicated in the methods.So please specify Method A consists of:; Method B consists of:.

References
The reference Scaturro et al. must be updated.Please find the exact reference at Frontiers in Microbiome: https://www.frontiersin.org/articles/10.3389/frmbi.2023.1170824/full The manuscript: The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies by Alessio Cavallaro, Marco Gabrielli, Frederik Hammes, William J. Rhoads , it is a valuable and interesting study that provides a great contribution to the detection of Legionella in water samples.As mentioned by the authors, an efficient DNA extraction is a crucial step for risk assessment analysis but also for understanding the ecology of Legionella within water and biofilm microbial communities.

Remarks 2.Material and methods
Please describe the two methodologies A and B better in the text in order to make it clearer to the reader the link to the Results section where he finds the methods A and B which are not so clearly indicated in the methods.So please specify Method A consists of:; Method B consists of:.June 6, 2024 1st Revision -Editorial Decision Re: Spectrum00713-24R1 (The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies) Dear Dr. Frederik Hammes: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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