Assessing sequencing-based pathogen surveillance of a recreational swimming area in Oslo, Norway
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Sequencing-based surveillance can enable rapid and sensitive detection of environmental pathogens. The Oslofjord inlet is relatively narrow and is exposed to substantial human activity, including occasional wastewater contamination. Restricted water exchange also allows for occasional summer heat spells with elevated water temperatures. Thus, infections stemming from wastewater contamination and seasonal opportunistic pathogens are potential health threats to recreational users of the fjord. In this pilot study, we assess the suitability of sequencing-based surveillance for the detection of pathogens at a popular urban location for recreational water activities, employing both long- and short-read sequencing platforms, paired with selective culturing. We find both metagenomic and full-length 16S sequencing to be promising tools for surveillance of seasonal opportunistic Vibrio pathogens. Furthermore, we identified Rhodoferax abundance to be a potentially attractive indicator of sewage contamination using low to medium-depth full-length 16S sequencing. Selective plating revealed minimal abundance of culturable extended-spectrum β -lactam-resistant bacteria, of which none were detected by metagenomic sequencing. Metagenomic analyses did, however, pick up several other β -lactamases in various bacterial taxa, including some that were closely related to those identified by selective plating and sequencing.
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Dear Dr Eldholm, Thank you for your edits, I'm please to say the manuscript has now been accepted. Thank you again for your patience during this process. All the best with you future works! Best wishes, John
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Dear Dr Eldholm, Thank you for your submission and apologies on the delay in turn around. I'm pleased to say that both independent reviews have minor amendment recommendations and i agree the manuscript is in a good state. Please address the comments attached. Best wishes, John.
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Comments to Author
Reviewer Report Manuscript Number - ACMI-D-25-00106 Manuscript entitled "Assessing sequencing-based pathogen surveillance of a recreational swimming area in Oslo, Norway." In this manuscript, the author evaluates sequencing-based environmental surveillance approaches for pathogen detection in a recreational marine setting. By integrating full-length 16S rRNA nanopore sequencing, Illumina shotgun metagenomics, and selective culturing, the authors provide a comprehensive comparison of the methodological strengths and limitations of these approaches. The focus on Vibrio spp., sewage contamination indicators, and antimicrobial resistance is highly relevant to climate change, urbanization, and public health monitoring. Overall, the study is technically sound, clearly written, and supported by extensive data …
Comments to Author
Reviewer Report Manuscript Number - ACMI-D-25-00106 Manuscript entitled "Assessing sequencing-based pathogen surveillance of a recreational swimming area in Oslo, Norway." In this manuscript, the author evaluates sequencing-based environmental surveillance approaches for pathogen detection in a recreational marine setting. By integrating full-length 16S rRNA nanopore sequencing, Illumina shotgun metagenomics, and selective culturing, the authors provide a comprehensive comparison of the methodological strengths and limitations of these approaches. The focus on Vibrio spp., sewage contamination indicators, and antimicrobial resistance is highly relevant to climate change, urbanization, and public health monitoring. Overall, the study is technically sound, clearly written, and supported by extensive data availability. The results generally support the conclusions. However, several aspects require clarification, stronger statistical treatment, and improved framing of limitations to enhance robustness and interpretability. I recommend that a minor revision be required before acceptance. Minor revision requires * Please define all abbreviations at first mention (e.g., eDNA, PCA). * Ensure consistent use of "plate-sweep" vs. "platesweep" throughout the text. * Indicate whether field or laboratory blanks were included during filtration and sequencing to control for contamination. * Clarify whether replicate PCRs or sequencing replicates were performed for 16S amplicons. * Figures are informative but dense; consider increasing font size in multi-panel figures (especially Figures 2 and 3). * Table 1 (AMR determinants) would benefit from a more precise separation between culture-based and metagenomic detections.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: Overall, the study is technically sound, clearly written, and supported by extensive data availability. The results generally support the conclusions. However, several aspects require clarification, stronger statistical treatment, and improved framing of limitations to enhance robustness and interpretability. I recommend that a minor revision be required before acceptance.
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Comments to Author
This manuscript reports the results of a pathogen surveillance study conducted in the Oslofjord using a variety of sequencing and culturing methods. The manuscript is well written, the data and analyses appear to be sound and well conducted, and the interpretation and discussion of results are appropriate, given the size and scope of the study. The main limitation of the study is its size and scope - the sampling is simply too limited and ad-hoc to enable thorough investigation into both the sequencing methods and also the impact of temperature and rainfall on bacterial composition. However, as a pilot study this is OK, and the study is a useful first step towards more sophisticated monitoring of the Oslofjord (compared with the current approach described in lines 43-48). Please see minor comments …
Comments to Author
This manuscript reports the results of a pathogen surveillance study conducted in the Oslofjord using a variety of sequencing and culturing methods. The manuscript is well written, the data and analyses appear to be sound and well conducted, and the interpretation and discussion of results are appropriate, given the size and scope of the study. The main limitation of the study is its size and scope - the sampling is simply too limited and ad-hoc to enable thorough investigation into both the sequencing methods and also the impact of temperature and rainfall on bacterial composition. However, as a pilot study this is OK, and the study is a useful first step towards more sophisticated monitoring of the Oslofjord (compared with the current approach described in lines 43-48). Please see minor comments and suggestions below: 183-184: How were the sampling timepoints chosen? 189-191: I would advise to rewrite this sentence - I only understood it after re-reading it a few times. Figure 1: It is very difficult to make out the locations of the two sampling locations from the map - a more zoomed in version would be better (with the river marked). 211-217: Given the likely diversity of the samples and the differences between the two approaches, it is not necessarily surprising that there is little overlap between the top 5 taxa. Was there more overlap between, say, the top 20? For surveillance, I would say that detection is most important, even if abundances aren't too well correlated. PCA would also give insight into the species composition according to the sequencing method. 238-250: It is nice to see a sober analysis of this finding! 298-300: Was this also true of the other correlated species (from the 16S analysis)? 319-321: 11 AMR genes from the eDNA samples is remarkably low given that the whole AMR database was used. It would be worth checking that the prior assembly did not remove many hits (due to poor assembly, for example). I recommend using Ariba with the CARD database on the raw reads (ARIBA does local assembly and works well on raw reads).
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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