Pilot-scale depuration demonstrates the suitability of non-pathogenic Vibrio parahaemolyticus as a surrogate for commercial-scale validation studies

Spencer L. Lunda; Samantha Kilgore; Jennifer M. Hesser; Joy G. Waite-Cusic; Carla B. Schubiger

doi:10.1371/journal.pone.0334240

Peer Review History

Original SubmissionApril 2, 2025
28 May 2025 Decision Letter - Brett Froelich, Editor Dear Dr. Lunda, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please make the changes or additions that are requested by myself and the other reviewers. Please submit your revised manuscript by Jul 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Brett Austin Froelich, Ph.D Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: [Taylor Shellfish Farms (Seattle, WA; https://www.taylorshellfishfarms.com) provided funding and the pilot-scale depuration system used in this study, as well as provided input on depuration parameters to evaluate. Additional funding provided by a United States Department of Agriculture National Institute of Food and Agriculture grant (2019-67017-29589; https://www.nifa.usda.gov) awarded to CBS supported these research efforts.]. Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: L 199-201. It seems the natural and artificial sea water terms are switched here, as natural seawater should not have a company associated with it. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Thank you for your time and for your interesting research review. Such a topic is very necessary Vibrio research oyster depuration application efforts. I do, however, suggest a few improvements should be made (see below). Overall This paper offers a valuable contribution to the body of knowledge concerning Vibrio mitigation. It is well written and structured, and successfully compares the effects of depuration across oyster species, evaluates non-pathogenic Vibrio as surrogates, and optimizes pilot-scale depuration. The results are particularly helpful for informing commercial depuration protocols for efforts to comply with NSSP standards for Vibrio reduction in oysters. However, the clarity of the figures detracts from the overall impact. Even when downloaded, I could not really view the figures. Please submit images with better resolution. Additionally, the rationale for selecting the specific temperatures of 5, 11, and 13°C for depuration trials is not clearly explained. Please include a brief explanation to help readers understand why these temperature parameters were selected. To further enhance the text, it would be useful to include data from the 2022 Campbell et al. review, Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters. This review could be referenced in lines 113-122 or in your discussion. The authors also mention: “Although many oyster depuration studies demonstrate significant variability when detailing the reduction of Vibrio vulnificus (VV) and Vibrio parahaemolyticus (VP), evaluation of these findings showed the greatest reductions of VV and VP were when processing time was from 4 to 6 days, water temperature was less than 20◦C, water salinity ranged from 25 to 32.2 ppt, and the water was flowing (non-static systems).” [Campbell, V. M., Chouljenko, A., & Hall, S. G. (2022). Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters. Comprehensive Reviews in Food Science and Food Safety, 21, 3480–3506. https://doi.org/10.1111/1541-4337.12969] Overall, this is an excellent study with practical applications that would benefit the oyster industry. Thank you. Reviewer #2: This manuscript reports pilot studies to better define the conditions to be used during larger-commercial scale depuration studies focusing on V. parahaemolyticus. The strains used as inoculum (pathogenic versus non-pathogenic), the inoculation approach (individual versus batch exposure), the autoclaved seawater used (artificial versus natural), the impact of the oyster species on the results as well as temperatures were evaluated during the study. I think the manuscript is well written and the experimental approach sound. My main concern relates to the method used to measure Vp concentrations (plating on TCBS) and I suggest discussing the limits of this approach and how this approach may be underestimating Vp concentrations. Ln 451,…: Does this mean that the only criterion for the Pacific Coast is that a reduction of >3.0 log is demonstrated? Is the < 30 MPN/g concentrations not used? Please clarify. Ln 228: Please include information relative to the number of oysters per chamber per species. Ln 253-256: Following NSSP, the method used to quantify the targeted Vibrio species during PHP validation is a MPN approach. Please justify the choice of a TCBS-based approach. Ln 253, …: Enumeration on TCBS can estimate total Vibrio counts, so how did Vp counts and concentrations were measured? Based on colonies color? Please include additional information. It seems that some sequencing was conducted but it is not clear how many colonies were sequenced per plate. These are critical information to assess if the concentrations reported are accurate. Please clarify. Ln 365,…: the Methods indicated that the natural Vp concentrations was assessed during this study (prior to inoculation), but these data are not presented. Please include these data. Ln 424: Such mortality is not expected in such a short time, any hypothesis regarding the factors involved? One can question the results of this trial since the other species were in the same tank. Were mortalities also observed in the other two species? ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org https://doi.org/10.1371/journal.pone.0334240.r001
Revision 1
19 Jul 2025 Author Response Additional Editor Comments: L 199-201. It seems the natural and artificial sea water terms are switched here, as natural seawater should not have a company associated with it. - Thank you for pointing this out, we have corrected this by adding the manufacturer following “…artificial seawater”. Reviewer #1: Thank you for your time and for your interesting research review. Such a topic is very necessary Vibrio research oyster depuration application efforts. I do, however, suggest a few improvements should be made (see below). Overall This paper offers a valuable contribution to the body of knowledge concerning Vibrio mitigation. It is well written and structured, and successfully compares the effects of depuration across oyster species, evaluates non-pathogenic Vibrio as surrogates, and optimizes pilot-scale depuration. The results are particularly helpful for informing commercial depuration protocols for efforts to comply with NSSP standards for Vibrio reduction in oysters. However, the clarity of the figures detracts from the overall impact. Even when downloaded, I could not really view the figures. Please submit images with better resolution. Additionally, the rationale for selecting the specific temperatures of 5, 11, and 13°C for depuration trials is not clearly explained. Please include a brief explanation to help readers understand why these temperature parameters were selected. - Thank you for these comments, we have provided higher-resolution figures that should allow for easier interpretation of results. We have also added a short explanation of why we tested these temperatures (after Line 230): “Trial temperatures were chosen to assess the efficacy of depuration at a range of temperatures below 15 °C, as V. parahaemolyticus is known to proliferate above that temperature threshold.” To further enhance the text, it would be useful to include data from the 2022 Campbell et al. review, Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters. This review could be referenced in lines 113-122 or in your discussion. The authors also mention: “Although many oyster depuration studies demonstrate significant variability when detailing the reduction of Vibrio vulnificus (VV) and Vibrio parahaemolyticus (VP), evaluation of these findings showed the greatest reductions of VV and VP were when processing time was from 4 to 6 days, water temperature was less than 20 °C, water salinity ranged from 25 to 32.2 ppt, and the water was flowing (non-static systems).” [Campbell, V. M., Chouljenko, A., & Hall, S. G. (2022). Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters. Comprehensive Reviews in Food Science and Food Safety, 21, 3480–3506. https://doi.org/10.1111/1541-4337.12969] Overall, this is an excellent study with practical applications that would benefit the oyster industry. Thank you. - Thank you for your comments, and for providing an additional review reference for citation. We have included this reference in Lines 113-122 to provide context of depuration parameters that are generally considered to be most effective for achieving targeted reductions: - “In a 2022 review of depuration parameters for reducing V. parahaemolyticus, the conditions found to be most effective were water temperatures < 20 °C, salinity from 25-32.2 ppt, depuration duration of four to six days, and non-static seawater conditions [28]. In the present study, we evaluated efficacy of depuration in a recirculating depuration system at temperatures from 5-13 °C, salinity of 35 ppt, and trial durations of five to seven days.” Reviewer #2: This manuscript reports pilot studies to better define the conditions to be used during larger-commercial scale depuration studies focusing on V. parahaemolyticus. The strains used as inoculum (pathogenic versus non-pathogenic), the inoculation approach (individual versus batch exposure), the autoclaved seawater used (artificial versus natural), the impact of the oyster species on the results as well as temperatures were evaluated during the study. I think the manuscript is well written and the experimental approach sound. My main concern relates to the method used to measure Vp concentrations (plating on TCBS) and I suggest discussing the limits of this approach and how this approach may be underestimating Vp concentrations. - Thank you for your comments and review; we have provided responses to your specific comments below. Ln 451,…: Does this mean that the only criterion for the Pacific Coast is that a reduction of >3.0 log is demonstrated? Is the < 30 MPN/g concentrations not used? Please clarify. - For harvest from areas closed due to reports of illness associated with V. parahaemolyticus, harvest may be allowed if depuration can achieve a > 3.0 log reduction, but oysters treated with this method would not have a labeling claim attached indicating that post-harvest processing methods have been used. For the labeling claim, the 3.52 log reduction and < 30 MPN/g is required. To clarify this point, we have added a sentence following the one that you highlighted: - “However, depuration did not achieve the targeted > 3.52 log CFU/g reduction of V. parahaemolyticus and reduction below the detection limit of 30 MPN/g that would be required for a PHP labeling claim on harvested oysters.“ Ln 228: Please include information relative to the number of oysters per chamber per species. - We have added a sentence to provide information on numbers of oysters used for, in line 243 in the original manuscript: “For each oyster species and V. parahaemolyticus treatment, 25 oysters were added for trials lasting five days, and 35 oysters for seven-day trials.” - In addition, to clarify about stocking we added “evenly” to the sentence: “To assess efficacy of depuration, inoculated oysters were evenly stocked into the system’s four holding chambers.” Ln 253-256: Following NSSP, the method used to quantify the targeted Vibrio species during PHP validation is a MPN approach. Please justify the choice of a TCBS-based approach. - Thank you for this comment; we are aware of the MPN approach included in the NSSP. We have performed side by side enumeration of inoculated and depurated oyster samples using both MPN and TCBS plate counts and results were similar for all samples between the two methods. Enumeration on TCBS consumes less resources (supplies, labor, and time); therefore, this method was selected for use in this study. Ln 253, …: Enumeration on TCBS can estimate total Vibrio counts, so how did Vp counts and concentrations were measured? Based on colonies color? Please include additional information. It seems that some sequencing was conducted but it is not clear how many colonies were sequenced per plate. These are critical information to assess if the concentrations reported are accurate. Please clarify. Background Vibrio populations in oysters were very low (<2 log CFU/g) and inoculation levels were very high (~6 log CFU/g); therefore, the risk of the natural Vibrio contamination influencing the enumeration was insignificant. Therefore, all colonies on TCBS from inoculated oysters displayed typical V. parahaemolyticus morphology and were enumerated. Uninoculated oyster samples of all species had low levels (<2 log CFU/g) of bacteria that were capable of growth on TCBS that displayed morphologies that were atypical (color, size) in comparison to V. parahaemolyticus. Due to this low level of background contamination, there were few colonies to assess. A total of 28 isolates were sequenced with their identities shown in Table 3. - In line 366, we have added the sentence: “A total of 28 isolates were sequenced”. Ln 365,…: the Methods indicated that the natural Vp concentrations was assessed during this study (prior to inoculation), but these data are not presented. Please include these data. - Thank you for this comment; the primary purpose of enumerating uninoculated oysters was to determine if V. parahaemolyticus was present. Presumptive natural Vibrio counts were very low (< 2 log CFU/g) in all sampled oysters, and we did not identify any colonies with the typical morphology for V. parahaemolyticus; all colonies were either yellow, green-black, irregularly shaped, or smaller than expected for V. parhaemolyticus. Ln 424: Such mortality is not expected in such a short time, any hypothesis regarding the factors involved? One can question the results of this trial since the other species were in the same tank. Were mortalities also observed in the other two species? - We were also surprised at the mortality that occurred in C. gigas in this trial, as we had not observed similar mortality in previous trials. In C. sikamea and C. virginica used for this trial, the only mortality that occurred was a single C. virginica individual from the non-pathogenic V. parahaemolyticus treatment. We hypothesize that the mortality of C. gigas may have been due to environmental stress (temperature, water quality, etc.) at the time and site of harvest, combined with stress from shipping to the depuration site. We have added sentences to the manuscript to elaborate on this for the readers: “Mortality of C. gigas was not observed in previous depuration trials, and mortality of the other oyster species in this trial was limited to one C. virginica individual from the non-pathogenic V. parahaemolyticus treatment. It is possible that the high mortality of C. gigas in this trial was due to environmental conditions such as temperature and water quality at the time and location of harvest, in addition to stress from shipping to the depuration site.” Attachments Attachment Submitted filename: PLOS One Depuration Manuscript Response to Reviewers 071825.docx https://doi.org/10.1371/journal.pone.0334240.r002
24 Sep 2025 Decision Letter - Mohammad Moniruzzaman, Editor <p>Pilot-scale depuration demonstrates the suitability of non-pathogenic Vibrio parahaemolyticus as a surrogate for commercial-scale validation studies PONE-D-25-17763R1 Dear Dr. Lunda, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Mohammad Moniruzzaman Academic Editor PLOS ONE Reviewer #1: The study is well-written and provides valuable data on optimizing depuration for Vibrio reduction in oysters. The experimental design is sound and the results are clearly presented. This work should be insightful for researchers and industry players. Thank you. Reviewer #2: The authors have taken into account the reviewers' comments. As a pilot study, this is a nice contribution to the field. ******** ******** https://doi.org/10.1371/journal.pone.0334240.r003
Formally Accepted
Acceptance Letter - Mohammad Moniruzzaman, Editor PONE-D-25-17763R1 PLOS ONE Dear Dr. Lunda, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Mohammad Moniruzzaman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0334240.r004

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