Quantitative bioluminescence assay for measuring Bacillus cereus nonhemolytic enterotoxin complex

Reuven Rasooly; Paula Do; Bradley Hernlem

doi:10.1371/journal.pone.0238153

Peer Review History

Original SubmissionMay 25, 2020
23 Jul 2020 Decision Letter - Maria Gasset, Editor PONE-D-20-15755 Quantitative Bioluminescence Assay for Measuring Bacillus cereus Nonhemolytic Enterotoxin Complex PLOS ONE Dear Dr. Reuven Rasooly, 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 mainly addressing the following points raised during the revision: 1. Update the information related to the impact of B. cereus as a food spoilage in human health. 2. Comment on how the new method compares with PCR/ELISA for cases with B. cereus presence and different toxin levels. 3. Comment on possible alternatives (kinetics, assay set-up, etc) Please submit your revised manuscript by september 1st. 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” If any authors received a salary from any of your funders, please state which authors and which funders. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. 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 requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: It is a very well written paper describing new approaches to detect and quantify the non-hemolytic enterotoxin of Bacillus cereus, a foodborne and highly dangerous toxin. The manuscript describes the design of not only a new assay, based on th inhibition of luciferase synthesis, but also describes the construction of a detection devise. I have no further comments to do and, in my opinion, the manuscript has enough merit and quality as to deserve publication as it is. Reviewer #2: The authors proposed a new strategy to detect active B.cereus toxin by inhibition of the luciferase synthesis in mammalian cells transfected with its gene. Similar techniques have been already used in vitro for the detection and evaluation of other membrane-active substances, including toxins. Also, a homemade cheap luminometer to carry out the evaluation of the active toxin in situ was described. The article is well written, with a right statistical approach, and original. Nevertheless the authors must answer important questions before approval for publication 1.- The scope of the article fits much better the scope of a Food technology journal rather than PLOS One, although it is a full decision from the Editor . 2.- The information provided in the Introduction about the impact of B.cereus as a food spoilage in human heath are outdated, the authors are advised to provide more recent information. 2,.- No doubt, the aim of the authors is to remark the practical implementation of their technique, and the advance provided by their approach. In order to do that, the drawbacks for the other techniques, animal model, PCR and ELISA were described, but they did not compared the sensitivity of at least PCR and ELISA with their method. From my point of view, just the presence of Bacillus cereus regardless of the level of toxin production is by itself a serious threat in food contamination. Does the new procedure challenge these two other techniques in terms of sensitivity? 3.- According to the protocol proposed transfection plus 72 h incubation with the toxin is required. Is this time frame affordable for groceries compared with ELISA or PCR?. Have the authors tried shorter time frames?. 4.- The authors described the problems associated to the practical use of ELISA or PCR in terms of the facilities required, but for this method a cell culture facility is needed , and also the t instability of the reagent solution for luciferase, especially for ATP, its substrate, that required to be frozen till its immediate use is obviated. In order to provide a fair scenario for this technique, these drawbacks must to be included, and if possible, their feasible solutions commented in the text. ****** 6. PLOS authors have the option to publish the peer review history of their article (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: Yes:** Álvaro Martínez-del-Pozo 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. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0238153.r001
Revision 1
5 Aug 2020 Author Response Dear Academic Editor Dr. Maria Gasset, Thank you for considering our manuscript entitled: “Quantitative Bioluminescence Assay for Measuring Bacillus cereus Nonhemolytic Enterotoxin Complex” (ID: PONE-D-20-15755) for publication as a research article in PLOS ONE. We have addressed the questions posed by the Reviewers and would like to thank Reviewers for their constructive comments. We feel that the manuscript has greatly benefited as a result. Reviewer #1 raised no objections to publication as is. Reviewer #2 raised the following points and we have addressed them as noted. … the authors must answer important questions before approval for publication 1.- The scope of the article fits much better the scope of a Food technology journal rather than PLOS One, although it is a full decision from the Editor. The authors recognize the broad scope of the journal PLOS ONE and the approach described may have broader analytic potential application. While the bioluminescence assay was demonstrated in the manuscript for measuring nonhemolytic enterotoxin activity, similar cell-based assays combined with low-cost optical sensing device can be adapted for the analysis of activity of other biomolecules and different type of toxins as an alternative to live animal testing. We added this important point to the discussion. We believe the broad potential application of the approach will be of interest to the journal’s readers. 2.- The information provided in the Introduction about the impact of B. cereus as a food spoilage in human heath are outdated, the authors are advised to provide more We have substantially expanded the introduction, specifically including more discussion of the impact of B. cereus and its toxins on human health and the need for improved alternatives to live animal testing for assessment of the toxic potential of isolates and contaminated foodstuffs. We have expanded the reference set accordingly with recent literature on the subject. Food is routinely heat treated to kill contaminant microbial pathogens and to inactivate their toxins. The main drawback of PCR and ELISA assays is their inability to differentiate between active toxin from inactivated toxin. PCR assay can detect the presence of the genetic material of non-viable microbial pathogens encoding toxin long after heat treatment already killed them. ELISA assay detects the presence of one of the three toxin subunits which poses no threat by itself. The activity bioassay that we developed distinguishes between the biological active form of the toxin, which poses a threat, from the inactive form, which poses no threat. 2,.- No doubt, the aim of the authors is to remark the practical implementation of their technique, and the advance provided by their approach. In order to do that, the drawbacks for the other techniques, animal model, PCR and ELISA were described, but they did not compared the sensitivity of at least PCR and ELISA with their method. From my point of view, just the presence of Bacillus cereus regardless of the level of toxin production is by itself a serious threat in food contamination. Does the new procedure challenge these two other techniques in terms of sensitivity? In terms of limit of detection, our bioluminescence assay can detect 7 ng/ml Nhe. This data compares with ELISA, PCR and lateral flow apparatus (LFA). ELISAs have a comparable detection limit to our assay with a range of 2-5 ng/ml and the LFA has a detection limit around 20 ng/ml [35]. It is difficult to determine a detection limit for PCR, but has been shown to produce a negative result in samples where the LFA will produce a positive result. This indicates that the detection limit of PCR is higher than that of 20 ng/ml. However, none of these methods demonstrate the presence of the biologically active form of Nhe and PCR and LFA produce only qualitative results. Further, commercially available tests only show the presence of one sub-unit of the Nhe complex. We have included further clarification of these points in the Introduction and Discussion. 3.- According to the protocol proposed transfection plus 72 h incubation with the toxin is required. Is this time frame affordable for groceries compared with ELISA or PCR?. Have the authors tried shorter time frames?. A shorter time frame will reduce the sensitivity of the assay. For the application of measuring toxin activity in food, the speed may not be so relevant because at the time of the measurement the presence of the toxin can already be established by immunologic screening assays, the relevant question remaining is whether the detected toxin is active and presents a health risk or whether the toxin is inactive. 4.- The authors described the problems associated to the practical use of ELISA or PCR in terms of the facilities required, but for this method a cell culture facility is needed , and also the t instability of the reagent solution for luciferase, especially for ATP, its substrate, that required to be frozen till its immediate use is obviated. In order to provide a fair scenario for this technique, these drawbacks must to be included, and if possible, their feasible solutions commented in the text. We have pointed out these additional requirement in the Discussion. However, it is worth stressing again that the method presented is more accurately described as an alternative to live animal testing for fully functional and active toxin and not as an alternative to PCR and ELISA which do not serve that purpose. We thank the reviewers for their constructive comments. Sincerely, Reuven Rasooly, Ph. D. Attachments Attachment Submitted filename: 4Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0238153.r002
11 Aug 2020 Decision Letter - Maria Gasset, Editor Quantitative Bioluminescence Assay for Measuring Bacillus cereus Nonhemolytic Enterotoxin Complex PONE-D-20-15755R1 Dear Dr. Reuven Rasooly, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Maria Gasset, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0238153.r003
Formally Accepted
15 Sep 2020 Acceptance Letter - Maria Gasset, Editor PONE-D-20-15755R1 Quantitative Bioluminescence Assay for Measuring Bacillus cereus Nonhemolytic Enterotoxin Complex Dear Dr. Rasooly: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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. Maria Gasset Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0238153.r004

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