Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains

Tamazight Cherifi; Julie Arsenault; Franco Pagotto; Sylvain Quessy; Jean-Charles Côté; Kersti Neira; Sylvain Fournaise; Sadjia Bekal; Philippe Fravalo

doi:10.1371/journal.pone.0236807

Peer Review History

Original SubmissionJune 3, 2020
18 Jun 2020 Decision Letter - Yung-Fu Chang, Editor PONE-D-20-16869 Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains PLOS ONE Dear Dr.Cherifi, 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. Your manuscript has been reviewed by two experts in your filed. A minor revision is suggested. Please submit your revised manuscript by two weeks. 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. 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In the interests of reproducibility, in your Methods, please state the names/locations of the slaughterhouses where samples were collected. If you are unable to share this information due to privacy agreements, please include an explanation in your Ethics Statement. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 4. Thank you for stating the following in the Competing Interests section: I have read the journal's policy and the authors of this manuscript have the following competing interests:Authors declare no competing of interest. Dr Sylvain Fournaise (from Olymel) contributed to the sampling design at the slaughterhouses and the interpretation of results, but had no role in laboratory analyses, statistical analyses or in reporting the results presented in this work. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. 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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: Partly ******** 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: Manuscript Number: PONE-D-20-16869 In this manuscript, the authors provided a deep analysis of L. monocytogenes distribution in the pork production chain from Quebec, Canada, demonstrating risk factors and relevance of production aspects on the presence of persistent strains, and also correlated with isolates previously obtained from other samples. Minor review is suggested: Please, provide results to support your comments. like in lines 33-34: how these proportions varied based on slaughterhouses and operation areas? Line 35-37: please, delete “..according to our definition (i.e isolated at least three times in the same slaughterhouse over the 16-month period, with a four- to five-month interval between each sampling visit)” Line 38: indicate p value for the significant finding Line 40: how much these genes were more frequent in persistent strains? Line 40-42: I would expect a much more interesting conclusion than this… this is already known… maybe you can add information related to resistance strains in this conclusion, a real important result. Line 50: replace Listeria by L. Line 62-65: this information is important! The authors need to provide further information about resistance of L. monocytogenes to sanitizers and how this is relevant to result in strain persistence. Then, you can address information about quaternary ammonium compounds and Listeria resistance. Line 66-69: this information could be better merged with the paragraph 53-61… in the present format is loose Line 102: replace Listeria by L. Line 112-113: please, indicate the target gene considered to confirm the isolates as L. monocytogenes Line 187: replace Listeria by L. Please, consider presenting the digestion profiles in figures, to allow the reader to visualize them and check their quality Discussion: please, indicate the figures and tables with the obtained results to support your discussion. Reviewer #2: The manuscript PONE-D-20-16869 “Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains” represents a very good application of typing methods and statistical logistic regression for the study of L. monocytogenes dissemination within slaughterhouses. It is also very interesting in discussing the persistency in relation to sample sources, type and presence of disinfectant resistance. However, I suggest some improvements below: Abstract: need specification on statistical methods used for assessing association between persistency and virulence/resistance Introduction: Line 55-57 need clarification Materials and Methods: Line 97: what do you mean by “put on ice”. Please, clarify Line 123-125: please clarify Line 158-163: redundancy with the previously presented statistical methods Line 195: “visually observed” needs clarification Results: Line 233-235: try to clarify the sentence to make numbers clearer. Line 242: check the table notes Line 302-304: no need, already stated in M and M Discussion: Line 372: check was/is Line 376-381: clarify sentence and explain better what do you mean by “subjectively observed …” what was observed to conclude that in one premise the cleaning practices were more effective? Check lists? Observation of some particular aspects? Line 396-399 need some more support, maybe a table, if not is just conjecture Line 406-412: too much speculation Line 415: ok, but you need to discuss more in depth the meaning of the underestimation of variability. Could it impair your results or conclusion? Line 432-437: need more explanation on the differences in cleaning Line 438-439: speculation (did you see these evidence during your visits?) Line 464-467: ok but mitigate: you have very few samples studied to say that you demonstrated something Line 468-470: speculation ****** 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: 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. 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Revision 1
10 Jul 2020 Author Response Response to Reviewer’s Comments - Manuscript PLOS D 20 16869 We would like to thank the reviewers for their time and constructive comments, which definitely help to improve the quality of our manuscript. Reviewer #1 In this manuscript, the authors provided a deep analysis of L. monocytogenes distribution in the pork production chain from Quebec, Canada, demonstrating risk factors and relevance of production aspects on the presence of persistent strains, and also correlated with isolates previously obtained from other samples. Minor review is suggested: 1. Please, provide results to support your comments. like in lines 33-34: how these proportions varied based on slaughterhouses and operation areas? Response: The range in proportions for slaughterhouses and operation areas were added in the Abstract (lines 34-35). 2. Line 35-37: please, delete “..according to our definition (i.e isolated at least three times in the same slaughterhouse over the 16-month period, with a four- to five-month interval between each sampling visit)” Response: This part of the sentence was deleted as suggested. 3. Line 38: indicate p value for the significant finding. Response: P-values were added for each of the two comparisons (lines 38-40). 4. Line 40: how much these genes were more frequent in persistent strains? Response: We modified the sentences in the Abstract to better present this association, including the frequency in each group (lines 39-41). 5. Line 40-42: I would expect a much more interesting conclusion than this… this is already known… maybe you can add information related to resistance strains in this conclusion, a real important result. Response: We improved the conclusion in the Abstract (lines41-44). 6. Line 50: replace Listeria by L. Response: The correction was done. We performed a full-text search and corrected another occurrence of the same error. 7. Line 62-65: this information is important! The authors need to provide further information about resistance of L. monocytogenes to sanitizers and how this is relevant to result in strain persistence. Then, you can address information about quaternary ammonium compounds and Listeria resistance. Response: We added more information according to the literature (lines 61-71). 8. Line 66-69: this information could be better merged with the paragraph 53-61… in the present format is loose. Response: True. We merged the two paragraphs in order to be more coherent (lines 61-71). 9. Line 102: replace Listeria by L. Response: Correction was provided within the text. 10. Line 112-113: please, indicate the target gene considered to confirm the isolates as L. monocytogenes. Response: Target genes are now listed in the text (prfA, prs, lmo0737, lmo1118, orf2819, orf2110, and flaA genes) (line 120). 11. Line 187: replace Listeria by L. Response: Correction was provided within the text. 12. Please, consider presenting the digestion profiles in figures, to allow the reader to visualize them and check their quality. Response: Thank you for your comment. We have added the gel images to the dendrogram in Figures 2A and 2B. 13. Discussion: please, indicate the figures and tables with the obtained results to support your discussion. Response: We have added references to the Tables and Figures throughout the Discussion, as suggested. Reviewer #2 Reviewer #2: The manuscript PONE-D-20-16869 “Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains” represents a very good application of typing methods and statistical logistic regression for the study of L. monocytogenes dissemination within slaughterhouses. It is also very interesting in discussing the persistency in relation to sample sources, type and presence of disinfectant resistance. However, I suggest some improvements below: 1. Abstract: need specification on statistical methods used for assessing association between persistency and virulence/resistance Response: An exact chi-square test was used for these analyses; this precision was added in the Abstract as suggested (lines 38, 41) . 2. Introduction: Line 55-57 need clarification. Response: Agreed. Some text was added to improve clarification (lines 55-57). 3. Materials and Methods: Line 97: what do you mean by “put on ice”. Please, clarify. Response: ”put on ice” has been replaced by “… kept in a cooler with ice packs…” (line 103). 4. Line 123-125: please clarify. Response: This sentence now reads as “One isolate per sample and per ribotype was selected for PFGE typing (i.e. if two isolates from a same sample had different ribotype, both were selected for PFGE typing; otherwise, one isolate was randomly selected). In addition, for a subset of randomly selected samples with two isolates sharing the same ribotype, the two isolates were typed.” We hope that the sentence is now clearer. 5. Line 158-163: redundancy with the previously presented statistical methods. Response: We replace “(…) with the visit included as a random effect. The GLIMMIX procedure of SAS v 9.4 (SAS Institute Inc., Cary, NC, USA) was used with Laplace estimation. Odds ratios were used to present results, with P-values adjusted for multiples comparisons using Tukey-Cramer tests” by “as described above or the analysis of L. monocytogenes-positive samples. “ (lines 174-175). 6. Line 195: “visually observed” needs clarification. Response: The sentence now reads as: “The MIC was defined as the lowest concentration of BAC at which the growth of the bacteria was inhibited based on visual assessment “. (lines 211-212) 7. Results: Line 233-235: try to clarify the sentence to make numbers clearer. Response: We understand that the direct reference to odds ratios can lead to cumbersome sentences. We rephrase them to put emphasis on the direction of the associations. (lines 281-292) 8. Line 242: check the table notes. Response: The superscript was removed when referring to an abbreviation and kept otherwise (all Tables were revised accordingly). 9. Line 302-304: no need, already stated in M and M Response: Agreed. This sentence was removed. 10. Discussion: Line 372: check was/is. Response: “is” was deleted. 11. Line 376-381: clarify sentence and explain better what do you mean by “subjectively observed …” what was observed to conclude that in one premise the cleaning practices were more effective? Check lists? Observation of some particular aspects? Response: Thank you for your comment. Because we had no additional information or data to confirm our observations, we chose to delete these sentences. 12. Line 396-399 need some more support, maybe a table, if not is just conjecture Response: Agreed. It was conjecture. We have chosen to delete this paragraph because we had no information to support these hypotheses. 13. Line 406-412: too much speculation Response: We agree. Thank you for raising the point. These sentences were removed as they could lead to over-interpretation. 14. Line 415: ok, but you need to discuss more in depth the meaning of the underestimation of variability. Could it impair your results or conclusion? Response: We added the following information in this paragraph: “The selection of one isolate by sample could have resulted in an underestimation of the pulsotype diversity and/or of the proportion of samples with persistent strains. However, this should not have biased our results related to the comparison of diversity and/or strain persistence between slaughterhouses, seasons or operation areas, as only one randomly selected isolate per sample was kept for theses analyses to ensure group comparability”. 15. Line 432-437: need more explanation on the differences in cleaning. Response: We added the following explanation in these differences:” The lairage and slaughtering and bleeding areas were located closed to each other and had similar cleaning and sanitation procedures. They were subjected to a weekly disinfection using alcohol (70 %; v/v) and potassium monopersulfate (1 %; m/v) based disinfectants. In the other operation areas (i.e. dehairing and evisceration, chilling and hanging, cutting and deboning), the disinfection was done daily using QACs (from 150 to 200 ppm), thus, L. monocytogenes strains were subjected to the same environmental conditions”. 16. Line 438-439: speculation (did you see these evidence during your visits?): Response: In the slaughterhouses we sampled, but also in food processing plants in general, the surfaces are always wet due to the daily cleaning and sanitation. This can lead to a dilution of the disinfectants in micro-places within the slaughterhouses or the food processing plants. 17. Line 464-467: ok but mitigate: you have very few samples studied to say that you demonstrated something Response: We substituted “demonstrated” by “observed”, and modified slightly the rest of the sentence to tone down our conclusion here 18. Line 468-470: speculation Response: We agree. Thank you for your comment. This sentence was removed. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0236807.r002
15 Jul 2020 Decision Letter - Yung-Fu Chang, Editor Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains PONE-D-20-16869R1 Dear Dr. Cherifi, 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, Yung-Fu Chang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0236807.r003
Formally Accepted
23 Jul 2020 Acceptance Letter - Yung-Fu Chang, Editor PONE-D-20-16869R1 Distribution, diversity and persistence of Listeria monocytogenes in swine slaughterhouses and their association with food and human listeriosis strains Dear Dr. Cherifi: 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. Yung-Fu Chang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236807.r004

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