Peer Review History
Original SubmissionMarch 17, 2021 |
---|
PONE-D-21-08856 Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani Population PLOS ONE Dear Dr. Bokhari, 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 submit your revised manuscript by Jul 05 2021 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. Please include the following items when submitting your revised manuscript:
If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. 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, Grzegorz Woźniakowski, Full professor, PhD, ScD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements.
https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 2.The research project was presented to the ethical review board, PIMS Islamabad. Informed written consent was solicited from enrolled patients.". Please amend your current ethics statement to confirm that your named institutional review board or ethics committee specifically approved this study. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: No Reviewer #2: Partly Reviewer #3: Partly ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: I Don't Know Reviewer #3: I Don't Know ********** 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: No Reviewer #2: Yes Reviewer #3: 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: No Reviewer #2: No Reviewer #3: No ********** 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: This study aims to characterize the colibactin production in E. coli isolated from patients with or without colorectal cancer. While this type of descriptive work is important the current study has a number of defects. Firstly, it is of low power: there are only 23 cancer patients and 17 healthy controls. There is little indication of any power analysis to demonstrate why this number was picked and whether any meaningful conclusions can be drawn from such a cohort. Secondly, the agarose gel electrophoresis presented in Figures 1 and 2 are poor quality, presented poorly and the conclusions drawn from them poorly integrated into the manuscript text. This makes it hard to understand what is being concluded from these figures. Thirdly, the reason for the antibiotic resistance profiling is unclear in the broader context of the study. It’s nice to know what the resistance profile is but why include it here? Fourthly, the cytotoxicity data in Figure 5 seems selective. Why look at these strains only? What do the bars represent? How many times have the experiments been performed? Are there meaningful differences between groups? Statements in the results section seem unsupported by data (for example: “Our results showed that all the strains irrespective of the phylogroup were cytolethal at MOI above 50” – where is the data supporting this?). These are just a few examples of the problems with this study which indicate that it is of low quality. Reviewer #2: Manuscript “Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani Population” is a very relevant study to show the association between specific Colibactin producing strain of E. coli with Colorectal Cancer. It is well designed epidemiological study accounting the dietary habit as well as hygiene practice in low socioeconomic backgrounds. Study mainly focused on strains harboring Pks Island which resides colibactin gene A and Q. Antibiogram profile of the isolates and ERIC-PCR Genotyping were among the tools used to type the strains. Overall, it is an informative study. Followings are my comments: Majors: 1. As main massage of this paper is to show the association of Colibactin positive E. coli strain with CRC, the data showed be compared and plotted using proper statistical tools to show this association. May be bar diagram: no. of CRC isolates vs healthy isolates with and without colibactin gene instead of Fig. 2. 2. It would be informative to analyze individual antibiotic profile with presence of colibactin gene with CRC in Fig. 3. 3. Figure 5 does not have SD or SEM. So, hard to make any conclusion as both colibactin positive E. coli strain as well as negative control showed toxicity above 85%. Better to use toxicity negative control strain which showed <10% toxicity when compared with colibactin positive E. coli strain. Minors: 1. Abstract is too long. 2. Table 1 primer list can be moved to supplement. 3. Figure quality in Fig. 1 and 2 low. Reviewer #3: The present work describing the “Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani Population” is original and interesting. However, it requires considerable improvements. General comments: - Next submission, please, insert pages and lines! - Abbreviations: define each abbreviation when it first appears in the text. After that, keep using the abbreviation. Sometimes words appear in abbreviated, sometimes in full formats (ex: CRC, pks, etc…) Standardize. Define “PIMS” when it first appears. - Standardization: For example: for polyketide synthase “–producing”, “-positive” E. coli, I suggest using “pks+ E. coli” throughout the text. The same for colibactin-positive E. coli (clb+ E. coli) - References: Instead of “XXX and his companions”, use “XXX et al.,”. - It is Ofloxacin and Nalidixic acid (and not Oflaxacin and Nilidixic acid) - In “Results and Discussion” session, authors present statistical data and association between hygiene and CRC development. However, there is no mention on how these data have been collected and analyzed. Provide details of methodology used. - English revision is recommended. Sentence structures and conjunctions are frequently misused. - Discussion and Conclusions could be better explored. Minor comments/suggestions: - Abstract, Line 6: “in the present study, colibactin positive E. coli…” - Abstract , penultimate sentence: (Suggestion) substitute “populace” with “population” - Introduction, Paragraph 4: “In 2006, Nougayrède et al. identified a meningitis E. coli strain IHE3034” / The sentence continues “…they named THIS toxin as colibactin”. Strain IHE3034 is not a toxin. Rephrase. // At the end of this paragraph: “… have shown an increased number of tumor in various CRC mouse models” - Introduction, Paragraph 5: “Recent studies have intimated (implied? suggested?) that some specific E. coli strains possess a cluster of genes named polyketide synthase (pks) island, which has been associated with development of CRC in humans (Arthur et al., 2012)” - Introduction, Paragraph 6: “HeLa cells” - Introduction, Paragraph 7: “The isolated E. coli strains…” - Sample collection: “Patients under 20 year of age suffering from severe (???) infection and bleeding were not included in this study.” Gastrointestinal infection and bleeding? general infection? - Bacterial culture and Biochemical identification: Confusing. Rewrite. Suggestion: A biopsy sample from each patient was collected, kept in 20% glucose solution and transported to the laboratory on the ice. Biopsies were then homogenized and the preparations were cultured on MacConkey agar plates incubated at 37°C for 18-24 hours for bacterial colonies isolation. Based on morphological characterization, single E. coli colonies were re-streaked and purified on MacConkey agar/LB agar incubated at 37°C overnight. The identification of E. coli colonies were confirmed by Gram’s staining and various biochemical tests (i.e. Triple Sugar Iron (TSI), Citrate Test, Catalase, Oxidase, Indole Test, Methyl red test and Urease test). Pure E. coli cultures confirmed by morphological and biochemical tests were processed and preserved in 20% glycerol and stored at -80 °C until further processing. - DNA extraction and quantification: Genomic DNA from E. coli strains were extracted by ethanol precipitation method using the method described by (Rasheed et al., 2011). Briefly, a single pure bacterial colony selected from each sample, was resuspended in 20 µL of 1% sodium dodecyl sulfate, 40 µL proteinase K (100 µg/mL), 80 µL of proteinase K buffer (4M NaCl, 0.5M EDTA; pH 7.5), and incubated at 55 °C for 1 hour. - Phylogrouping: Genomic DNA extracted from each E. coli isolate was used for…. // The primers used for determination of phylogroups (chuA, yjaA, TspE4.C2, and arpA) are listed in Table 1. - Detection of Colibactin Genes: The primers used for the determination of colibactin producing E.coli isolates (ClbA and ClbQ) are shown in Table 1. - Antibiotics Susceptibility Profiling: E. coli strains were classified as resistant, susceptible, intermediate by comparison of diameter of inhibition according to the CLSI, 2017 guidelines. - ERIC-PCR Genotyping: To determine the evolutionary relationship among the 40 E. coli isolates studied, genotyping was performed using ERIC fingerprinting assay. - Viability/ Cytoxicity of Bacterial Culture against HeLa Cell Lines: Correct “CO2” and “Co2”: CO2 // “Six well plates containing HeLa cells...”. // “Bacteria were suspended in PBS and OD adjusted to 1.0 (2x107 CFU/mL).” - Results and Discussion, second paragraph: observe comments above. How was these data accessed? How was the hygiene and potato intake accessed? Was the consumption of other food also accessed? // “Whereas, among female population 50% of the females were cancerous while 50% of females appeared normal.” Appeared” (??) vague - Phylogroup Identification using Quadraplex PCR: “According to recent studies (REF), the majority of pathogenic E. coli strains belong to B2 and D phylogroups, and these strains tend to possess more virulence factors in comparison to commensal strains of E. coli.” - Colibactin Gene Identification: “Our results showed that nine (9) out of ten (10/23) B2 phylogroup cancer-associated isolates were found as colibactin possessing E. coli.” Rewrite this sentence to make it clearer. “9 out of 10… (10/23)” is a bit confusing… // It would be easier if authors use “clb+” or “clb- E.coli” notation. It is important to observe that it is not the patient or heathy individual the one who “has positive/negative colibactin gene, but the E. coli strains. “Seventeen healthy individuals presented clb- E. coli strains, while among 23 CRC patients, 14 and 9 respectively presented clb- and clb+ E. coli strains.” - In vivo viability/cytotoxicity activity of Colibactin Positive E. coli Strains: “Our results showed that ALL the strains irrespective of the phylogroup were cytolethal at MOI above 50.” But, as described in Material and Methods, this “all” strains refers to only 2 clb+ and 1 clb- E.coli (and 2 positive controls). This must be clear in this paragraph. Have you checked if these strains also produce other cyclomodulins like CDT and CNF? I think that a cytotoxin negative control should have been included in this experiment.// Revise: “(2 positive controls and 2 E. coli strains isolated in this study)” - Table 1: The following primers are missing: ClbQ R, ClbB F and R. - Figure 1, legend: Clearly state that this figure refers to the 23 CRC related E. coli strains. - Figure 2, legend: Clearly state that this figure refers to the 10 E. coli strains belonging to the B2 phylogroup which were isolated from CRC cases. Figure 3: “Graphical representation for antibiotic profiling of E. coli isolates among colon cancer patients and healthy individuals”. This not represents antibiotic profile of E. coli. This represents the frequency of antibiotic resistance of this group of E. coli for each antibiotic tested. - Figure 4: “Dendrogram showing the phylogenetic grouping” of the 40 E. coli strains studied. - Conclusions: Authors concluded that “Cancer-associated E. coli isolates were found to be multidrug-resistant and genetically diverse.” Would it be the equivalent to say that “clb+ E.coli are multidrug-resistant and genetically diverse”? Regarding antibiotic resistance experiment, the only result presented was the frequency of resistant isolates for individual antibiotics. Would be very interesting to compare resistance profiles presented by clb+ and clb- E. coli groups, and see if clb+ strains are in fact more “multidrug”-resistant than clb- strains. Likewise, it would be interesting to see the distribution of both groups in dendrogram presented in Figure 4. ********** 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 Reviewer #3: 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. |
Revision 1 |
Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani Population PONE-D-21-08856R1 Dear Dr. Bokhari, 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, Grzegorz Woźniakowski, Full professor, PhD, ScD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: |
Formally Accepted |
PONE-D-21-08856R1 Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani Population Dear Dr. Bokhari: 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 Prof. Grzegorz Woźniakowski Academic Editor PLOS ONE |
Open letter on the publication of peer review reports
PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.
We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.
Learn more at ASAPbio .