Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates

Noriaki Ide; Miki Kawada-Matsuo; Mi Nguyen-Tra Le; Junzo Hisatsune; Hiromi Nishi; Toshinori Hara; Norikazu Kitamura; Seiya Kashiyama; Michiya Yokozaki; Hiroyuki Kawaguchi; Hiroki Ohge; Motoyuki Sugai; Hitoshi Komatsuzawa

doi:10.1371/journal.pone.0280676

Peer Review History

Original SubmissionSeptember 30, 2022
28 Dec 2022 Decision Letter - Guadalupe Virginia Nevárez-Moorillón, Editor PONE-D-22-27105Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolatesPLOS ONE Dear Dr. Komatsuzawa, 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, consider the suggestions of both reviewers, and send the document back for evaluation. Please submit your revised manuscript by Feb 11 2023 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. 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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: The work is well thought out, and carried out the related tests. The perspectives that the author proposes are appropriate to the results found. I have brief doubts, which I will write here considering the line number: Line 18: replace gut microflora with gut microbiota Line 121: include a “.” after “Table 1” Line 125: Missing to include the country of Becton Dickinson and Company Table 1. Why are the recent isolates written as Clostridium, considering that they are now called Clostridioides? Line 15. Metronidazole should be lowercase in this case. Line 176: mentions that qPCR, as previously indicated, however, does not include any reference. Line 182: what is the reason for only using the two indicated strains (JCM5243 and H39)? Line 190: what was the maximum time before the use of the spores? Line 225: remove spaces between Table 2 and throughout the text, and check the space between words. Line 293: unify “Fig.” throughout the text; sometimes, it is not written the same. Table 4: Is there any parameter according to the CLSI to determine the multi-resistance or susceptibility of C. difficile? Line 313: from here on, there are values between “( )” bibliographical references that clarify and homogenize the text. Line 450: replace flora with microbiota Line 515. Homogenize references; here, the year appears in parentheses, and in the others it does not. Reviewer #2: In recent years, the problem of drug-resistant bacteria has been the most important issue in the therapeutic treatment of bacterial infections. In the case of Clostridium spp. infection, the occurrence of drug-resistant bacteria has also become serious problem, and the response to this problem needs to be addressed.　In this paper, using 11 clinical isolates of Clostridium difficile, the authors examined the susceptibility to several bacteriocins, including nisin A, and antibiotics. The results of the nisin A susceptibility test showed that the isolates were classified into two major groups, and the classification was similar for the other bacteriocin susceptibility except Mutacin IV. Additionally, no correlation was found between the expression levels of cpr or dlt genes, a group of molecules involved in nisin A resistance, and its susceptibility. Thus, the authors performed a comparative analysis of the whole genome sequences among the isolates to determine the cause of the differences in nisin A susceptibility. As results, the exact same two group formations seen in the susceptibility were confirmed by the genetic information analysis. Furthermore, the authors found that the amino acid sequences of Cpr molecules are unique to each group of bacteria, and speculated that the differences in the amino acid sequences affects the susceptibility to nisinA. The reviewer considers the current manuscript to be well organized and sufficient for a publication of PLOS ONE if the author will provide suitable responses to the following minor comments; 1. The authors test susceptibility to several bacteriocins in the manuscript. In most bacteriocin susceptibilities, there is a significant difference between the two groups formed by nisinA susceptibility, but not in one. It is strongly expected that this may be caused by structural features of the bacteriocins. However, the current manuscript lacks information on the structure of bacteriocins and their structure-activity relationships. The reviewer recommend that the authors provide a figure showing bacteriocin structure in this regard. 2. Related to comment #1. What would happen to the susceptibility of isolates if other bacteriocins with less structural homology to nisinA, as like mutacin IV, were used in the test? If the authors have any information on this point, they should provide it or discuss it. 3. The pathogenicity of C. difficile has been well studied, and the typical toxins Tcds and CDT are known to be closely related to its virulence. In this study, the authors have divided the isolates into two groups according to nisinA susceptibility, are there any commonalities in the virulence factors in each group? The authors could add an explanation on this point based on whole genome information, which would further strengthen the value of this paper. 4. The authors need to provide more detailed information regarding the source of the bacteria used in this paper. 5. The authors use taurocholate in the experiments. The purpose of its use should be stated in the Methods section. 6. (Line121) Add period after “Table 1”. 7. (Line176) The authors should add a reference paper regarding qPCR experiment. ****** 6. 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Revision 1
4 Jan 2023 Author Response Reviewer #1: The work is well thought out, and carried out the related tests. The perspectives that the author proposes are appropriate to the results found. I have brief doubts, which I will write here considering the line number: Line 18: replace gut microflora with gut microbiota Our response: According to the suggestion, we revised it. Line 121: include a “.” after “Table 1” Our response: According to the suggestion, we revised it. Line 125: Missing to include the country of Becton Dickinson and Company Our response: According to the suggestion, we revised it. Table 1. Why are the recent isolates written as Clostridium, considering that they are now called Clostridioides? Our response: According to the suggestion, we revised it. Line 15. Metronidazole should be lowercase in this case. Our response: According to the suggestion, we deleted this sentence. Line 176: mentions that qPCR, as previously indicated, however, does not include any reference. Our response: According to the suggestion, we put the reference about qPCR method. Line 182: what is the reason for only using the two indicated strains (JCM5243 and H39)? Our response: According to the suggestion, we revised it to explain the reason. Line 190: what was the maximum time before the use of the spores? Our response: According to the suggestion, we revised it. Line 225: remove spaces between Table 2 and throughout the text, and check the space between words. Our response: According to the suggestion, we revised it. Line 293: unify “Fig.” throughout the text; sometimes, it is not written the same. Our response: According to the suggestion, we revised it throughout the text. Table 4: Is there any parameter according to the CLSI to determine the multi-resistance or susceptibility of C. difficile? Our Response: There are MIC breakpoints of several antibiotics according to CLSI. However, among antibiotics used in this study, vancomycin and metronidazole are included in the MIC breakpoints of C. difficile by CLSI, but other antibiotics are not included. However, we wanted to compare the MIC value between 2 groups and found no correlation between 2 groups. Line 313: from here on, there are values between “( )” bibliographical references that clarify and homogenize the text. Our response: According to the suggestion, we revised it. Line 450: replace flora with microbiota Our response: According to the suggestion, we revised it. Line 515. Homogenize references; here, the year appears in parentheses, and in the others it does not. Our response: According to the suggestion, we revised it. Reviewer #2: In recent years, the problem of drug-resistant bacteria has been the most important issue in the therapeutic treatment of bacterial infections. In the case of Clostridium spp. infection, the occurrence of drug-resistant bacteria has also become serious problem, and the response to this problem needs to be addressed.　In this paper, using 11 clinical isolates of Clostridium difficile, the authors examined the susceptibility to several bacteriocins, including nisin A, and antibiotics. The results of the nisin A susceptibility test showed that the isolates were classified into two major groups, and the classification was similar for the other bacteriocin susceptibility except Mutacin IV. Additionally, no correlation was found between the expression levels of cpr or dlt genes, a group of molecules involved in nisin A resistance, and its susceptibility. Thus, the authors performed a comparative analysis of the whole genome sequences among the isolates to determine the cause of the differences in nisin A susceptibility. As results, the exact same two group formations seen in the susceptibility were confirmed by the genetic information analysis. Furthermore, the authors found that the amino acid sequences of Cpr molecules are unique to each group of bacteria, and speculated that the differences in the amino acid sequences affects the susceptibility to nisinA. The reviewer considers the current manuscript to be well organized and sufficient for a publication of PLOS ONE if the author will provide suitable responses to the following minor comments; 1.The authors test susceptibility to several bacteriocins in the manuscript. In most bacteriocin susceptibilities, there is a significant difference between the two groups formed by nisinA susceptibility, but not in one. It is strongly expected that this may be caused by structural features of the bacteriocins. However, the current manuscript lacks information on the structure of bacteriocins and their structure-activity relationships. The reviewer recommend that the authors provide a figure showing bacteriocin structure in this regard. Our responses: According to the suggestion, we showed the structure of bacteriocins including nisin A and its structural related bacteriocins which were discussed in Discussion section as the supplemental figure (Supplemental Fig. 5). 2. Related to comment #1. What would happen to the susceptibility of isolates if other bacteriocins with less structural homology to nisinA, as like mutacin IV, were used in the test? If the authors have any information on this point, they should provide it or discuss it. Our responses: According to the suggestion, we investigated the susceptibility of other bacteriocins with no structural relation to nisin A. As the result, we found no tendency as nisin A susceptibility. Therefore, the different amino acid sequences of CprA-C only affected the susceptibility of nisin A and its-related bacteriocins. We revised Table 3 and Fig. 3 by adding two bacteriocins, and also discussed it in Discussion section. 3. The pathogenicity of C. difficile has been well studied, and the typical toxins Tcds and CDT are known to be closely related to its virulence. In this study, the authors have divided the isolates into two groups according to nisinA susceptibility, are there any commonalities in the virulence factors in each group? The authors could add an explanation on this point based on whole genome information, which would further strengthen the value of this paper. Our responses: We investigated the genes coding for Cdt and Txoin AB (TcdAB), and found a tendency of cdt genes in Group I and II, but did not find the tendency of tcdAB. We added these results in Discussion section. 4. The authors need to provide more detailed information regarding the source of the bacteria used in this paper. Our responses: According to the suggestion, we added the detailed information of the strains in Methods section. Also, the strain name of JCM1296 was corrected to JCM1296T. 5. The authors use taurocholate in the experiments. The purpose of its use should be stated in the Methods section. Our responses: According to the suggestion, we describe the purpose of taurocholate in Material and Methods section. 6. (Line121) Add period after “Table 1”. Our responses: According to the suggestion, we revised it. 7. (Line176) The authors should add a reference paper regarding qPCR experiment. Our responses: According to the suggestion, we put the reference. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0280676.r002
6 Jan 2023 Decision Letter - Guadalupe Virginia Nevárez-Moorillón, Editor Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates PONE-D-22-27105R1 Dear Dr. Komatsuzawa, 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, Guadalupe Virginia Nevárez-Moorillón, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0280676.r003
Formally Accepted
12 Jan 2023 Acceptance Letter - Guadalupe Virginia Nevárez-Moorillón, Editor PONE-D-22-27105R1 Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates Dear Dr. Komatsuzawa: 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. Guadalupe Virginia Nevárez-Moorillón Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280676.r004

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