Isolation of a Virulent Clostridium perfringens Strain from Elaphurus davidianus and Characterization by Whole-Genome Sequence Analysis
, Siyuan Li 1, Yuhang Fu 1, Yan Li 1, Shah Nawaz 1, Jing Chen 2, Guoxiang Yang 2, Jiakui Li 1,*,†and Daoliang Shi 2,3,*,†Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have provided a very extensive article reporting mainly the genomic features of a pathogenic strain of Clostridium perfringens.
The article is too extensive reporting a total of 16 figures plus 11 tables. This is too much. Authors need to reduce the manuscript, namely the amount of tables and figures included in the main manuscript. The high length of the manuscript makes it very hard to follow, with a lack of focus on truly relevant results. Please also consider using figures with different panels.
The methodology needs to be better explained, controls need to be clearly stated, and references are missing since I believe the authors did not develop any of the protocols specifically for this article.
Genomic islands and pseudogenes are simply reported with no further investigation regarding the genes present in the genomic islands or the meaning of the found pseudogenes.
The authors perform comparative genomes, nevertheless, the phylogenomic comparison is not made, only a phylogenetic comparison of the 16s rRNA gene; why not a whole-genome comparison based on SNPs?
the authors start with several reference genomes but the ones used throughout the article are not always the same; no justification is presented.
The results section represents a total of 14 pages, but the discussion is resumed to a total of 2 pages, with low to no result integration between all the findings reported. It is a resume of the result section with some comparison with former literature.
Minor comments:
Lines 27 to 33 - Please add references.
Line 37 - Please replace "flora" with "microbiota".
Line 43 - Please remove the "1".
Lines 50 to 55 - Please add references.
Lines 60 to 62 - Please add references.
Lines 100 to 101 - This information should be stated in the statistical section.
Line 164 - what does the "3" mean before the Figure 2 reference?
Tables 2 and 3 can easily become a figure expressing these results more appealingly.
Figures 4, 5, and 6 are unnecessary in the main article.
Section 3.5 is too big and too fragmented, with a lack of connection between uncovered findings.
Figure 7 - did the authors use nanopore and Illumina technology to sequence this genome? if so, the method section is not clear regarding this aspect. Furthermore, this figure together with Table 4, is not needed in the main manuscript.
Table 5 - Once again, this table is not the focus of the study, since it aims to report pathogenicity/virulence-related features. The overall genomic information is necessary to be reported as a sequencing statistic and quality control measure, nevertheless, not this extensively in the main manuscript.
Figure 8 is again unnecessary and pie charts need to be avoided since they tend to mislead the reader. Additionally, the pink color is not labeled in the figure legend.
Figure 9 - the comparison between databases is also not the main focus of this report, thus this should be removed or allocated to supplementary material.
Lines 280 - there is a hyperlink associated with the text. Please remove it.
Lines 320 - repetition.
Figure 14 and Table 10 have the same data; repetitive.
Comments on the Quality of English LanguageMinor editing of English language required.
Author Response
Dear Editor,
We really appreciate you and the reviewer for valuable comments and suggestions regarding our manuscript entitled ‘Screening of elk-source Clostridium perfringens and whole-genome sequencing analysis of virulent strain CX1-4’, which remarkably helped us to improve the manuscript accordingly.
All the modifications were tracked in the revised manuscript, and detailed point-to-point responses to each comment are provided in below section.
Reviewer 1
- The article is too extensive reporting a total of 16 figures plus 11 tables. This is too much. Authors need to reduce the manuscript, namely the amount of tables and figures included in the main manuscript. The high length of the manuscript makes it very hard to follow, with a lack of focus on truly relevant results. Please also consider using figures with different panels.
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- The methodology needs to be better explained, controls need to be clearly stated, and references are missing since I believe the authors did not develop any of the protocols specifically for this article.
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- Genomic islands and pseudogenes are simply reported with no further investigation regarding the genes present in the genomic islands or the meaning of the found pseudogenes.
Response: We appreciate the reviewer's comment regarding the lack of further investigation into the genes present in genomic islands and the discovered pseudogenes. Our study aimed to identify and characterize the most virulent strain of Clostridium perfringens type A from elk, focusing on establishing a foundational genomic framework, including basic genomic features, phylogenetic relationships, and virulence factors. Detailed functional analyses of the genes within genomic islands and the roles of pseudogenes were beyond the initial scope and required extensive resources, time, and specific experimental setups. These investigations often involve complex bioinformatics analyses and functional genomics experiments that were not feasible within the current study's timeframe. Our findings on genomic islands and pseudogenes are preliminary and provide a basis for future research to explore their potential roles in virulence and pathogenicity.
- The authors perform comparative genomes, nevertheless, the phylogenomic comparison is not made, only a phylogenetic comparison of the 16s rRNA gene; why not a whole-genome comparison based on SNPs?
Response: We appreciate the opportunity to address your concerns and further clarify our methodology. Regarding your query about utilizing 16S rRNA gene sequencing instead of whole-genome comparison based on SNPs, we acknowledge the significance of whole-genome analyses in elucidating detailed genomic variations. However, in our study, we opted for 16S rRNA gene sequencing as a pragmatic and efficient approach to assess genetic relatedness among Clostridium perfringens strains. Its well-established utility in bacterial phylogenetics informed the decision to employ 16S rRNA gene sequencing. This molecular marker is a cornerstone for taxonomic classification and phylogenetic inference, offering valuable insights into evolutionary relationships within bacterial populations. By leveraging 16S rRNA gene sequences, we efficiently evaluated the genetic relatedness among strains and identified phylogenetic patterns within the Clostridium perfringens species.
We hope this explanation addresses your concerns satisfactorily.
- The authors start with several reference genomes but the ones used throughout the article are not always the same; no justification is presented.
Response: Our laboratory previously isolated and identified five strains of C. perfringens in ill elk. After our experiments, we found that CX1-4 strains had the strongest virulence effect and were very representative, so we only carried out whole genome sequencing analysis of CX1-4 strains.
- The results section represents a total of 14 pages, but the discussion is resumed to a total of 2 pages, with low to no result integration between all the findings reported. It is a resume of theresult section with some comparison with former literature.integration between all the findings reported. It is a resume of the result section with some comparison with former literature.
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- Lines 27 to 33 - Please add references.
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- Line 37 - Please replace "flora" with "microbiota".
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- Line 43 - Please remove the "1".
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- Lines 50 to 55 - Please add references.
Response: We added the corresponding references. - Lines 60 to 62 - Please add references.
Response: Thank you for your feedback. Regarding your request to add references to lines 60 to 62, I acknowledge the need for appropriate citations. However, a paucity of existing literature focuses explicitly on the complete genome of Clostridium perfringens isolated from elk. Our study aims to contribute to this area by conducting a comprehensive genomic analysis of C. perfringens strains sourced from elk. As such, we appreciate your understanding of the limited availability of relevant references for this particular aspect of our research.
- Lines 100 to 101 - This information should be stated in the statistical section.
Response: The changes have been made as suggested.
- Line 164 - what does the "3" mean before the Figure 2 reference?
Response: It was a typo error. We have removed the "3".
- Tables 2 and 3 can easily become a figure expressing these results more appealingly.
Response: Thank you for your advice. We have been present Tables 2 and 3 in the manuscript in the form of pictures, Figure 4 and Figure 5, respectively.
- Figures 4, 5, and 6 are unnecessary in the main article.
Response: We have removed Figures 4, 5 and 6 from the manuscript.
- Section 3.5 is too big and too fragmented, with a lack of connection between uncovered findings.
Response: We combined parts 3.5.1, 3.5.2, and 3.5.3 into one section.
- Figure 7 - did the authors use nanopore and Illumina technology to sequence this genome? if so, the method section is not clear regarding this aspect. Furthermore, this figure together with Table 4, is not needed in the main manuscript.
Response: We appreciate your feedback and concerns. We employed nanopore and Illumina sequencing methodologies regarding the sequencing technology used for this genome. We apologize for any clarity regarding this aspect in the method section. We have revised the method section to provide a more detailed explanation of the sequencing techniques utilized in our study. Furthermore, we acknowledge your suggestion regarding deleting Figure 7 and Table 4 in the main manuscript. Thank you for bringing these points to our attention.
- Table 5 - Once again, this table is not the focus of the study, since it aims to report pathogenicity/virulence-related features. The overall genomic information is necessary to be reported as a sequencing statistic and quality control measure, nevertheless, not this extensively in the main manuscript.
Response: The changes have been made as suggested.
- Figure 8 is again unnecessary and pie charts need to be avoided since they tend to mislead the reader. Additionally, the pink color is not labeled in the figure legend.
Response: The changes have been made as suggested.
- Figure 9 - the comparison between databases is also not the main focus of this report, thus this should be removed or allocated to supplementary material.
Response: The changes have been made as suggested.
- Lines 280 - There is a hyperlink associated with the text. Please remove it.
Response: We carefully considered the reviewer's suggestions and revised the manuscript accordingly.
- Lines 320 - repetition.
Response: The changes have been made as suggested.
- Figure 14 and Table 10 have the same data; repetitive.
Response: The changes have been made as suggested.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe objective of the work was the study of Clostridium populations from elk.
The manuscript has significant flaws, which preclude publication of the manuscript. Some notes are herebelow.
1. The origin of the bacteria used in the study is not described. There are no details on how these bacteria had been isolated, e.g., region of the country, medical history of animals from which isolated, antibiotic susceptibility patterns etc.
2. Mouse test. No details are provided regarding this part of the work, e.g., number of mice, route for inoculation of the bacteria, end-points for inoculated mice.
3. There is no description of controls for any of the procedures described in the entirety of the study, e.g., control bacterial isolates, control animals in the pathogenicity study, control chemicals in the various assays performed.
Overall. The manuscript shows significant flaws, both in technical aspects during carrying out the experimental part of the work and in writing up the manuscript. As it is, the manuscript cannot be accepted.
Comments on the Quality of English LanguageExtensive editing of English language required.
Author Response
Dear Editor,
We really appreciate you and the reviewer for valuable comments and suggestions regarding our manuscript entitled ‘Screening of elk-source Clostridium perfringens and whole-genome sequencing analysis of virulent strain CX1-4’, which remarkably helped us to improve the manuscript accordingly.
All the modifications were tracked in the revised manuscript, and detailed point-to-point responses to each comment are provided in below section.
Reviewer 2
Here are some of my comments;
- The origin of the bacteria used in the study is not described. There are no details on how these bacteria had been isolated, e.g., region of the country, medical history of animals from which isolated, antibiotic susceptibility patterns etc.
Response: Our laboratory has done this work in the early stage, and it was published in the Chinese Journal of Wildlife, The specific details of the isolation of the bacteria are illustrated in this article. Fu Yuhang, Wang Mingquan, Gao Gao, et al. Comprehensive diagnosis of Clostridium perfringens disease in Shishou Elk [J]. Journal of Wildlife, 2022,43(01): 202-206.DOI:10.19711/j.cnki.issn2310-1490.2022.01.029.
- Mouse test. No details are provided regarding this part of the work, e.g., number of mice, route for inoculation of the bacteria, end-points for inoculated mice.
Response: We have revised and supplemented the manuscript accordingly.
- There is no description of controls for any of the procedures described in the entirety of the study, e.g., control bacterial isolates, control animals in the pathogenicity study, control chemicals in the various assays performed.
Response: We appreciate your attention to detail and recognize the importance of including descriptions of controls in our study procedures.
For the Mouse LD50 test, we implemented a rigorous experimental design that included experimental and control groups. Specifically, mice in the experimental group were intraperitoneally injected with different concentrations of bacterial solution (0.5ml/mice), while mice in the control group received intraperitoneal injections of normal saline (0.5ml/mice). This approach allowed us to assess the effects of the bacterial solution relative to a baseline saline control, ensuring the validity and reliability of our results. Similarly, in the Pathogenicity study of the most virulent strain, we adhered to the same experimental framework by establishing experimental and control groups. Mice in the experimental group were subjected to intraperitoneal injections of CX1-4 bacterium liquid (0.5 ml/mice), while mice in the control group received intraperitoneal injections of normal saline (0.5 ml/mice). This control setup enabled us to isolate the effects of the bacterium liquid and evaluate its pathogenicity compared to the saline control group.
We understand the importance of transparency and rigor in experimental design, and we will ensure that detailed descriptions of control procedures are included in the manuscript to provide clarity and enhance the reproducibility of our findings. Thank you for your constructive feedback, and we remain committed to addressing all concerns raised during the review process.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have provided an improved manuscript. Nevertheless, several questions were not answered satisfactorily:
1. The article is still too extensive, both in figures and tables. I advise the authors to produce figures with several panels instead of single figures. Additionally, the changes in methodology are still minor, considering the points mentioned in the previous revision round (no references for the methodological component of this work were added).
2. I agree with the authors regarding the time-consuming and resource-consuming aspect of in vitro testing the function of genes encountered on genomic islands and pseudogenes. However, the bioinformatic function prediction of such genes is a simple process that would enrich the present manuscript.
3. The authors used the 16S rRNA gene to perform a comparative phylogenetic analysis of several strains from the same species. The 16S gene is not a good predictor of such a phylogenetic relationship, since this gene is only able to differentiate to the genus taxonomical level. This can even be seen in the analysis of the low bootstrapped values reported by the authors. Please read https://doi.org/10.1038/s41598-024-59667-3 for further information. If the author calculated the genome ANI, using 16S rRNA for phylogenetic analysis is a downgrade of the analysis with no justifiable rationale.
4. The authors did not justify why the number of reference genomes changed throughout the manuscript. This is, in Table 1, the authors have 8 reference strains, while in Figure 9, they have 9 strains, and in Figures 10 and 11, they have 8 strains again, but in Table 6 and Figure 12, only 3 strains are shown.
5. Comparative genomics is performed against several reference genomes, with no rationale behind its selection.
6. Lines 60 to 62 – The authors state “Only a limited number of studies have been carried out…”, this means that there are at least two studies carried out on the topic, thus, those studies need to be cited.
7. The discussion section is still missing several references. Please verify. Furthermore, the lack of result integration as previously mentioned is still lacking.
Author Response
Reviewer 1
- The article is still too extensive, both in figures and tables. I advise the authors to produce figures with several panels instead of single figures. Additionally, the changes in methodology are still minor, considering the points mentioned in the previous revision round (no references for the methodological component of this work were added).
Response: Thank you for your feedback on our revised manuscript. We have addressed your concerns by consolidating figures and tables and adding the necessary references to support the methodological aspects of our work. These changes have resulted in a more concise and focused manuscript, and we hope you find the updated version suitable for publication. Please let us know if you have any further questions or comments.
- I agree with the authors regarding the time-consuming and resource-consuming aspect of in vitro testing the function of genes encountered on genomic islands and pseudogenes. However, the bioinformatic function prediction of such genes is a simple process that would enrich the present manuscript.
Response: We agreed that bioinformatic function prediction for genes on genomic islands and pseudogenes is a straightforward process that can enhance the manuscript. We incorporated this information to provide a more comprehensive understanding of the genes studied.
- The authors used the 16S rRNA gene to perform a comparative phylogenetic analysis of several strains from the same species. The 16S gene is not a good predictor of such a phylogenetic relationship, since this gene is only able to differentiate to the genus taxonomical level. This can even be seen in the analysis of the low bootstrapped values reported by the authors. Please read https://doi.org/10.1038/s41598-024-59667-3(https://www.sci-hub.ee/10.1038/s41598-024-59667-3) for further information. If the author calculated the genome ANI, using 16S rRNA for phylogenetic analysis is a downgrade of the analysis with no justifiable rationale.
Response: We appreciate your valuable feedback on our revised manuscript. You raised a valid concern regarding using the 16S rRNA gene for phylogenetic analysis, and we have carefully considered your suggestions. After reviewing your literature, we agree that the 16S rRNA gene is not a suitable marker for differentiating phylogenetic relationships at the species level, as it can only be distinguished at the genus taxonomic level. We noted the low bootstrap values reported in our previous analysis, supporting your recommendation. As a result, we have decided to remove the 16S rRNA gene-related experiments from the manuscript and instead focus on the more robust genome-based analysis, such as the Average Nucleotide Identity (ANI) approach, as you suggested. This change will strengthen the phylogenetic component of our study and provide a more accurate representation of the relationships between the strains. Thank you again for your insightful feedback, which has helped us improve the quality and reliability of our manuscript.
- The authors did not justify why the number of reference genomes changed throughout the manuscript. This is, in Table 1, the authors have 8 reference strains, while in Figure 9, they have 9 strains, and in Figures 10 and 11, they have 8 strains again, but in Table 6 and Figure 12, only 3 strains are shown.
Response: We appreciate your feedback on our revised manuscript. You pointed out that the number of reference genomes varied throughout the manuscript, which we acknowledge as inconsistent. To clarify, we selected eight reference strains from the NCBI database representing different species and countries. We used nine strains, including CX1-4 and the eight reference strains, for the genomic ANI analysis. For the genomic collinearity analysis, we aligned the genomes of CX1-4 and the eight reference strains using Geneious Prime software. We also refer to several studies for comparative analysis methods, including those by Nowell et al. (2012), Saadh et al. (2022), Myers et al. (2006), Ferreira et al. (2002), Gubash et al. (1997), Teraguchi et al. (1995), Park et al. (2013), Liu et al. (2020), Li et al. (2017), Saito et al. (2018), and Talukdar et al. (2022). These studies provide valuable insights into the genetic and functional characteristics of Clostridium perfringens strains.
- Comparative genomics is performed against several reference genomes, with no rationale behind its selection.
Response: We understand your concern regarding selecting reference genomes for comparative genomics. We selected the reference genomes based on their relevance to the study of Clostridium perfringens and their availability in the NCBI database. The selected reference genomes provide a representative set for comparative analysis and help to understand the genetic characteristics of our isolate better. We have added a detailed description of the methods and references used in our study to provide a clear understanding of our approach and the sources we drew upon.
- Lines 60 to 62 – The authors state “Only a limited number of studies have been carried out…”, this means that there are at least two studies carried out on the topic, thus, those studies need to be cited.
Response: Respected reviewer, we understand your point regarding the statement about the limited number of studies on the topic. To clarify, there are no reports on using whole genome sequencing to study C. perfringens from elk, and we have revised the original presentation in the manuscript to reflect this accurately.
- The discussion section is still missing several references. Please verify. Furthermore, the lack of result integration as previously mentioned is still lacking.
Response: Respected reviewer, we have added references in the discussion and completed the integration of the results.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have made the requested changes and have clarified all potential issues. I have no further comments.
Author Response
Reviewer 2 don't asks anymore question after round of revision so first round comments should be considered as final
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have provided an imporved version of the revised manuscript in which they have attended all comments and suggestion previously raised.
I would just like to point out that in figure 1, panel e) is mislabeled. Please take this opportunity to check all figures and tables, including legends and text references, to avoid any mismatch.
Comments on the Quality of English LanguageMinor editing of English language required.
Author Response
- The authors have provided an improved version of the revised manuscript in which they have attended all comments and suggestions previously raised.
Response: We are very honored to thank the editors and reviewers for their recognition.
- I would just like to point out that in figure 1, panel e) is mislabeled. Please take this opportunity to check all figures and tables, including legends and text references, to avoid any mismatch.
Response: We have modified figure 1 and checked all the other figures keeping in mind your suggestions and updated.
- Minor editing of English language required.
Response: Comment has addressed accordingly.
