Genomic Characterization of an O-Antigen-Deficient, Hydrogen Sulfide-Negative Salmonella enterica Serovar Senftenberg Isolated from Cooked Mussels

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Genomic characterization of an atypical hydrogen sulfide-negative Salmonella enterica serovar Senftenberg strain lacking somatic antigen expression isolated from cooked mussels" is written well but needs some improvements to enhance the quality of the manuscript. The improvement points are listed below, and author should address all the comments. 

1. Entericashould be italic in the whole manuscript such as in line 20 is non-italic.
2. The following sentence Line 40-41“Salmonella enterica is one of the most common pathogens responsible for foodborne 40 illnesses worldwide”is very true and should be strengthen with recent and relevant references. Please cite recent that indicate food-origin salmonella contamination including this.  https://doi.org/10.47278/journal.ijvs/2025.025.
3. A recent study also reported the colistin resistance in Salmonella which can be cited from Line 59-60to strengthen the http://dx.doi.org/10.29261/pakvetj/2025.118 
4. The authors compared the phsoperon of SF1060 with Typhimurium LT2. But these are different serovars, so many SNPs differences are naturally expected even if both strains were normal. Therefore, it cannot be concluded that these mutations are related to the H₂S-negative phenotype because they may simply reflect normal serovar differences. So, I recommend comparing SF1060 with a closely related H₂S-positive S. Senftenberg strain. Then analyze phs, ars, and cysJIH operons, and identify mutations present only in SF1060. Accordingly, also should be corrected in Results/Discussion section.
5. The phylogenetic tree (Figure 4) lacks bootstrap values and node support statistics. The phylogenetic clustering, specifically the close relationship between SF1060 and the 2015 regional strain CFSAN080379 is a major conclusion supporting the endemicity of this lineage, so it is critical to demonstrate the statistical robustness of these clades. Thus, modifying Figure 4 along with mentioning bootstrap method in Methods section should be performed.
6. The methods describe 24 representative genomes were selected to create a "representative dataset". However, authors did not explain about search queries, inclusion/exclusion thresholds, or sampling methods used to filter the thousands of available NCBI genomes down to this specific subset. So, providing a reproducible genome selection workflow in Methods seems concerning regarding selection bias.
7. The authors mentioned CLC Genomics Workbench v20 and Geneious Prime v2025.0.3 were used for the comparative genomic alignments shown in Figure 2 and Figure 3, respectively. But, did not specified that which alignment algorithm and non-default/modified parameters were used, because without this analysis is not reproducible. So, mentioning exact alignment algorithm, setting/parameters used in Methods section should be done.
8. The strain Senftenbergcame from cooked mussels presenting its for exceptional heat resistance compared to other Salmonella serovars. Even the authors have complete genome data, did not check for LHR (Locus of Heat Resistance) and/or the clpK gene, which are known heat-resistance determinants. So, analyzing the genome for these markers or other heat-resistance genes would significantly complete the study's scope and provide mechanistic context for the pathogen's survival in a cooked product.
9. The investigation into the H₂S-negative phenotype is incomplete given the availability of a fully closed genome. The authors limit their analysis to searching for stop codons and coding mutations inside the phs, cysJIH, and ars But the gene expression can also be affected by promoter mutations and regulatory-region insertions/deletions. So, since the authors have a complete closed genome, should also analyze the promoter sequences, upstream regulatory regions and transcription factor binding regions for mutations. And, also regulatory-region analysis should be added in Methods and Results.
10. The authors listed amino acids substitutions, but did not explain that whether these substitutions are important, do they lie in catalytic domains, and whether similar mutations were previously linked to H₂S-negative strains. Mutations are currently biologically uncontextualized. So, discussion on protein domains, catalytic sites, structural significance and literature evidence for similar mutations is of great importance.
11. The authors claimed that this specific atypical ST14 lineage shows "endemic persistence", but only have newly sequenced SF1060 strain with a single historical isolate (CFSAN080379) from 2015 which are insufficient to strongly prove "endemic persistence". So, the authors should temper this conclusion or clearly acknowledge the limited sample size of this specific lineage as a constraint on generalizability.

Author Response

Comment 1: The manuscript "Genomic characterization of an atypical hydrogen sulfide-negative Salmonella enterica serovar Senftenberg strain lacking somatic antigen expression isolated from cooked mussels" is written well but needs some improvements to enhance the quality of the manuscript. The improvement points are listed below, and author should address all the comments. 

Response 1: We thank the reviewer for their thoughtful comments and constructive suggestions. We appreciate the time and effort invested in reviewing our manuscript. We believe that addressing these comments significantly improved the quality and clarity of our manuscript.

Comment 2: Enterica should be italic in the whole manuscript such as in line 20 is non-italic.

Response 2: done.

Comment 3: The following sentence Line 40-41“Salmonella enterica is one of the most common pathogens responsible for foodborne 40 illnesses worldwide”is very true and should be strengthen with recent and relevant references. Please cite recent that indicate food-origin salmonella contamination including this.  https://doi.org/10.47278/journal.ijvs/2025.025.

Response 3: Agreed and done.

Comment 4: A recent study also reported the colistin resistance in Salmonella which can be cited from Line 59-60to strengthen the http://dx.doi.org/10.29261/pakvetj/2025.118 

Response 4: Agreed and done

Comment 5: The authors compared the phs operon of SF1060 with Typhimurium LT2. But these are different serovars, so many SNPs differences are naturally expected even if both strains were normal. Therefore, it cannot be concluded that these mutations are related to the H₂S-negative phenotype because they may simply reflect normal serovar differences. So, I recommend comparing SF1060 with a closely related H₂S-positive S. Senftenberg strain. Then analyze phs, ars, and cysJIH operons, and identify mutations present only in SF1060. Accordingly, also should be corrected in Results/Discussion section.

Response 5: Thank you for this comment. We would like to clarify that we did not intend to claim that the missense mutations identified in the phs or related operons were responsible for the H₂S-negative phenotype. Our conclusion was instead the opposite: because no premature stop codons or obvious disruptive mutations were identified in the phs, ars, or cysJIH operons, the H₂S-negative phenotype is unlikely to be explained by the classical inactivating mutations previously described in these pathways.

We agree, however, that the comparison with S. Typhimurium LT2 could be misinterpreted as implying phenotype association. To avoid this confusion, we have revised the Results and Discussion sections to clarify that the comparison with LT2 was used only as a reference sequence and not to infer causality of the observed SNPs. We now explicitly state that these sequence differences may simply reflect normal divergence between serovars and that no evidence was presented linking them to the phenotype.

Revised text in the Results section:

“The phs operon of strain SF1060, responsible for reduction of thiosulfate to hydrogen sulfide, did not contain premature stop codons or other obvious disruptive mutations. Comparison with the reference S. Typhimurium LT2 sequence identified several SNP differences in phsA and phsC; however, these likely reflect normal sequence divergence between serovars and were not interpreted as causative of the H₂S-negative phenotype.”

Revised text in the Discussion section:

“No nonsense mutations or other obvious inactivating mutations were detected in the phs, ars, or cysJIH operons, suggesting that the H₂S-negative phenotype of SF1060 is not explained by the classical gene-disrupting mechanisms previously reported for atypical Salmonella strains. Although several missense differences were observed relative to S. Typhimurium LT2, these comparisons involved different serovars and were not used to infer causality.”

We also removed the sentence “It is possible that the missense mutations identified affect protein folding, substrate binding, or catalytic efficiency, or alternatively, that regulatory mechanisms controlling operon expression are disrupted at the transcriptional or post-translational level, which will require future transcriptomic analysis to resolve.”

Comment 6: The phylogenetic tree (Figure 4) lacks bootstrap values and node support statistics. The phylogenetic clustering, specifically the close relationship between SF1060 and the 2015 regional strain CFSAN080379 is a major conclusion supporting the endemicity of this lineage, so it is critical to demonstrate the statistical robustness of these clades. Thus, modifying Figure 4 along with mentioning bootstrap method in Methods section should be performed.

 

Response 6: Thank you for this valuable suggestion. We agree that statistical support for the phylogenetic clustering is important, particularly because the close relationship between SF1060 and strain CFSAN080379 supports our conclusion regarding the persistence of this lineage in the Galician marine environment. Accordingly, we revised the phylogenetic analysis and the manuscript as follows: (i) the original allele-based wgMLST neighbor-joining tree was moved to the Supplementary Material (Supplementary Figure 1);
(ii) a new Figure 4 was generated using a core genome SNP alignment derived from the wgMLST analysis; (iii) phylogenetic reconstruction was performed using the Maximum Likelihood method with the Kimura 2-parameter substitution model and 1,000 bootstrap replicates in MEGA12.1; and (iv) bootstrap support values were added to the nodes in the revised Figure 4. The Materials and Methods section was also updated to describe the SNP extraction process, phylogenetic reconstruction method, and bootstrap analysis in detail. The revised analysis confirms the close clustering between SF1060 and strain CFSAN080379 with strong statistical support, reinforcing the hypothesis of long-term persistence of this ST14 lineage in the regional aquaculture environment.

 

Comment 7: The methods describe 24 representative genomes were selected to create a "representative dataset". However, authors did not explain about search queries, inclusion/exclusion thresholds, or sampling methods used to filter the thousands of available NCBI genomes down to this specific subset. So, providing a reproducible genome selection workflow in Methods seems concerning regarding selection bias.

Response 7: Thank you for this important comment. We agree that transparency in genome selection criteria is important to minimize potential selection bias and improve reproducibility. We would like to clarify that our intention was not to generate a comprehensive population structure analysis of all publicly available S. Senftenberg genomes, but rather to contextualize strain SF1060 within a geographically and temporally diverse dataset, while specifically assessing its relationship to strains previously isolated from mussels in Galicia, Spain.

Comment 8: The authors mentioned CLC Genomics Workbench v20 and Geneious Prime v2025.0.3 were used for the comparative genomic alignments shown in Figure 2 and Figure 3, respectively. But, did not specified that which alignment algorithm and non-default/modified parameters were used, because without this analysis is not reproducible. So, mentioning exact alignment algorithm, setting/parameters used in Methods section should be done.

 

Response 8: Thank you for this comment. We agree that specifying the alignment algorithms and parameters improves reproducibility. In our analyses, the alignments shown in Figures 2 and 3 were generated using the default settings of the respective software packages, and no custom or modified parameters were applied. To clarify this, we have updated the Materials and Methods section to explicitly state the algorithms and settings used: “Pairwise whole-genome alignments used for comparative genomic analysis (Figure 2) were performed in CLC Genomics Workbench v20 (QIAGEN, Redwood City, CA, USA) using the default whole-genome alignment settings. Gene alignments shown in Figure 3 were generated in Geneious Prime v2025.0.3 (Biomatters Ltd., Auckland, New Zealand) using the default global alignment algorithm and default parameters. No non-default or manually modified alignment parameters were applied in either analysis.” We have also indicated in the corresponding figure legends that the analyses were performed using the default settings of each software package.

 

Comment 9: The strain Senftenberg came from cooked mussels presenting its for exceptional heat resistance compared to other Salmonella serovars. Even the authors have complete genome data, did not check for LHR (Locus of Heat Resistance) and/or the clpK gene, which are known heat-resistance determinants. So, analyzing the genome for these markers or other heat-resistance genes would significantly complete the study's scope and provide mechanistic context for the pathogen's survival in a cooked product.

 

Response 9: Thank you for this insightful suggestion. We agree that the presence of heat-resistance determinants such as the LHR (Locus of Heat Resistance) and the clpK gene would be relevant in the context of a Salmonella strain isolated from a cooked seafood product, particularly given the known association of S. Senftenberg with enhanced thermal tolerance. However, the primary objective of this study was to characterize the atypical phenotypic features of strain SF1060 related to the absence of H₂S production and somatic antigen expression, rather than to investigate mechanisms of heat resistance or survival after cooking. Therefore, analysis of heat-resistance loci was outside the original scope of the study.

 

 

Comment 10: The investigation into the H₂S-negative phenotype is incomplete given the availability of a fully closed genome. The authors limit their analysis to searching for stop codons and coding mutations inside the phs, cysJIH, and ars But the gene expression can also be affected by promoter mutations and regulatory-region insertions/deletions. So, since the authors have a complete closed genome, should also analyze the promoter sequences, upstream regulatory regions and transcription factor binding regions for mutations. And, also regulatory-region analysis should be added in Methods and Results.

 

Response 10: Thank you for this suggestion. We agree that regulatory-region mutations could potentially contribute to the H₂S-negative phenotype. However, the aim of this study was to determine whether the classical gene-disrupting mechanisms previously associated with H₂S-negative Salmonella strains, such as nonsense mutations or major coding-region disruptions in the phs, ars, and cysJIH operons, were present in SF1060. Since no such mutations were identified, we concluded only that the phenotype is not explained by these commonly reported mechanisms. A detailed analysis of promoter regions and regulatory elements was beyond the scope of the present study and would require complementary functional validation (e.g., transcriptomics or promoter activity assays). We have now clarified this limitation in the Discussion section.

 

Comment 11: The authors listed amino acids substitutions, but did not explain that whether these substitutions are important, do they lie in catalytic domains, and whether similar mutations were previously linked to H₂S-negative strains. Mutations are currently biologically uncontextualized. So, discussion on protein domains, catalytic sites, structural significance and literature evidence for similar mutations is of great importance.

 

Response 11: Thank you for this comment. We agree that the biological significance of the observed amino acid substitutions cannot be determined solely from sequence comparison. However, our intention was not to imply that these mutations are functionally important or associated with the H₂S-negative phenotype, but rather to report that no obvious inactivating mutations were identified in the analyzed operons. Because no functional, structural, or experimental analyses were performed, and similar substitutions have not been previously linked to H₂S-negative Salmonella strains, we believe that further interpretation would be speculative and beyond the scope of this study. To avoid overinterpretation, we have revised the text to clarify that these substitutions are descriptive observations only and that their functional relevance remains unknown.

 

Comment 12: The authors claimed that this specific atypical ST14 lineage shows "endemic persistence", but only have newly sequenced SF1060 strain with a single historical isolate (CFSAN080379) from 2015 which are insufficient to strongly prove "endemic persistence". So, the authors should temper this conclusion or clearly acknowledge the limited sample size of this specific lineage as a constraint on generalizability.

 

Response 12: Thank you for this important comment. We agree that the term “endemic persistence” may be too strong given the limited number of closely related isolates currently available. Our conclusion was based on the observation that SF1060 and strain CFSAN080379, isolated 10 years apart from mussels in the same geographic region, showed extremely close genomic relatedness. However, we acknowledge that two isolates alone are insufficient to definitively demonstrate long-term endemic persistence of this specific lineage. Accordingly, we have tempered the wording throughout the manuscript and now refer more cautiously to the “possible persistence” or “continued circulation” of this ST14 lineage in the regional marine aquaculture environment. We also added a statement acknowledging the limited sample size as a study limitation.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents WGS results of an atypical Salmonella Senftenberg isolate that did not produce black colonies on XLD agar (commonly used for this pathogen) and could not be serotyped by conventional methods. The topic is highly relevant given the importance of this pathogen as a cause of foodborne illness, which supports the scientific merit of the study.

The manuscript is very well organized and written, with no methodological flaws, and I have only minor recommendations to further improve its quality.

Minor remarks

L20: Double-check the use of italics for all scientific names throughout the manuscript.

L22 and 67: Provide the full name of the culture medium before using the abbreviation. After line 67, the abbreviation can be used.

L86: Specify the concentration of the peptone water.

L143: The reference is duplicated.

The plates shown in Figure 1, although understandable, are of relatively low quality, and the lettering is excessively large. I suggest improving the image resolution and overall visual presentation.

L217: Double-check the use of italics for all genes throughout the manuscript. In this section, also specify which antimicrobial classes the detected genes are associated with.

L415: The authors should clearly indicate which antimicrobial classes the isolate harbored antimicrobial resistance genes (ARGs) against.

Author Response

Comment 1: The manuscript presents WGS results of an atypical Salmonella Senftenberg isolate that did not produce black colonies on XLD agar (commonly used for this pathogen) and could not be serotyped by conventional methods. The topic is highly relevant given the importance of this pathogen as a cause of foodborne illness, which supports the scientific merit of the study. The manuscript is very well organized and written, with no methodological flaws, and I have only minor recommendations to further improve its quality.

Response 1: We thank the reviewer for their thoughtful comments and constructive suggestions. We appreciate the time and effort invested in reviewing our manuscript. We believe that addressing these comments significantly improved the quality and clarity of our manuscript.

Comment 2: L20: Double-check the use of italics for all scientific names throughout the manuscript.

Response 2: correct, and fixed.

Comment 3: L22 and 67: Provide the full name of the culture medium before using the abbreviation. After line 67, the abbreviation can be used.

Response 3: done and fixed.

Comment 4: L86: Specify the concentration of the peptone water.

Response 4: done, the concentration used was 2%.

Comment 5: L143: The reference is duplicated.

Response 5: fixed it. Thanks.

Comment 6: The plates shown in Figure 1, although understandable, are of relatively low quality, and the lettering is excessively large. I suggest improving the image resolution and overall visual presentation.

Response 6: Agreed and done. We have created a new figure of higher quality and reduce the text in the figure.

Comment 7: L217: Double-check the use of italics for all genes throughout the manuscript. In this section, also specify which antimicrobial classes the detected genes are associated with

Response 7: fixed it. Thanks

.

Comment 8: L415: The authors should clearly indicate which antimicrobial classes the isolate harbored antimicrobial resistance genes (ARGs) against.

Response 8: we have added “such as colistin” after the last-resort antibiotic comment. 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents an interesting study on the characterization of an atypical Salmonella enterica serovar strain isolated from cooked mussels. The combination of phenotypic characterization and hybrid whole-genome sequencing is used for this characterization which is provides a strength to the study and demonstrates the value of WGS in resolving „problematic“ isolates that may escape conventional detection methods. The topic is timely and important for food safety surveillance, particularly in seafood-associated environments where atypical Salmonella variants may persist.

Overall, the manuscript is well structured, scientifically sound, and suitable for publication after minor revision. The genomic analyses are convincing, and the discussion and the discussion adequately highlights the implications of surveillance for atypical strains. I particularly appreciated the attempt to connect genomic findings to practical diagnostic challenges in routine laboratories.

The study is scientifically valuable, relevant for food microbiology and genomic surveillance, and overall suitable for publication.

There are only few small issues that would improve the clarity and overall quality of the manuscript.

The discussion about the H₂S-negative phenotype is balanced, but currently somewhat speculative. Since no disruptive mutations were identified in the major operons involved in H₂S production, the authors should more clearly acknowledge that the identified missense mutations are only hypothetical contributors to the phenotype. This has already been partially discussed, but the wording sometimes sounds more convincing than the data to support it.

A genomic explanation for the loss of somatic antigen expression is plausible and well supported by a rearrangement in the rfb operon. In contrast, the mechanism explaining the H₂S negativity remains unresolved. Therefore, the manuscript would have benefited from explicitly stating this as a limitation earlier in the discussion section.

The figures are overall well prepared, although some text and numerical labels are quite small and difficult to read. Increasing the font size slightly would improve clarity.

 

there are several grammatical inconsistencies and small editing issues throughout the text:

Latin names (even abbreviated) should be in italic, there is a lot of inconsistency throughout the text so please correct this.

Line 43. “cultured-based methods” should be “culture-based methods”

Line 122. I presume it should state “Filtlong”.

Line 142. please remove duplicated citation

Author Response

Comment 1: The manuscript presents an interesting study on the characterization of an atypical Salmonella enterica serovar strain isolated from cooked mussels. The combination of phenotypic characterization and hybrid whole-genome sequencing is used for this characterization which is provides a strength to the study and demonstrates the value of WGS in resolving „problematic“ isolates that may escape conventional detection methods. The topic is timely and important for food safety surveillance, particularly in seafood-associated environments where atypical Salmonella variants may persist.

Overall, the manuscript is well structured, scientifically sound, and suitable for publication after minor revision. The genomic analyses are convincing, and the discussion and the discussion adequately highlights the implications of surveillance for atypical strains. I particularly appreciated the attempt to connect genomic findings to practical diagnostic challenges in routine laboratories. The study is scientifically valuable, relevant for food microbiology and genomic surveillance, and overall suitable for publication. There are only few small issues that would improve the clarity and overall quality of the manuscript.

 

Response 1: We thank the reviewer for their thoughtful comments and constructive suggestions. We appreciate the time and effort invested in reviewing our manuscript. We believe that addressing these comments significantly improved the quality and clarity of our manuscript.

 

Comment 2: The discussion about the H₂S-negative phenotype is balanced, but currently somewhat speculative. Since no disruptive mutations were identified in the major operons involved in H₂S production, the authors should more clearly acknowledge that the identified missense mutations are only hypothetical contributors to the phenotype. This has already been partially discussed, but the wording sometimes sounds more convincing than the data to support it. A genomic explanation for the loss of somatic antigen expression is plausible and well supported by a rearrangement in the rfb operon. In contrast, the mechanism explaining the H₂S negativity remains unresolved. Therefore, the manuscript would have benefited from explicitly stating this as a limitation earlier in the discussion section.

 

Response 2: Agreed and we have modified the paragraph as follows: “…While the genomic basis for the loss of somatic antigen expression is supported by the identified rearrangement within the rfb operon, the molecular mechanism underlying the H₂S-negative phenotype remains unresolved. Although several missense mutations were identified in genes associated with sulfur metabolism, their contribution to the phenotype remains hypothetical and was not functionally validated in this study. Together, these traits illustrate the complexity of S. enterica phenotypic variation, and the difficulties associated with isolation and identification using conventional phenotypic methods.”

Comment 3: The figures are overall well prepared, although some text and numerical labels are quite small and difficult to read. Increasing the font size slightly would improve clarity.

 Response 3: Thank you for this helpful comment. We agree that improving the readability of the figures enhances the overall clarity of the manuscript. In response, we have revised all figures by increasing the font size of text and numerical labels to ensure they are easier to read and more visually clear throughout the manuscript.

Comment 4: there are several grammatical inconsistencies and small editing issues throughout the text:

Latin names (even abbreviated) should be in italic, there is a lot of inconsistency throughout the text so please correct this.

Response 4: correct, and fixed.

Comment 5: Line 43. “cultured-based methods” should be “culture-based methods”

Response 5: correct, and fixed.

Comment 6: Line 122. I presume it should state “Filtlong

Response 6: correct, and fixed.

Comment 7: Line 142. please remove duplicated citation

Response 7: correct, and fixed.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have i proved the manuscript but still i think there is need of little modifications listed below:

1. Authors need to remove the unnecessary dots in the subheadings of methods and results sections.
2. I think the title needs to be modified. I think "lacking somatic antigen expression" should be deleted from the title.
3. There are some unnecessary large spaces between paragraphs that should be corrected in final version. 
4. Figure 2 contains duplicated figures. Please keep latest version and delete earlier. 
5. The results section 3.5. contains figure but there is no legend and also needs citations in the text. 
6. Figure 4 in section 3.6. contains two figures that can be labelled as figure 4a and 4b. Update the in-text citations accordingly. 
7. The Discussion heading is merged with behind text that should be separated into line and needs to be bold. 

Author Response

Comment 1:  Authors need to remove the unnecessary dots in the subheadings of methods and results sections.

Response 1: Agreed and done.

Comment 2: I think the title needs to be modified. I think "lacking somatic antigen expression" should be deleted from the title.

Response 2: We appreciate the reviewer's feedback regarding the length and phrasing of the title. We agree that 'lacking somatic antigen expression' was overly wordy. However, because the loss of the somatic antigen is a key defining feature of this atypical strain, we felt it was important to retain the concept. To address the reviewer's concern while maintaining scientific precision, we have condensed the phrasing and revised the title to: 'Genomic characterization of an O-antigen-deficient, hydrogen sulfide-negative Salmonella enterica serovar Senftenberg isolated from cooked mussels'.

Comment 3: There are some unnecessary large spaces between paragraphs that should be corrected in final version. 

Response 3: Agreed and fixed.

Comment 4:  Figure 2 contains duplicated figures. Please keep latest version and delete earlier. 

Response 4: Agreed and done.

Comment 5: The results section 3.5. contains figure but there is no legend and also needs citations in the text. 

Response 5: It was misaligned in the markup version. We have included only one final version without markup changes to avoid this kind of event. The section 3.5 contain both a figure, legend and citation in the text.

Comment 6: Figure 4 in section 3.6. contains two figures that can be labelled as figure 4a and 4b. Update the in-text citations accordingly. 

Response 6: Same as response 5.

Comment 7: The Discussion heading is merged with behind text that should be separated into line and needs to be bold. 

Response 7: Agreed and fixed.

Author Response File: Author Response.docx