Genotypes and Phylogenetic Analysis of Helicobacter pylori Clinical Bacterial Isolates

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This in an interesting topic about Helicobacter pylori, this manuscript in general form is manuscript is very well written, well-structured and with sufficient information.

The introduction is adequate, with the enough information and well-written.

In statistical section please add a text if used any type of statistical analysis.

All figures are ok, but the figure 5 is small, please increase the size of this figure.

In figure 5 there are a bold square, please indicate if that means something.

In discussion section please add a short text at the beginning with a little context, so as not to go directly into the discussion of results. 

In line 341 please the word in vitro in italics text.  In vitro.

Comments on the Quality of English Language

okay

Author Response

Dear Reviewer,

We appreciate the time and effort you put into providing us with feedback on our manuscript and we appreciate the valuable comments and improvements you made. We have incorporated the suggestions made by the reviewers. The comments and responses are listed below.

1. This in an interesting topic about Helicobacter pylori, this manuscript in general form is manuscript is very well written, well-structured and with sufficient information. The introduction is adequate, with the enough information and well-written.

Response: We appreciate your comments and your positive observations.

2. In statistical section please add a text if used any type of statistical analysis.

Response: Only a clustering analysis was performed to cluster the strains according to the presence or absence of virulence genes (line 158).

3. All figures are ok, but the figure 5 is small, please increase the size of this figure.

Response: We thank you for your observation, the size of the figure was increased.

4. In figure 5 there are a bold square, please indicate if that means something.

Response: The bold square indicates the bacterial isolates from the present study, this was noted in the figure caption (line 313).

5. In discussion section please add a short text at the beginning with a little context, so as not to go directly into the discussion of results. 

Response: We appreciate this suggestion. Accounting for this given suggestion, we have now add a short text at the beginning (line 334).

6. In line 341 please the word in vitro in italics text.  In vitro.

Response: The word has already been changed (line 358).

Reviewer 2 Report

Comments and Suggestions for Authors

The work is devoted to a thorough and painstaking study of H. pylori isolates obtained from biopsy material of patients with various diseases of the digestive system. The isolates were studied by microscopic, microbiological and molecular biology methods in order to establish the presence of certain genetic markers that determine the virulence of these pathogens.

Dear authors, it is necessary to note the high methodological level of this work; all studies were carried out using a complex of modern methods and approaches. 

There are a number of questions regarding the work.

The goal of the work was to study the level of virulence of isolated isolates and its correlation with various phenotypic and genetic characteristics of bacteria. However, the level of virulence was established only through theoretical research, based on published works. While no experiments were conducted, the work does not even provide clinical data on the condition of the patients from whom the isolates were obtained. Thus, the goal of the work was not achieved; it is necessary to reformulate the goal or supplement the work with an experimental assessment of the virulence of the isolated isolates.

There are also a few smaller comments.

Section 2.1 should indicate whether informed consent was obtained from all patients and whether the work was carried out in accordance with the Declaration of Helsinki. These points are extremely important when performing experiments using human biopsy material. This section also requires to indicate the manufacturer of the antibiotics used in the experiments, the CO2 incubator, and the source of the blood of adult goats.

Section 2.2.1 contains an insufficiently detailed description of Gram staining.

Section 2.2.2 contains an insufficient description of the urease test, one of the fundamental methods for determining the presence of H. pylori.

Section 2.2.3 does not indicate the source of PCR primers.

In Figure 6, the red background makes it impossible to distinguish the codes representing the isolates, and it remains unclear which isolates are more genetically similar.

Despite the comments made, the work makes a very good impression, especially with regard to the review of the literature on the problem and the discussion of the results obtained. Nevertheless, without experimental verification of the obtained calculations, the work cannot be considered completely completed.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Reviewer,

We appreciate the time and effort you put into providing us with feedback on our manuscript and we appreciate the improvements you made. We have incorporated most of the suggestions made by the reviewers. The comments and responses are listed below.

1. The goal of the work was to study the level of virulence of isolated isolates and its correlation with various phenotypic and genetic characteristics of bacteria. However, the level of virulence was established only through theoretical research, based on published works. While no experiments were conducted, the work does not even provide clinical data on the condition of the patients from whom the isolates were obtained. Thus, the goal of the work was not achieved; it is necessary to reformulate the goal or supplement the work with an experimental assessment of the virulence of the isolated isolates.

Response: We appreciate your comment, the objective has already been reformulated emphasizing that the aim of the work was to identify the virulence factors associated with diseases and not to experimentally verify the virulence of each strain (line 89).

2. Section 2.1 should indicate whether informed consent was obtained from all patients and whether the work was carried out in accordance with the Declaration of Helsinki. These points are extremely important when performing experiments using human biopsy material.

Response: In the original document, this paragraph is included in the Informed Consent Statement section (line): “Patient consent was waived due to the biopsy samples provided by the Civil Hospital of Guadalajara "Fray Antonio Alcalde" were not obtained directly from patients but were provided by the hospital from biopsies that were performed on patients at the direction of their physician to diagnose the presence of H. pylori, regardless of the purposes of the present study and only a portion of each biopsy was taken to perform isolation bacterial” (line 428).

According to the Declaration of Helsinki, research should not present a risk to the patient. In this study, there was no risk to the patients since there was no interaction with them and no procedure was performed other than that indicated by their physician (line 98-102).

In addition, the Declaration of Helsinki mentions that the privacy and confidentiality of the patients must be protected, which effectively occurred in this study, since no personal data, other than age and sex, was provided by the physicians to the researchers. The Declaration of Helsinki also mentions that the project and the experimental method must be reflected in a protocol and this must be reviewed and approved by an ethics evaluation committee, which was also done, the Research Ethics Committee of the Old Civil Hospital of Guadalajara "Fray Antonio Alcalde" approved the research project entitled "Alternatives for the control of Helicobacter pylori: bacteriophages and plant extracts of axihuitl" with official letter number HCG / CEI-0027 / 22, this is included in the original document in the Institutional Review Board Statement section (line 425) (document of approval by the ethics committee is attached).

3. This section also requires to indicate the manufacturer of the antibiotics used in the experiments, the CO2 incubator, and the source of the blood of adult goats.

Response: We thank you for your observation, the missing data has already been added. The blood was purchased from a local supplier (hemo-proveedores, batch#S-1276) (line 109-113).

4. Section 2.2.1 contains an insufficiently detailed description of Gram staining.

Response: We had thought that since it is a widely used technique it was not necessary to add the details, however, because you consider it necessary this information was added (line 120).

5. Section 2.2.2 contains an insufficient description of the urease test, one of the fundamental methods for determining the presence of H. pylori.

Response: In the same way as in the previous methodology, this information was added, thank you for emphasizing these details (line 129).

6. Section 2.2.3 does not indicate the source of PCR primers.

Response: The manufacturer of the primers was added (line 146).

7. In Figure 6, the red background makes it impossible to distinguish the codes representing the isolates, and it remains unclear which isolates are more genetically similar.

Response: We appreciate this observation, you are right, it is difficult to see the codes because of the red background, the figure has already been modified and we hope this change allows for better visualization.

8. Despite the comments made, the work makes a very good impression, especially with regard to the review of the literature on the problem and the discussion of the results obtained. Nevertheless, without experimental verification of the obtained calculations, the work cannot be considered completely completed.

Response: In agreement with your comment about reformulating the objective, we believe that by reformulating it we managed to give more clarity to the work in general and in this way the work is no longer considered incomplete.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

- Consider improving the conclusion in the abstract and the conclusion of the article itself, emphasizing the relevance and perspectives of the findings,

- Update references used, recent reviews must be considered, especially when discussing mechanisms,

- Introduction can be summarized, various information could be included in the discussion,

- Emphasize the objective of the study,

-The summary must also present numerical results.

- Figure 1 - indicate something that helps to measure the size of the colony, indicate incubation days in the figure caption,

- Table 4 mixes data and figures, that could be better described

- There was a predominance of females in the study, and samples from patients over 60 years old, do the authors identify any correlation?

Author Response

Dear Reviewer,

We appreciate all your valuable comments on our work. We have revised our manuscript according to the comments, questions, and suggestions of the reviewers and we have incorporated most of the suggestions. The comments and responses are listed below.

1. Consider improving the conclusion in the abstract and the conclusion of the article itself, emphasizing the relevance and perspectives of the findings,

Response: We thank you for this observation, the conclusion was improved by adding relevant information regarding the isolated genotypes and emphasizing the contribution of these genotypes to disease development (line 409).

2. Update references used, recent reviews must be considered, especially when discussing mechanisms,

Response: We know that it is important to have up-to-date information, so we have updated the references to the cagA mechanism. However, since the information in the other references is still current, and several of these references are the original reference, citing a newer reference would not make sense because the most recent reference in turn cites the old reference, so it was decided to keep these references (line 64).

3. Introduction can be summarized, various information could be included in the discussion,

Response: We appreciate your comment, the introduction has already been summarized. However, no information was moved to the discussion because we consider it important that the introduction provides a broad overview of the context of the work and all the information contained in it seemed relevant to us.

4. Emphasize the objective of the study,

Response: The objective has been reformulated emphasizing that the aim of the work was to identify the virulence factors associated with diseases and not to experimentally verify the virulence of each strain. In this way, the objective is clearer (line 89).

5. The summary must also present numerical results.

Response: The number of samples and strains was included to complement the abstract (line 20).

6. Figure 1 - indicate something that helps to measure the size of the colony, indicate incubation days in the figure caption,

Response: Error bars were placed. The incubation days are 3 and are shown in the figure caption in hours (72 h).

7. Table 4 mixes data and figures, that could be better described

Response: Because two of the editors besides yourself pointed out this table and requested that the images be removed, they were removed leaving only the text (line 250).

8. There was a predominance of females in the study, and samples from patients over 60 years old, do the authors identify any correlation?

Response: This cannot be concluded due to the small sample size. This information was added to the text emphasizing that further studies are necessary to conclude a relationship (line 349-353).

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “GENOTYPES AND PHYLOGENETIC ANALYSIS OF Helicobacter pylori CLINICAL BACTERIAL ISOLATES” aimed to isolate and identify H. pylori from clinical samples of human stomach biopsies and characterize the isolates according to its colonial and microscopic morphology, phenotypic characteristics, and molecular genotyping (MLST, 16S rRNA and other genes sequencing) to determine the bacterial isolate virulence.

The research is well planned, but the results show provide more details regarding MLST analysis. Unfortunately, the number of samples (n=22) and isolated strains (n=3) studied was small, so maybe the article could be reduced and published as a “short communication”.

Abstract:

L15-16: This sentence is not necessary in the abstract.

L17 – Include the number of samples and strains isolated in the abstract.

MLST and ST identified are not presented in the abstract.

L23 – “16S” must not be in italic format.

Keywords: remove “Mexican strains”

Introduction

L69 – Describe the abbreviation “kb” (first mention)

Material and Methods

L118 – Delete “from the Antiguo Hospital Civil de Guadalajara”

L139 – Consider presenting Tables 1 and 2 as supplementary material.

L169, 184 – “16S” must not be in italic format.

Results

L236 – Delete “from the Antiguo Hospital Civil de Guadalajara”

L244 – Remove the figures inside the Table 4.

L259 – Figure 3 is unnecessary. Its is better to present the results combining Tables 4 and 5.

L280 - Do not use “our” is not an appropriate scientific language

L287, 296, 300 – “16S” must not be in italic format.

The authors should provide more information regarding 16S rRNA sequencing, for example, the size (pb) of the fragment evaluated for each strain. For evaluation the results, I suggested the authors to use the curated database EzBioCloud (Yoon SH, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017 May;67(5):1613-1617. doi: 10.1099/ijsem.0.001755. Only species that presented an identification percentage ≥ 98.7 % were considered valid (Yoon et al, 2017). Results that presented more than one possible species within the same genus were considered as identified at the genus level.

L314 – If the authors find new alleles (and consequently new STs) the alleles must be analyzed and validate by the database curator. The new STs numbers and identification of the deposited strains must be also provided.

Discussion

L376-380 - This paragraph doesn't seem to be “discussion section”, since in no point it mentions or compare the information’s with the results obtained in the present study.

L385 - Do not use “our” is not an appropriate scientific language

Discussion section should include the new STs found and clonal complex identified.

Author Response

Dear Reviewer,

We greatly appreciate the comprehensive comments you provided on the submitted article. We ensured that each of the reviewers' comments was carefully addressed and the article was revised accordingly. The comments and responses are listed below.

Abstract:

1. L15-16: This sentence is not necessary in the abstract.

Response: The sentence was removed.

2. L17 – Include the number of samples and strains isolated in the abstract.

Response: The number of samples and strains was included to complement the abstract (line 20).

3. MLST and ST identified are not presented in the abstract.

Response: MLST analysis was added to the abstract (line 19, 28), however, we consider that the sequence numbers obtained in pubMLST should not be included in the abstract since these are accession numbers (like those generated in genbank) and these codes have no place in the abstract.

4. L23 – “16S” must not be in italic format.

Response: All "16S" in the document were reviewed and modified.

5. Keywords: remove “Mexican strains”

Response: The keyword “Mexican strain” was removed (line 30).

Introduction

6. L69 – Describe the abbreviation “kb” (first mention)

Response: The abbreviation has already been described (line 66).

Material and Methods

7. L118 – Delete “from the Antiguo Hospital Civil de Guadalajara”

Response: The name of the hospital has already been removed from the 2.2 subtitle (line 117).

8. L139 – Consider presenting Tables 1 and 2 as supplementary material.

Response: We appreciate your comment, this information is not really necessary in the main document, so both tables will be presented as supplementary material.

9. L169, 184 – “16S” must not be in italic format.

Response: All "16S" in the document were reviewed and modified.

Results

10. L236 – Delete “from the Antiguo Hospital Civil de Guadalajara”

Response: The name of the hospital has already been removed from the 3.3 subtitle (line 243).

11. L244 – Remove the figures inside the Table 4.

Response: The figures were removed leaving only the text (line 250).

12. L259 – Figure 3 is unnecessary. Its is better to present the results combining Tables 4 and 5.

Response: We thank you for your comment, however, we decided that the figure remains due to it is the evidence of our results from the genotypic characterization of the different isolated strains.

13. L280 - Do not use “our” is not an appropriate scientific language

Response: The word was changed and the general language was revised, thank you for noticing this detail (line 286).

14. L287, 296, 300 – “16S” must not be in italic format.

Response: All "16S" in the document were reviewed and modified.

15. The authors should provide more information regarding 16S rRNA sequencing, for example, the size (pb) of the fragment evaluated for each strain. For evaluation the results, I suggested the authors to use the curated database EzBioCloud (Yoon SH, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017 May;67(5):1613-1617. doi: 10.1099/ijsem.0.001755. Only species that presented an identification percentage ≥ 98.7 % were considered valid (Yoon et al, 2017). Results that presented more than one possible species within the same genus were considered as identified at the genus level.

Response: The size of each sequenced fragment was added next to its GenBank accession number in the results section (line 304-305). On the other hand, the sequences were analyzed in the EzBioCloud database, where it was determined that the isolated strains indeed belong to Helicobacter pylori, with a similarity ≥ 98.7 %, and show a lower similarity with other species of the same genus (line 305-308), (the results are attached).

16. L314 – If the authors find new alleles (and consequently new STs) the alleles must be analyzed and validate by the database curator. The new STs numbers and identification of the deposited strains must be also provided.

Response: The sequences were submitted and analyzed by the curator of the pubMLST database and were assigned allele numbers, which have already been added to the paper in the results section (line 326).

Discussion

17. L376-380 - This paragraph doesn't seem to be “discussion section”, since in no point it mentions or compare the information’s with the results obtained in the present study.

Response: Thank you for this important observation, the paragraph was modified and moved to make more sense (line 392-394).

18. L385 - Do not use “our” is not an appropriate scientific language

Response: The word was changed and the general language was revised (line 401).

19. Discussion section should include the new STs found and clonal complex identified.

Response: The new allele sequences were added in the discussion as well as in results (line 399).

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

Ríos-Sandoval et al. analyzed some clinically isolated H.pylori strains from human stomach biopsies, they tested the virulence of each strain through colonial morphology, Gram staining, urease, catalase and oxidase tests and identification of virulent genes and alleles using PCR.

1. What are those images in Fig.1, hemolysis on the culture plates? You need a lower mag to show hemolysis around the colonies and put scale bars.

2.  Fig. 2, separate higher mag images for the morphological characteristics you indicate

3. Table 4, you don't need images here.

4. Fig. 3, put the corresponding bp length and name for each amplification here near the gel photo, also (+/-).

5. Fig. 5, maybe you should include some common strains here that are well studied in the lab?

6. Together with Fig. 5, maybe you can compare the characteristics between well-studied strains and the clinical isolates you have?

5. 

Comments on the Quality of English Language

Minor edits are required for writing.

Author Response

Dear Reviewer,

The authors would like to thank you for your valuable time and valuable comments. We have taken all comments into account carefully. The comments and responses are listed below.

1. What are those images in Fig.1, hemolysis on the culture plates? You need a lower mag to show hemolysis around the colonies and put scale bars.

Response: It is not hemolysis in the culture, it is just bacterial colonies, each little ball is a colony. The scale bars were placed.

2. 2, separate higher mag images for the morphological characteristics you indicate

Response: Zoom images of characteristic bacterial morphologies have been added, we hope the magnification is sufficient.

3. Table 4, you don't need images here.

Response: We appreciate your observation, the figures were removed leaving only the text (line 250).

4. Fig. 3, put the corresponding bp length and name for each amplification here near the gel photo, also (+/-).

Response: The corresponding bp length and name for each amplification were included in the figure.

5. Fig. 5, maybe you should include some common strains here that are well studied in the lab?

Response: Throughout the work, the ATCC 43504 strain is used as a control; this reference strain is a well-studied strain in the laboratory.

6. Together with Fig. 5, maybe you can compare the characteristics between well-studied strains and the clinical isolates you have?

Response: In all experiments the reference strain ATCC 43504 was used to compare the characteristics.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, thank you for the changes you have made. The results described in the text correspond to the stated goal. The methods and techniques used to solve it are highly informative and up to date. I recommend accepting the article in its present form.

Author Response

Dear authors, thank you for the changes you have made. The results described in the text correspond to the stated goal. The methods and techniques used to solve it are highly informative and up to date. I recommend accepting the article in its present form.

Response: Dear reviewer, we greatly appreciate your response and your recommendation to accept the article.

Reviewer 3 Report

Comments and Suggestions for Authors

In this version, the authors presented several changes I consider appropriate to finalize and publish the article.

Author Response

In this version, the authors presented several changes I consider appropriate to finalize and publish the article.

Response: Dear reviewer, we greatly appreciate your response and your recommendation to publish the article.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “GENOTYPES AND PHYLOGENETIC ANALYSIS OF Helicobacter pylori CLINICAL BACTERIAL ISOLATES” was revised and many points were revised and/or answered by the authors. However, the MLST results are still confused and not clear. The ST is the combination of the seven alleles of the genes: atpA, efp, mutY, ppa, trpC, ureI, and yphC. The authors should provide the allele sequence of each gene. If the sequence(s) is(are) new, it must be sent to the curator and the new allele sequencing number is assigned. Consequently, a new ST number is also assigned. These information’s must be provided in the text with access link of the isolates (to prove that the isolate, allele and ST was deposited). In addition, the new STs described must be evaluate to Clonal Complex formed in the database to perform a real epidemiology analysis of the strains. (see some examples of MLST results description and discussion: Costa PV, et al. Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil. Food Res Int. 2020 Mar;129:108805. doi: 10.1016/j.foodres.2019.108805. Epub 2019 Nov 21. PMID: 32036894. / Luz IDS, et al. Assessment of the microbiological quality of natural mineral waters according to the manufacturing time of 20 L returnable packs in Brazil. FEMS Microbiol Lett. 2020 Aug 1;367(15):fnaa120. doi: 10.1093/femsle/fnaa120. PMID: 32678435.).

If the authors decided to keep gel figures, it should be better to send as supplementary material, since they are unnecessary and take a lot of space of the manuscript. In EZBioCloud analysis for identification, the authors must select the “Valid names only”.

Author Response

Dear Reviewer,

The authors would like to thank you for your valuable time and valuable comments. We have made the new corrections. The corresponding comments and responses are listed below.

1. The manuscript “GENOTYPES AND PHYLOGENETIC ANALYSIS OF Helicobacter pylori CLINICAL BACTERIAL ISOLATES” was revised and many points were revised and/or answered by the authors. However, the MLST results are still confused and not clear. The ST is the combination of the seven alleles of the genes: atpA, efp, mutY, ppa, trpC, ureI, and yphC. The authors should provide the allele sequence of each gene. If the sequence(s) is(are) new, it must be sent to the curator and the new allele sequencing number is assigned. Consequently, a new ST number is also assigned. These information’s must be provided in the text with access link of the isolates (to prove that the isolate, allele and ST was deposited). In addition, the new STs described must be evaluate to Clonal Complex formed in the database to perform a real epidemiology analysis of the strains. (see some examples of MLST results description and discussion: Costa PV, et al. Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil. Food Res Int. 2020 Mar;129:108805. doi: 10.1016/j.foodres.2019.108805. Epub 2019 Nov 21. PMID: 32036894. / Luz IDS, et al. Assessment of the microbiological quality of natural mineral waters according to the manufacturing time of 20 L returnable packs in Brazil. FEMS Microbiol Lett. 2020 Aug 1;367(15):fnaa120. doi: 10.1093/femsle/fnaa120. PMID: 32678435.).

The allele sequences were submitted and accepted in the pubMLST database, so the assigned accession numbers (for both alleles and ST) were included in the results and discussion. With the accession numbers from the multi-locus analysis, the graph in figure 6 was made again, wich presented some changes. In addition, in the discussion some phrases were added between lines 401-403.

2. If the authors decided to keep gel figures, it should be better to send as supplementary material, since they are unnecessary and take a lot of space of the manuscript.

We appreciate your comment, however, we consider this image as an important part of our research, so we prefer to keep the image within the article.

3. In EZBioCloud analysis for identification, the authors must select the “Valid names only”.

We have already taken care of this detail and the result is the same as previously reported, evidence of the analysis is attached.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

Authors have answered questions in the last revision.

Comments on the Quality of English Language

N/A

Author Response

Authors have answered questions in the last revision.

Response: Dear reviewer, we appreciate your time and your response.