Salivary Oral Microbiota in Patients with Prediabetes Undergoing Intragastric Balloon Surgery
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsReview Report,
The manuscript `Salivary Oral Microbiota in Patients with Diabetes Undergoing Intragastric Balloon Surgery' investigates the changes in salivary microbiota among obese patients with type 2 diabetes undergoing intragastric balloon (IGB) placement. The topic is highly relevant, bridging oral microbiology, bariatric therapy, and metabolic disorders. The methodology is generally sound, and the bioinformatic pipeline is well described. However, there are methodological and statistical limitations that affect the strength of the conclusions. The study has potential, but major revision is required to enhance scientific validity and interpretability.
Comment 1
The Abstract should be restructured according to MDPI format: Background, Methods, Results, Conclusions.
Comment 2
The Introduction is overly descriptive; reduce the epidemiological background and focus on the scientific rationale
Comment 3
Sample size and statistical representativeness – The study cohort (20 patients, 18 analyzed) is very small given that approximately 45% of Kuwaiti adults are obese. It is unclear whether a power analysis was performed to determine the minimum required sample size. The authors must clarify how the sample size was calculated and whether the study was designed as a pilot. Without such justification, the representativeness of the sample is questionable, and the generalizability of the findings is limited. Suggestion: Add a sub-section titled 'Sample size and statistical power' and indicate if an effect size estimation was considered.
Comment 4
Short follow-up period – The 3–5-week post-IGB follow-up is too brief to assess microbiota stability. This limitation should be explicitly acknowledged in the Discussion and Conclusions.
Comment 5
Uncontrolled confounding factors – Oral hygiene status, periodontal condition, and dietary habits were not described. These are critical variables that can significantly influence the salivary microbiota and should either be controlled for or discussed as limitations.
Comment 6
Statistical analysis – Please specify whether correction for multiple comparisons (FDR or Bonferroni) was applied. Without such correction, the reported p-values ​​may lead to false positives.
Comment 7
Presentation of results - Figures 5 and 6 are overcrowded and difficult to interpret. It is recommended to add a summary table listing the genera and species that showed significant differences (with direction of change and p-values). Numerical values ​​of Shannon and Simpson indices should be provided.
Comment 8
Discussion and conclusions – The Discussion is detailed but somewhat repetitive. I consider condensing the section and critically discussing how the small sample size and short duration may limit the interpretation of results.
Comment 9
Ensure reference numbering and punctuation follow MDPI style.
Comment 10
A professional English language proofreading is recommended.
Recommendation
Major Revision
Author Response
Reviewer 1
The manuscript `Salivary Oral Microbiota in Patients with Diabetes Undergoing Intragastric Balloon Surgery' investigates the changes in salivary microbiota among obese patients with type 2 diabetes undergoing intragastric balloon (IGB) placement. The topic is highly relevant, bridging oral microbiology, bariatric therapy, and metabolic disorders. The methodology is generally sound, and the bioinformatic pipeline is well described. However, there are methodological and statistical limitations that affect the strength of the conclusions. The study has potential, but major revision is required to enhance scientific validity and interpretability.
Response: Our sincere thanks to the reviewers for their time and efforts in critically reviewing our manuscript. We have put all our efforts in revising the manuscript and hope that the revision is satisfactory.
Comment 1
The Abstract should be restructured according to MDPI format: Background, Methods, Results, Conclusions.
Response: Abstract is organized into sections.
Comment 2
The Introduction is overly descriptive; reduce the epidemiological background and focus on the scientific rationale
Response: The introduction has been thoroughly modified to address the above issue.
Comment 3
Sample size and statistical representativeness – The study cohort (20 patients, 18 analyzed) is very small given that approximately 45% of Kuwaiti adults are obese. It is unclear whether a power analysis was performed to determine the minimum required sample size. The authors must clarify how the sample size was calculated and whether the study was designed as a pilot. Without such justification, the representativeness of the sample is questionable, and the generalizability of the findings is limited. Suggestion: Add a sub-section titled 'Sample size and statistical power' and indicate if an effect size estimation was considered.
Response: We thank the reviewer for raising this critical issue. This was considered a pilot study and therefore we did not do power calculations. It has now been mentioned in the methods as well as under limitations in the discussion section.
Comment 4
Short follow-up period – The 3–5-week post-IGB follow-up is too brief to assess microbiota stability. This limitation should be explicitly acknowledged in the Discussion and Conclusions.
Response: The follow-up period 3-5 weeks was an error. It should have been 6 weeks and corrections have been made in all relevant places in the revised manuscript. The above concern has been addressed as a limitation in the discussion section as well as in the conclusions.
Comment 5
Uncontrolled confounding factors – Oral hygiene status, periodontal condition, and dietary habits were not described. These are critical variables that can significantly influence the salivary microbiota and should either be controlled for or discussed as limitations.
Response: The above concern has been addressed as a study limitation.
Comment 6
Statistical analysis – Please specify whether correction for multiple comparisons (FDR or Bonferroni) was applied. Without such correction, the reported p-values ​​may lead to false positives.
Response: We conducted a simple two-group comparison (baseline vs follow-up) using Mann Whitney U test, to test the hypothesis. Since we did not do any multiple comparisons, corrections such as FDR or Bonferroni were not required.
Comment 7
Presentation of results - Figures 5 and 6 are overcrowded and difficult to interpret. It is recommended to add a summary table listing the genera and species that showed significant differences (with direction of change and p-values). Numerical values ​​of Shannon and Simpson indices should be provided.
Response: Table 1 presenting significant differences has been added in the revised manuscript. Numerical values of Shannon and Simpson indices are added in the text.
Comment 8
Discussion and conclusions – The Discussion is detailed but somewhat repetitive. I consider condensing the section and critically discussing how the small sample size and short duration may limit the interpretation of results.
Response: Discussion section has been condensed now removing repetitive text. Small sample size and a short follow-up period have been discussed in the limitations.
Comment 9
Ensure reference numbering and punctuation follow MDPI style.
Response: Thank you. Reference numbering and punctuation have been adjusted as per the journal guidelines.
Comment 10
A professional English language proofreading is recommended.
Response: The revised manuscript has been thoroughly checked for grammar and typos.
Recommendation
Major Revision
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript addresses the important issue of the impact of bariatric procedures on the oral microbiota in patients with obesity and type 2 diabetes. This topic is clinically relevant. The authors utilize modern NGS methods and bioinformatic analyses. The article is largely clear and methodologically sound, but requires several important additions and corrections:
- Small study sample size (n=20)
- Lack of a control group, e.g., patients with obesity who did not undergo intragastric balloon [IGB] placement.
- At what point in the study did patients receive oral hygiene instruction? Was it at the time of the first saliva sample collection? Could the changes in the numbers of specific bacterial genera and species compared to baseline be due to changes in oral hygiene habits?
- Short observation time . It is unclear when the balloon was removed in the patients. Was the second saliva sample collection related to the balloon removal? If not, why was the collection performed after 3-5 weeks, and what was the exact time of collection? Was saliva collected for each patient after the same time interval from the first collection or at different times?
- Please clarify how saliva samples were collected from each patient: at the same time of day, by what method, and whether patients avoided eating or oral hygiene activities before collection – these are factors that influence the oral microbiome.
- There is no information about the duration of the disease in individual patients, the level of diabetes control, and medications used.
- Did the patients undergo oral examinations for periodontal disease, which has a significant impact on the oral microbiome?
- The Mann–Whitney U test, which is not a paired-sample test, was used in the statistical analyses.
- The data are presented descriptively in the results; it would be better to present them in tables. Why don't the authors address clinical parameters (BMI, HbA1c)?
- The Materials and Methods should describe the groups – the results section describes the baseline and follow-up groups, which were not previously described.
- The nomenclature of bacterial species should be standardized.
- Please correct any linguistic errors.
Author Response
Reviewer 2
This manuscript addresses the important issue of the impact of bariatric procedures on the oral microbiota in patients with obesity and type 2 diabetes. This topic is clinically relevant. The authors utilize modern NGS methods and bioinformatic analyses. The article is largely clear and methodologically sound, but requires several important additions and corrections:
Small study sample size (n=20)
Lack of a control group, e.g., patients with obesity who did not undergo intragastric balloon [IGB] placement.
Response: The above concerns have been addressed as limitations of the study.
At what point in the study did patients receive oral hygiene instruction? Was it at the time of the first saliva sample collection? Could the changes in the numbers of specific bacterial genera and species compared to baseline be due to changes in oral hygiene habits?
Response: Oral hygiene instructions were given to the patients two weeks before they arrived for the balloon surgery. Thus, it is unlikely that the changes in the abundance levels of a few specific bacterial species were due to oral hygiene.
Short observation time . It is unclear when the balloon was removed in the patients. Was the second saliva sample collection related to the balloon removal? If not, why was the collection performed after 3-5 weeks, and what was the exact time of collection? Was saliva collected for each patient after the same time interval from the first collection or at different times?
Response: The balloon was removed from the patients after 6 months. But the samples were collected 6 weeks after the surgery. The follow-up samples were collected from all patients after the same time interval i.e., 6 weeks. We apologize that 3-5 weeks was a mistake.
Please clarify how saliva samples were collected from each patient: at the same time of day, by what method, and whether patients avoided eating or oral hygiene activities before collection – these are factors that influence the oral microbiome.
Response: Thank you for the important comment. We have now added info about the above in the methods part.
There is no information about the duration of the disease in individual patients, the level of diabetes control, and medications used.
Response: All patients were pre-diabetic. As mentioned in the inclusion criteria, the patients did not take any antibiotics during the past 6 months.
Did the patients undergo oral examinations for periodontal disease, which has a significant impact on the oral microbiome?
Response: It was not possible to conduct oral examination on the patients. This has been included in the study limitations.
The Mann–Whitney U test, which is not a paired-sample test, was used in the statistical analyses. The data are presented descriptively in the results; it would be better to present them in tables.
Response: Significant differences in relative abundances of genera and species are now presented in Table 1.
Why don't the authors address clinical parameters (BMI, HbA1c)?
Response: Mean BMI and HbA1c data from the study participants has now been added in the results part.
The Materials and Methods should describe the groups – the results section describes the baseline and follow-up groups, which were not previously described.
Response: The groups have now been described in the methods.
The nomenclature of bacterial species should be standardized.
Response: Manuscript has been thoroughly checked for inconsistencies concerning bacterial species nomenclature, italicized names etc.
Please correct any linguistic errors.
Response: The revised manuscript has been thoroughly checked for grammar and typos.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors, the study has scientific merit and relevant results, but some points need critical review:
-Introduction: Although it provides a good foundation on obesity and DM2, there is a lack of updated data on:
the prevalence of oral diseases in these patients;
the clinical impact of oral dysbiosis in obese and diabetic individuals;
the relevance of saliva as a diagnostic marker;
what is already known about oral microbiota in individuals who have undergone bariatric surgery.
The inclusion of these elements will make the study's rationale more complete.
-Methodology: It is well detailed in terms of laboratory aspects, but essential dental information is lacking:
presence or absence of periodontal disease;
standardization of oral hygiene before collection;
collection times and salivary flow control;
use of mouthwashes or medications that alter oral microbiota.
Could any of these methodological factors modify the results?
Discussion: Integrate the findings with possible clinical repercussions (risk of caries, periodontitis, halitosis, oral inflammation).
Conclusion: Suggest dental follow-up protocols for patients after intragastric balloon placement; and to further emphasize the importance of the dentist as part of a multidisciplinary team, clarify how the results can assist dentists in preventive care.
Author Response
Reviewer 3
Dear authors, the study has scientific merit and relevant results, but some points need critical review:
Response: We sincerely thank the reviewer for an overall positive feedback and critical comments.
-Introduction: Although it provides a good foundation on obesity and DM2, there is a lack of updated data on:
the prevalence of oral diseases in these patients;
the clinical impact of oral dysbiosis in obese and diabetic individuals;
the relevance of saliva as a diagnostic marker;
what is already known about oral microbiota in individuals who have undergone bariatric surgery.
The inclusion of these elements will make the study's rationale more complete.
Response: We totally agree with the reviewer. The introduction has been thoroughly modified now.
-Methodology: It is well detailed in terms of laboratory aspects, but essential dental information is lacking:
presence or absence of periodontal disease;
Response: Unfortunately, it was not possible for us to do an oral examination. This is now added as a limitation in the discussion part.
standardization of oral hygiene before collection;
Response: Oral hygiene instructions were given to all patients before two weeks of their arrival for the bariatrc surgery.
collection times and salivary flow control;
Response: The details are added in the method section.
use of mouthwashes or medications that alter oral microbiota.
Could any of these methodological factors modify the results?
Response: The patients did not take any medication or antibiotics for at least 6 months before the surgery.
Discussion: Integrate the findings with possible clinical repercussions (risk of caries, periodontitis, halitosis, oral inflammation).
Response: This issue has been addressed in the discussion.
Conclusion: Suggest dental follow-up protocols for patients after intragastric balloon placement; and to further emphasize the importance of the dentist as part of a multidisciplinary team, clarify how the results can assist dentists in preventive care.
Response: Conclusion has been modified accordingly.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have satisfactorily addressed all concerns raised. The revised version presents improved methodological clarity and more balanced discussion of limitations.
I am satisfied with the revision and recommend acceptance of the manuscript.
Author Response
We sincerely thank the reviewer for their valuable time and efforts on critically reviewing our manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsI would like to thank the authors for responding to the comments submitted and for preparing a revised version of the manuscript. Below I present additional issues that require further refinement.
- The title should be modified – instead of: "in patients with diabetes" it should be written: "in patients with prediabetes". It should also be added that this is a pilot study.
- In methods – instead of the expectoration method, there should be a spitting method for collecting saliva.
- Are the mean Shannon index values ​​(baseline value = 5.62, follow-up period = 0.49) reported correctly in the text?
- The Mann–Whitney U test, which is not a paired-sample test, was used in the statistical analyses – why was this test chosen?
- References 42 to 47 are given in the discussion, but they do not appear in the bibliography.
- Was the lack of a control group included in the study limitations?
Author Response
I would like to thank the authors for responding to the comments submitted and for preparing a revised version of the manuscript. Below I present additional issues that require further refinement.
Response: Our sincere thanks to the reviewer for their precious time on reviewing our manuscript.
- The title should be modified – instead of: "in patients with diabetes" it should be written: "in patients with prediabetes". It should also be added that this is a pilot study.
Response: The title has been modified accordingly.
2. In methods – instead of the expectoration method, there should be a spitting method for collecting saliva.
Response: The method has been changed as suggested.
3. Are the mean Shannon index values ​​(baseline value = 5.62, follow-up period = 0.49) reported correctly in the text?
Response: We apologize for the error. It has been corrected as follows: Mean diversity index values from observed, Shannon (baseline=5.62, follow-up=5.49) or Simpson
4. The Mann–Whitney U test, which is not a paired-sample test, was used in the statistical analyses – why was this test chosen?
Response: The following explanation is added in the statistics part of methods:”…. determined using the non-parametric Mann–Whitney U-test, since the data was not normally distributed as assessed by Shapiro-Wilk test and histograms.”
5. References 42 to 47 are given in the discussion, but they do not appear in the bibliography.
Response: We apologize for the error. The missing references have now been added.
6. Was the lack of a control group included in the study limitations?
Response: The following text has been added in the study limitations paragraph of the discussion: Another key limitation of this study is the absence of a control group without intragastric balloon placement. Such a control would have enabled the differentiation of intervention-specific effects from background temporal variations in the gut microbiota, thereby allowing a more precise attribution of observed effect sizes to the balloon procedure.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
I congratulate you on your dedication in developing this work and writing the manuscript, which will certainly contribute significantly to the advancement of microbiology and dentistry.
Author Response
We sincerely thank the reviewer for their valuable time and efforts on critically reviewing our manuscript.
