Integrative Analysis of Fungal and Bacterial Microbiomes Across Skin, Blood, and Stool in Rosacea Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

At first very interesting topic!! 

Comments:

Report eligible criteria of the Rosacea patin5s and controls

Were topical treatment status reported in those patients for example topical ivermectin may alter the results Also demodex status should also be reported and correlated with the microorganisms 

Well presented figures and tables as well as statistics 

Were rosacea subtypes or severity correlated with skin microbioma;

Detailed manuscript with minor need of revisions

Author Response

We would like to sincerely thank the reviewer for taking the time to carefully read our manuscript and provide thoughtful and constructive feedback. We truly appreciate the effort and valuable suggestions, which have helped us to improve the quality and clarity of our work. Please find our point-by-point responses below.

1. Report eligible criteria of the Rosacea patients and controls.

Thank you very much for pointing out that the inclusion and exclusion criteria were not described in sufficient detail. As stated in the manuscript, patients with a first-time diagnosis of rosacea were included in the study. Exclusion criteria were: use of antibiotics or probiotics within the past six months; any topical or systemic treatment for skin conditions within the last four weeks; gastrointestinal symptoms within the last four weeks; and pregnancy. Healthy controls without any dermatological conditions were included. The exclusion criteria for the control group were identical to those applied to the rosacea patients. We have added this information to the Materials and methods section of the manuscript.

2. Were topical treatment status reported in those patients for example topical ivermectin may alter the results. Also Demodex status should also be reported and correlated with the microorganisms 

Thank you very much for this important question. Patients who had received topical therapy within the past four weeks were excluded from the study.

In the present study, we did not consider the density of Demodex mites on the skin of either rosacea patients or the control group. According to the literature, a higher abundance of these mites is typically found on the skin of rosacea patients; however, this aspect was beyond the scope of our current investigation. This limitation has been duly acknowledged in the limitations section.

At the same time, we would like to express our gratitude for your excellent suggestion, which has inspired a future research direction. We plan to target a comparative analysis of the microbiome and mycobiome composition of Demodex mites isolated from the skin of rosacea patients and healthy controls.

3. Were rosacea subtypes or severity correlated with skin microbioma.

Thank you very much for this insightful comment. In the current study, we did not distinguish between different rosacea subtypes or severity levels. Our primary focus was on comparing patients with rosacea to healthy controls. However, we agree that stratifying patients by subtype and disease severity could yield important insights and would be a valuable direction for future research.

Reviewer 2 Report

Comments and Suggestions for Authors

This was a well-performed study of rosacea patients. This is not a well-studied area and the results are intriguing.

1. The microbiome is widely divergent among different populations, and this study only included a small number of rosacea patients from a single county. The authors should identify this as a major limitation.
2. The authors have found alterations in the fungal microbiome in the gut and skin of rosacea patients. Is there any evidence that treatment for these fungal organisms alters disease severity?
3. Can the authors speculate as to why patients may have lower skin alpha diversity than controls?

Author Response

We would like to sincerely thank the reviewer for taking the time to carefully read our manuscript and provide thoughtful and constructive feedback. We truly appreciate the effort and valuable suggestions, which have helped us to improve the quality and clarity of our work. Please find our point-by-point responses below.

1. The microbiome is widely divergent among different populations, and this study only included a small number of rosacea patients from a single county. The authors should identify this as a major limitation.

Thank you very much for the valuable comment. We have added this information to the limitations section of the manuscript.

2. The authors have found alterations in the fungal microbiome in the gut and skin of rosacea patients. Is there any evidence that treatment for these fungal organisms alters disease severity?

Thank you for this interesting and thought-provoking question. To date, antifungal treatment is not part of the standard treatment in rosacea, although recent studies have highlighted the significant role of mycobiome dysbiosis in skin health.(1) Currently, no studies have specifically investigated the effects of antifungal treatment in rosacea. However, antifungal treatment has been shown to alleviate symptoms in conditions such as seborrheic dermatitis, where Malassezia is the predominant fungi involved.(2) Regarding the gut microbiome, several studies have established a link between rosacea and gastrointestinal diseases such as gastrooesophageal reflux disease, irritable bowel syndrome, and small intestine bacterial overgrowth.(3-6) Notably, treatment of SIBO has been associated with improvement in rosacea symptoms.(7, 8) It is also hypothesized that antimicrobial therapy may indirectly influence the fungal microbiome. Further research is warranted to determine whether antifungal treatment could reduce the severity of rosacea and potentially become part of the standard therapeutic approach.

3. Can the authors speculate as to why patients may have lower skin alpha diversity than controls?

Thank you for this insightful question. The reduction in microbial diversity among patients with rosacea is likely associated with an imbalance in the skin microbiome. In our current mycobiome study, we did not observe a statistically significant difference in alpha diversity, consistent with the findings of Wang et al.(9). However, in our previous study investigating the bacterial skin microbiome, we identified a significant decrease in alpha diversity (10), a finding also reported by Thompson et al.(11). In contrast, studies by Rainer et al. (12), which did not reach statistical significance, and Wang et al.(9) demonstrated increase alpha diversity in the skin microbiome of patients with rosacea compared to healthy controls. These inconsistencies across studies may be partly explained by small sample sizes, which can limit statistical power and the ability to detect meaningful differences.

1. Nguyen UT, Kalan LR. Forgotten fungi: the importance of the skin mycobiome. Curr Opin Microbiol. 2022;70:102235.
2. Jung WH. Alteration in skin mycobiome due to atopic dermatitis and seborrheic dermatitis. Biophys Rev (Melville). 2023;4(1):011309.
3. Holmes AD, Spoendlin J, Chien AL, Baldwin H, Chang ALS. Evidence-based update on rosacea comorbidities and their common physiologic pathways. J Am Acad Dermatol. 2018;78(1):156-66.
4. Wu CY, Chang YT, Juan CK, Shieh JJ, Lin YP, Liu HN, et al. Risk of inflammatory bowel disease in patients with rosacea: Results from a nationwide cohort study in Taiwan. J Am Acad Dermatol. 2017;76(5):911-7.
5. Egeberg A, Weinstock LB, Thyssen EP, Gislason GH, Thyssen JP. Rosacea and gastrointestinal disorders: a population-based cohort study. Br J Dermatol. 2017;176(1):100-6.
6. Drago F, De Col E, Agnoletti AF, Schiavetti I, Savarino V, Rebora A, et al. The role of small intestinal bacterial overgrowth in rosacea: A 3-year follow-up. J Am Acad Dermatol. 2016;75(3):e113-e5.
7. Parodi A, Paolino S, Greco A, Drago F, Mansi C, Rebora A, et al. Small intestinal bacterial overgrowth in rosacea: clinical effectiveness of its eradication. Clin Gastroenterol Hepatol. 2008;6(7):759-64.
8. Drago F, Ciccarese G, Parodi A. Effects of the treatment for small intestine bacterial overgrowth on rosacea. J Dermatol. 2017;44(12):e321.
9. Wang R, Farhat M, Na J, Li R, Wu Y. Bacterial and fungal microbiome characterization in patients with rosacea and healthy controls. Br J Dermatol. 2020;183(6):1112-4.
10. Joura MI, Jobbágy A, Dunai ZA, Makra N, Bánvölgyi A, Kiss N, et al. Characteristics of the Stool, Blood and Skin Microbiome in Rosacea Patients. Microorganisms. 2024;12(12).
11. Thompson KG, Rainer BM, Antonescu C, Florea L, Mongodin EF, Kang S, et al. Comparison of the skin microbiota in acne and rosacea. Exp Dermatol. 2021;30(10):1375-80.
12. Rainer BM, Thompson KG, Antonescu C, Florea L, Mongodin EF, Bui J, et al. Characterization and Analysis of the Skin Microbiota in Rosacea: A Case-Control Study. Am J Clin Dermatol. 2020;21(1):139-47.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript compares the mycobiomes of skin, blood, and stool samples between rosacea patients and healthy controls. The sample size is relatively small, but the figures are clear. 

Comments:

1. As mentioned in Lines 108–110, five participants were excluded from data analysis. Please revise Table 1 to present the information for only the included patients and healthy controls.
2. Gut flora can be influenced by many factors such as diet, alcohol consumption, smoking, and BMI. Please include more clinical characteristic information in Table 1.
3. Please specify how the skin samples were collected.
4. Figure 5 focuses on Saccharomyces and Candida. Please explain in detail the rationale for selecting these two fungi.
5. As shown in Figure 5, Prevotella (p = 0.01) and Agathobacter (p = 0.006) displayed significant differences. Could you also present separate figures for Prevotella and Agathobacter? Currently, the results indicate significant differences, but the extent of these differences is unclear.

Author Response

We would like to sincerely thank the reviewer for taking the time to carefully read our manuscript and provide thoughtful and constructive feedback. We truly appreciate the effort and valuable suggestions, which have helped us to improve the quality and clarity of our work. Please find our point-by-point responses below.

1. As mentioned in Lines 108–110, five participants were excluded from data analysis. Please revise Table 1 to present the information for only the included patients and healthy controls.

Thank you very much for the helpful suggestion. We have revised the table accordingly.

2. Gut flora can be influenced by many factors such as diet, alcohol consumption, smoking, and BMI. Please include more clinical characteristic information in Table 1.

Thank you very much for this valuable comment. We have expanded the table to include additional information such as BMI, smoking status, alcohol consumption, and diet-related symptom exacerbation.

3. Please specify how the skin samples were collected.

Thank you for pointing out that this important information was missing from the manuscript. Skin swabs were taken from both cheeks. Participants were instructed not to wash their face for 24 hours prior to sampling. Sterile foam-tipped swabs were used, and each region was vigorously swabbed for 30 seconds while rotating the swab. We have added this information to the revised version of the manuscript.

4. Figure 5 focuses on Saccharomyces and Candida. Please explain in detail the rationale for selecting these two fungi.

We have revised the relevant section of the manuscript to provide a more precise explanation as to why only Saccharomyces and Candida—among the fungal genera showing significant differences in the stool of rosacea patients—were selected for further investigation. The manuscript now includes the following passage: Stool samples from rosacea patients demonstrated a significantly higher median relative abundance of multiple fungal genera compared to skin samples, including Ascomycota, Candida, Chaetothyrales, Didymosphaeria, Penicillium, Rinodina, Russula, Saccharomyces, and Tomentella. However, notable differences emerged in the distribution of these fungi across biological compartments: Ascomycota, Didymosphaeria, and Tomentella exhibited greater relative abundance in blood samples than in stool, implying that their DNA may originate from sources other than, or in addition to, the gastrointestinal tract. Moreover, the median relative abundances of Chaetothyrales, Rinodina, and Russula in stool were consistently below 5%, indicating a limited presence.

In contrast, Saccharomyces and Candida were among the most abundant genera detected in stool samples and are well-established as relevant to human mycobiome dynamics. Taken together, these data justify focusing subsequent analyses exclusively on Saccharomyces and Candida, as they represent the most pertinent fungal taxa for exploring potential links between the gut mycobiome and rosacea pathogenesis.

5. As shown in Figure 5, Prevotella (p = 0.01) and Agathobacter (p = 0.006) displayed significant differences. Could you also present separate figures for Prevotella and Agathobacter? Currently, the results indicate significant differences, but the extent of these differences is unclear.

Thank you for bringing to our attention that the significant differences are not clearly visible in Figure 5. In response, we have prepared a new Figure 6, which demonstrates the abundance of Agathobacter and Prevotella in the stool of rosacea patients depends on the abundance of Saccharomyces and Candida.