Differences in the Biliary Microbiome Between Biliary Tract Cancer and Benign Biliary Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The bile microbiomes of individuals with biliary tract cancer (BTC) and biliary benign disorders (BBDs) were compared in this study. 16S rRNA sequencing was used to examine the V3–V4 region of bacterial DNA. Patients with BTC tended to have lower microbial alpha diversity than those with BBDs, although this difference was not statistically significant. Approximately 85% of the bacterial species were shared by the two groups, but 6% and 9% of the species were specific to BBDs and BTC, respectively.

Overall, the results indicate that different bacterial species distributions were linked to malignant versus benign biliary diseases, despite the small changes in global microbial diversity. These findings suggest that the bile microbiome may play a part in biliary tract illness and call for more research into its diagnostic and treatment implications.

But before it is fit for publishing, the author must fix a few issues, which I listed below.

1. Healthy controls for bile analysis
   The authors examined bile juice samples from BTC and BBD patients for microbial analysis; however, the rationale for not including healthy control subjects is unclear. A clear explanation should be provided to justify the absence of healthy controls, particularly given their importance for baseline microbial comparison.
2. Choice of V3–V4 region of 16S rRNA
   The authors selected the V3–V4 region of the 16S rRNA gene for microbial profiling. The scientific rationale for choosing this specific region should be clearly explained, including its advantages in terms of taxonomic resolution, coverage, and reliability. Appropriate references should be cited to support this choice.
3. Consideration of diet in patient grouping
   Among the parameters used for patient grouping, it is unclear whether dietary factors were considered. Diet is a significant variable that can influence bile secretion, bile composition, and microbial communities. The authors should clarify whether dietary intake was assessed or controlled, and discuss its potential impact if it was not included.
4. Clarification of “total microbial abundance” (Line 161)
   In line 161, the authors refer to “total microbial abundance” and compare it with their findings. It is unclear whether this term refers to the total microbial population in the human body, within the gastrointestinal tract, or specifically within bile juice. This needs to be clearly defined to avoid ambiguity.
5. Confusion in Figure 2 and percentage calculations
   The presentation of Figure 2 and the associated percentage calculations are confusing. For example, it is unclear why 5,433 species represent only 5.9%, 803 species represent 9.3%, and 311 shared species account for 84.4%. The reference or denominator used to calculate these percentages should be clearly stated. Additionally, the percentages mentioned in lines 163–164 do not match those shown in the figure. This inconsistency needs to be corrected and clarified.
6. Explanation of the Shannon diversity index
   The manuscript mentions the use of the Shannon method for diversity analysis but does not explain what this method is. The authors should clearly describe the Shannon diversity index, explain why it was chosen, and discuss its acceptance and relevance in microbial ecology studies. Proper citations should be included to support its use.

Author Response

1. Healthy controls for bile analysis

The authors examined bile juice samples from BTC and BBD patients for microbial analysis; however, the rationale for not including healthy control subjects is unclear. A clear explanation should be provided to justify the absence of healthy controls, particularly given their importance for baseline microbial comparison.

Response 1. We appreciate the Reviewer’s insightful comments regarding the inclusion of a healthy control group. We agree that this group would have provided a clearer biliary microbiome baseline. However, obtaining bile from healthy individuals necessitates invasive procedures like endoscopic retrograde cholangiopancreatography and percutaneous transhepatic biliary drainage, which pose serious risks, including pancreatitis, bleeding, and perforation. In accordance with the Declaration of Helsinki, subjecting healthy volunteers to these risks without direct clinical benefit is unethical. Consequently, following the established standards in biliary research, we used bile samples from patients with benign biliary diseases as a surrogate control group. We have added a statement regarding this practical limitation in the Discussion section (page 12, lines 311–314 of the revised manuscript).

2. Choice of V3–V4 region of 16S rRNA

The authors selected the V3–V4 region of the 16S rRNA gene for microbial profiling. The scientific rationale for choosing this specific region should be clearly explained, including its advantages in terms of taxonomic resolution, coverage, and reliability. Appropriate references should be cited to support this choice.

Response 2. We appreciate the Reviewer’s constructive comments on the selection of the 16S rRNA hypervariable region. We targeted the V3–V4 region because it is widely recognized that it provides superior taxonomic resolution and higher alpha diversity coverage compared to those afforded by the properties of other regions (V1–V9). Scientific evidence suggests that this specific region offers the most accurate species-level identification and reliable microbial community profiling, as demonstrated in previous benchmarking studies [Refs 8–10 in the manuscript]. Accordingly, we have added a detailed scientific rationale and supporting references to the Materials and Methods section (page 3, lines 106–110) to clarify the reliability and advantages of this choice.

3. Consideration of diet in patient grouping

Among the parameters used for patient grouping, it is unclear whether dietary factors were considered. Diet is a significant variable that can influence bile secretion, bile composition, and microbial communities. The authors should clarify whether dietary intake was assessed or controlled, and discuss its potential impact if it was not included.

Response 3. We agree with the Reviewer that dietary habits can influence biliary microbiome. Although strict dietary control was not enforced, most patients were provided with standardized hospital meals during their stay. In accordance with the standard clinical protocols, all patients underwent a minimum 8-hour fasting period before their procedure or surgery; therefore, we believe that the potential influence of immediate dietary intake on biliary microbiome samples was effectively minimized. Furthermore, we believe that observing the microbial community under real-world clinical conditions — rather than in a strictly controlled environment — enhanced the generalizability of our findings and increased their value for future diagnostic and therapeutic applications. We have added a statement acknowledging this limitation and the clinical relevance of our approach in the Discussion section (page 13, lines 328–333).

4. Clarification of “total microbial abundance” (Line 161)

In line 161, the authors refer to “total microbial abundance” and compare it with their findings. It is unclear whether this term refers to the total microbial population in the human body, within the gastrointestinal tract, or specifically within bile juice. This needs to be clearly defined to avoid ambiguity.

Response 4. We thank the Reviewer for pointing out this ambiguity. The term “total microbial abundance” refers specifically to the total microbial population within the collected and analyzed bile juice. To ensure clarity and avoid confusion, we have revised the wording in the manuscript (page 7, lines 163–170) to define the context explicitly.

5. Confusion in Figure 2 and percentage calculations

The presentation of Figure 2 and the associated percentage calculations are confusing. For example, it is unclear why 5,433 species represent only 5.9%, 803 species represent 9.3%, and 311 shared species account for 84.4%. The reference or denominator used to calculate these percentages should be clearly stated. Additionally, the percentages mentioned in lines 163–164 do not match those shown in the figure. This inconsistency needs to be corrected and clarified.

Response 5. We sincerely apologize for the confusion caused by the inaccuracies in Figure 2 and the associated text. We have identified a clerical error in the labeling and description of the percentages, and thoroughly revised Figure 2 and the corresponding results (page 7, lines 166–171, Figure 2 image) for consistency.

6. Explanation of the Shannon diversity index

The manuscript mentions the use of the Shannon method for diversity analysis but does not explain what this method is. The authors should clearly describe the Shannon diversity index, explain why it was chosen, and discuss its acceptance and relevance in microbial ecology studies. Proper citations should be included to support its use.

Response 6. We thank the Reviewer for the valuable suggestion to clarify the use of the Shannon diversity index.

The Shannon diversity index is a widely accepted ecological metric that quantifies microbial alpha diversity by considering species richness and evenness. We have selected this index because it is one of the most commonly used and validated diversity measures in microbiome studies based on 16S rRNA gene sequencing. The Shannon index allows meaningful comparisons of within-sample microbial diversity between clinical groups.

In response to the Reviewer’s comment, we have added a clear explanation of the Shannon diversity index, including its definition and the rationale for its selection, to the Materials and Methods under “Alpha diversity analysis” (Section 2.3; page 4, lines 123–132).

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, metagenomic sequencing of bile was achieved in 35 of 56 carcinoma samples and in 24 of 85 samples from patients with biliary benign diseases (BBDs). Mean alpha-diversity was 2.788±2.833 in BBD and 2.319±1.355 in BTC, showing a non-significant trend toward reduction in cancer (P = 0.399). Core analysis revealed 84.8 % of species were shared; 9 % were unique to BTC and 6 % to BBD. Bacteroides coprocola, Prevotella copri and B. plebeius were enriched in BTC bile, whereas B. vulgatus and B. uniformis predominated in BBD. Thus, although overall diversity does not differ, specific bile microbes are associated with malignant versus benign biliary disease. The study is well designed and of interest. There are only several suggestions for the authors.

1. The authors only provides a landscape of metagenomic feature in BBD and BTC. Is there any evidence that they identified the key roles that is critical in tumorigenesis?
2. Is there any experimental validation of the functions of the specific bacteria in BTC?
3. How did the authors avoid external bacteria pollution during sample collection and sequencing?

Author Response

1. The authors only provides a landscape of metagenomic feature in BBD and BTC. Is there any evidence that they identified the key roles that is critical in tumorigenesis?

Response 1. We appreciate the Reviewer’s comments regarding the potential role of specific microbial taxa in biliary tract tumorigenesis.

The primary aim of this study was to characterize the differences in bile microbial composition between samples from patients with BTC and BBD, rather than to establish a causal role for individual bacteria. Given the descriptive nature of taxonomy-based 16S rRNA sequencing and limited sample size, this study was not sufficiently powered to determine whether specific taxa act as critical drivers of cancer development. Instead, our analysis focused on identifying the differentially abundant taxa and community-level microbial patterns associated with malignant biliary conditions. We have clarified this point and the corresponding limitations in the Discussion (page 12, lines 304–308).

 

2. Is there any experimental validation of the functions of the specific bacteria in BTC?

Response 2. We appreciate the Reviewer’s insightful suggestion regarding the experimental validation of functions of specific bacteria in BTC. Although we agree that such validation is highly important, it was not feasible in the current study because of the rarity of BTC, which makes it challenging to collect a sufficient volume of bile samples from a large patient cohort in a short period of time. Given that it would take more than 5 years of additional collection to secure enough samples for experimental validation, we focused on profiling in this study and plan a follow-up study with a larger sample size and a longer timeframe to address these functional aspects. Accordingly, we have acknowledged this limitation and described the necessity for future validation studies in the Discussion section (page 13, lines 342–349).

 

3. How did the authors avoid external bacteria pollution during sample collection and sequencing?

Response 3. We thank the reviewer for raising the important concern regarding potential external bacterial contamination.

To minimize contamination, bile samples were aspirated directly from the bile duct using sterile single-use catheters immediately after selective guide wire-assisted cannulation and before contrast injection or therapeutic intervention. Samples were promptly transferred to sterile containers and stored at −80 °C until DNA extraction. These procedural details have been clarified in the Materials and Methods (page 3, lines 69–80).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

There are minor inconsistencies in the use of abbreviations throughout the manuscript. The abbreviations biliary tract cancer (BTC) and benign biliary diseases (BBDs) are defined in line 17; however, their full forms are repeated again in lines 48 and 59. Similarly, QC-PCR is defined earlier but its full form is repeated later in the manuscript. The authors are advised to define each abbreviation only at its first occurrence and use the abbreviated form consistently thereafter.

Author Response

Comment 1: There are minor inconsistencies in the use of abbreviations throughout the manuscript. The abbreviations biliary tract cancer (BTC) and benign biliary diseases (BBDs) are defined in line 17; however, their full forms are repeated again in lines 48 and 59. Similarly, QC-PCR is defined earlier but its full form is repeated later in the manuscript. The authors are advised to define each abbreviation only at its first occurrence and use the abbreviated form consistently thereafter.

 

Response 1: Thank you for your valuable feedback. As suggested, we have removed redundant words and repetitive expressions throughout the manuscript to improve clarity and flow. Please refer to the revised manuscript for these changes.