Review Reports - Biliary Microbiota in Health and Disease: Clinical Implications in Lithiasis, Infection, and Antimicrobial Resistance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review very well describes the biliary microbiota, presence of bacteria in gut liver axis and antimicrobial resistance.
Under section 2 and section 3 information could have been made more specific by including data from recent works notably bacterial information upto species level, as the author mentions work has been done 16S rRNA gene sequencing and metagenomics. If possible data specific to detection of bacterial species in different diseased conditions.
The authors very well describe role of bacteria in production of EPS and biofilm but there seems a repetition of this information.
After reviewing manuscript, it seems that the authors could have compiled the data in a crisp form. Though the investigations and compilations are extensive but seems more of dispersed information.
The authors could have depicted the information in flowchart or a kind of crosstalk of the microbiomes as well as their implications.
The numbering of the last subsection of section 3 is missing.

Comments on the Quality of English Language

Instead of very long sentences the authors could have used short and crisp sentences to depict the information. Grammatically it would be more sound.

Author Response

Comments and Suggestions for Authors

The review very well describes the biliary microbiota, presence of bacteria in gut liver axis and antimicrobial resistance.

We wish to thank the reviewer for finding this review to very well describe biliary microbiota.

Under section 2 and section 3 information could have been made more specific by including data from recent works notably bacterial information up to species level, as the author mentions work has been done 16S rRNA gene sequencing and metagenomics. If possible data specific to detection of bacterial species in different diseased conditions.

In the revised version of the manuscript, more specific informations regarding bacterial species detected in different conditions have been reported. In particular it has now been reported as follows: “The most prevalent gram-negative isolates are Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Among gram-positive pathogens, the species most frequently identified are Enterococcus faecium, Enterococcus avium, and Enterococcus faecalis. With regard to fungal isolates Candida albicans, C. tropicalis and C. parapsilosis seems to be the more frequently isolated” and “Current evidence indicates that the phyla most frequently detected in the biliary tract of individuals with choledocholithiasis include Firmicutes, Proteobacteria, Bacteroidota and Actinobacteriota, although their relative abundance may vary across studies due to methodological and clinical heterogeneity”. Over the revised text, other more specific informations have been added (highlighed), as suggested.

The authors very well describe role of bacteria in production of EPS and biofilm but there seems a repetition of this information.

We wish to thank the reviewer for giving un the opportunity to further improve our manuscript. As suggested, in the revised version of the manuscript, repetitions of the role of bacteria in production of EPS and biofilm have been deleted.

After reviewing manuscript, it seems that the authors could have compiled the data in a crisp form. Though the investigations and compilations are extensive but seems more of dispersed information. The authors could have depicted the information in flowchart or a kind of crosstalk of the microbiomes as well as their implications.

We wish to thank the reviewer for this helpful comment. In order to improve readability and to summarize informations, table 1 has been modified and simplified. Morover, a Table 2 has been added, reporting the more common microbes isolated in different pathologic conditions.

The numbering of the last subsection of section 3 is missing.

Numbering of the manusctipt subsections has been corrected.

Comments on the Quality of English Language

Instead of very long sentences the authors could have used short and crisp sentences to depict the information. Grammatically it would be more sound.

We wish to thank the reviewer for this comment. As suggested, in the revised version of the manuscript sentences have been shortened and simplified to improve readability.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a review of the health and clinical implications of the bile microbiota, focusing on the mechanisms underlying pathologies such as lithiasis and infectious processes, and with a particular emphasis on antimicrobial resistance. The work also takes into account the integration of microbiological, immunological, and clinical aspects, and achieves its intended goal. However, some changes should be made to improve the text's readability and comprehension.
First of all, the bibliography should be updated, since it appears somewhat dated, considering that this is a review.
In Table 1, the "Adaptations" have been specified in the "Taxonomic level" and "Dominant groups" columns. They should be inserted in a separate column, corresponding to the related microorganisms.
In lines 101 and 102, the same sentence is repeated.
In lines 228-229 and 282-284, the same concept is repeated.
The name of the bacteria should be italicized in the abstract, text, and figure legends.
Close the quotation marks in line 66.
Remove the words 'Benedetto e Dario' from the title of chapter 3.
In line 308, the paragraph numbering is missing (perhaps 3.3 ?).
The final period is missing from the sentence in line 371.
Standardize the spelling of the name of the bacteria. The authors have written the full name of the microorganisms in many places, and this should be done where it was used for the first time.
In chapter 5, renumber the paragraphs.

Author Response

Comments and Suggestions for Authors

First of all, the bibliography should be updated, since it appears somewhat dated, considering that this is a review.

We wish to thank the reviewer for this helpful commentary. As requested, bibliography has been expanded (now featuring 117 citations) and updated with more recent evidences.

In Table 1, the "Adaptations" have been specified in the "Taxonomic level" and "Dominant groups" columns. They should be inserted in a separate column, corresponding to the related microorganisms.
As suggested, in the revised table the adaptation line was deleted, as it was misleading considering the role of the table itself.

In lines 101 and 102, the same sentence is repeated.

Repetition has been deleted as suggested.

In lines 228-229 and 282-284, the same concept is repeated.

Repetition has been deleted as suggested.

The name of the bacteria should be italicized in the abstract, text, and figure legends.

The names of bacteria have been italicized as suggested all over the manuscript.

Close the quotation marks in line 66.

Done as suggested.

Remove the words 'Benedetto e Dario' from the title of chapter 3.

Done as suggested.

In line 308, the paragraph numbering is missing (perhaps 3.3 ?).

Corrected as suggested.

The final period is missing from the sentence in line 371.

The period has been added in the right place as suggested.

Standardize the spelling of the name of the bacteria. The authors have written the full name of the microorganisms in many places, and this should be done where it was used for the first time.

The spelling of the names of the bacteria has been standardized as suggested.

In chapter 5, renumber the paragraphs.

The paragraphs of chapter 5 have been renamed.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a review of current knowledge regarding the biliary microbiota and its importance in health and disease, with particular emphasis on interactions within the gut-liver-bile duct axis, the mechanisms of pathogenesis of gallstones and biliary tract infections, and the problem of increasing antibiotic resistance. The work's topic is timely and relevant, contributing to the rapidly evolving field of microbiome research and its clinical significance.

The main strengths of this work include its broad substantive scope and its attempt to integrate data from various fields – microbiology, immunology, hepatology, and clinical medicine. The authors systematically present the composition of the biliary microbiota under physiological conditions, demonstrating a shift away from the traditional view of bile sterility. Of particular value is the discussion of the role of bile acids as factors modulating the microbiota and the description of bacterial adaptation mechanisms to an environment rich in bile salts. Also noteworthy is the extensive section on the gut-liver-biliary axis, which effectively synthesizes current concepts regarding the interrelationships between the intestinal microbiota and the hepatobiliary system.

Another strength of the manuscript is its detailed presentation of the role of microorganisms in the pathogenesis of gallstones, including the involvement of biofilms and bacterial enzymes such as β-glucuronidase and phospholipase. The authors aptly emphasize the importance of microbiota in the processes of lithogenesis and disease recurrence. The section on antibiotic resistance is current and well-placed in the clinical context, pointing to the growing importance of ESBL, VRE, and MDR strains in biliary tract infections.

Despite its numerous strengths, the manuscript has significant limitations that require revision. Primarily, the work is primarily descriptive and review-based, lacking a clear critical approach to the cited studies. In many places, there is no assessment of the quality of the source data, including methodological limitations of microbiome studies, such as low biomass of bile samples or the risk of contamination. The authors mention these issues but do not elaborate sufficiently, which weakens the analytical value of the work.

Another significant problem is a certain amount of redundancy and repetition, particularly in the descriptions of the microbiota composition and the mechanisms of bile acid action. The structure of the text sometimes lacks coherence, and some sections are excessively extensive (e.g., detailed descriptions of the mechanisms of lithogenesis), while other, potentially crucial issues – such as the use of microbiota as biomarkers – are The lack of a clear distinction between correlation and causation is also problematic. Many passages suggest a role for microbiota in the pathogenesis of biliary tract diseases, but without clearly emphasizing that most of the available data is observational in nature. It would be worthwhile to more clearly emphasize the limitations of interpretation and the need for mechanistic studies.

In the section on clinical applications and future research directions, the authors rightly point to the potential of microbiota profiling, but these considerations remain relatively general. More specific implementation proposals, for example, in the context of diagnostics, targeted therapy, or microbiota modulation, are lacking.

The text requires linguistic and editorial corrections. Numerous typographical errors are evident (e.g., missing spaces, typos, repeated taxon names), formatting inconsistencies, and minor grammatical errors. Several areas also contain imprecise wording that may complicate the reading experience.

References include 111 references.

Author Response

Comments and Suggestions for Authors

We wish to thank the reviewer for giving us the opportunity to further improve our review. As requested, the quality of the source data has been commented when cited along the text. Moreover, this concept has been further highlighted in the revised Clinical Implications and Future Directions section (However, evidence regarding the resident bile microbiota in healthy subjects is still accumulating, and further research is still needed to reach a deeper understanding of its role. Indeed, available studies are burdened by methodological limitations, including small sample sizes, various and non-standardized bile collection techniques). For example, in the “bile: basic concepts” subsection it has now been more clearly reported as follows: “However, the reliability of invasive sampling methods has always represented a relevant limitation to the investigation of bile microbiota. Bile collecting techniques such as en-doscopic retrograde cholangiopancreatography (ERCP), percutaneous biliary drainage and intra-operatory sampling, are all burdened by a high risk of contamination. This methodological limitation largely accounts for the predominant focus of current literature on the role of biliary microbiota in pathological conditions, such as cholelithiasis and biliary tract infections [15-17].”.

Another significant problem is a certain amount of redundancy and repetition, particularly in the descriptions of the microbiota composition and the mechanisms of bile acid action. The structure of the text sometimes lacks coherence, and some sections are excessively extensive (e.g., detailed descriptions of the mechanisms of lithogenesis), while other, potentially crucial issues – such as the use of microbiota as biomarkers – are the lack of a clear distinction between correlation and causation is also problematic. Many passages suggest a role for microbiota in the pathogenesis of biliary tract diseases, but without clearly emphasizing that most of the available data is observational in nature. It would be worthwhile to more clearly emphasize the limitations of interpretation and the need for mechanistic studies.

As suggested, the redundancy and repetition, particularly in the description of the microbiota composition and the mechanisms of bile acid action, have been addressed in the revised version of the manuscript. Limitations of the quality and of the study design have also been reported.

We wish to thank the reviewer for this intresting comment. The current knowledge regarding microbiota profile oriented treatments is still very limited. Therefore, in the clinical applications and future directions section we opted to report only general hypotheses on how the reaserch on suh themes may be translated into clinical practice. To further enhance this concept, in the revised version of the manuscript it has now been reported as follows: “Moreover, high quality research to investigate the efficacy of the currently available therapeutic armamentarium to influence the bile microbiota in both healthy and non-infectious disease is needed. A deeper knowledge of the gut–liver–biliary axis may also lead to the development of new therapies or repurposing of already existent ones, hitting targets different from pathogens such as microfilm and mucus, bile acids and bile compositions.”

As suggested, the manuscript has undergone grammatic and spelling revision to improve the reading experience.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The changes requested by the Reviewer were made by the Authors.