Review for 10.1186/s12876-025-04159-5 (2025)

Title: Relationship between gut microbiota dysbiosis and bile acid in patients with hepatitis B-
induced cirrhosis
Version: 2 Date: 22 Jun 2025
Reviewer's report:
This manuscript investigates the interplay between gut microbiota composition and bile acid
(BA) levels in patients with hepatitis B-induced cirrhosis (HBC), using 16S rRNA sequencing.
The study is well-structured, and the authors stratify patients into BA-normal and BA-high
subgroups to explore microbial diversity and specific taxa alterations. The work offers valuable
insights into potential microbiota-based biomarkers and therapeutic targets in HBC. However,
several critical methodological, analytical, and interpretational concerns should be addressed.
Major Concerns
1. Causality vs. Association
While the study demonstrates associations between microbiota profiles and BA levels in
HBC, the language occasionally implies causation (e.g., “direct role of BAs in driving HBC
progression”). As this is a cross-sectional study, causality cannot be inferred. The authors must
revise such claims throughout the text to reflect associative findings.
2. Sample Size and Statistical Power
The relatively small sample size (n=46 HBC, divided into two subgroups) may limit the power
of subgroup analyses and raise concerns regarding overinterpretation of differences, especially
with multiple comparisons. The authors should acknowledge this as a limitation and consider
validation in a larger cohort or external dataset.
3. Definition of BA Thresholds
The criteria used to classify patients into BA-normal and BA-high groups are not clearly defined.
What clinical or laboratory thresholds were used to delineate these groups? Without transparent
definitions, reproducibility and clinical relevance are weakened.
4. Confounding Variables
Although the exclusion criteria are comprehensive, dietary intake, antiviral treatment status,
disease duration, and severity of portal hypertension are not discussed as potential confounders.
These could influence both bile acid levels and microbiota composition.
5. Functional Prediction Missing
The study focuses on taxonomic differences but lacks any prediction or analysis of microbial
function (e.g., PICRUSt, KEGG pathways). This would enhance biological interpretation,
particularly regarding the impact on BA metabolism or immune modulation.
6. Overinterpretation of Streptococcus and Lactobacillales
While the enrichment of these taxa is noted, the proposed mechanistic pathways (e.g.,
modulation of hepatic immune responses) are speculative and not directly tested in this study.
These hypotheses should be clearly presented as such and ideally reserved for discussion rather
than conclusions.
7. Inconsistent Terminology
The manuscript uses multiple terms interchangeably (e.g., “BA” and “TBA”) without clear
definition or consistency. Clarification is needed to avoid confusion between total bile acids and
specific bile acid profiles.
Minor Concerns
1. Grammar and Style
Some sentences are overly complex or grammatically incorrect (e.g., “This phenomenon implies
that Lactobacillales may contribute…” # consider rephrasing). The text would benefit from
professional English editing to improve clarity and conciseness.
2. Figures and Tables
Some figures (e.g., LEfSe plots) lack clear labeling or explanation in the legend. Taxonomic
levels (family vs. genus) should be clearly indicated. Additionally, Figure 4 mentions
“Streptococcus” as both increased and decreased—please clarify.
3. Reference Accuracy
The reference list appears current and relevant, but some in-text citations (e.g., “[3]”) are not
consistently matched to their references. Please ensure accurate citation formatting according
to journal guidelines.
4. Data Availability Statement
Although the manuscript states that data are available on request and in GSA, a direct link or
accession code is missing from the abstract and methods. Ensure this information is clearly
included.
Unacknowledged Limitations
• Absence of longitudinal data or follow-up analysis, which limits the understanding of
microbiota dynamics over time.
• Lack of control for liver disease severity (e.g., MELD score, HVPG) which may influence
findings.
• Functional BA profiling (primary vs. secondary) is not reported, limiting mechanistic
interpretation.
• Absence of antibiotic usage history over a longer period (beyond 3 months) may underestimate
long-term microbiota disruption.
Language and Reference Revisions
• Improve clarity and precision across the manuscript; avoid passive or redundant phrases.
• Ensure consistent use of terminology (e.g., “gut microbiota,” “microbiome,” “intestinal flora”).
• Replace vague language such as “new insights” with more specific descriptors.
• References at the end of sentences should be repositioned to follow journal-specific style, and
citation formatting should be harmonized.