This observational study aims to investigate the impact of polyethylene glycol (PEG) laxatives used for bowel preparation on the gut microbiota of patients who have undergone cholecystectomy. Emerging evidence suggests that both cholecystectomy and high-dose PEG exposure can independently alter the intestinal microbial ecosystem. However, whether the microbiota of post-cholecystectomy patients is more vulnerable to PEG-induced perturbation and exhibits delayed recovery remains unknown.

Approximately 10 adults with prior cholecystectomy scheduled for colonoscopy and 10 age-matched controls without cholecystectomy will be enrolled. All participants will undergo standard colonoscopy preparation with PEG-based laxatives. Stool samples will be collected at five time points: before bowel preparation, at the first non-watery stool after colonoscopy, and at 1, 3, and 6 months post-colonoscopy. Metagenomic shotgun sequencing will be performed to characterise the taxonomic and functional profiles of the gut microbiome. Alpha diversity, beta diversity, differential abundance, and metabolic pathway alterations will be compared within and between groups over time.

The findings are expected to reveal whether cholecystectomised individuals are more susceptible to long-term gut dysbiosis after PEG exposure, and to inform future strategies for microbiota restoration in this specific population.

Gut microbiota dysbiosis has been implicated in various gastrointestinal and metabolic disorders. Polyethylene glycol (PEG)-based laxatives, routinely administered for bowel cleansing before colonoscopy, induce transient osmotic diarrhoea and have been shown in animal models to cause prolonged alterations in microbial composition and function. Meanwhile, cholecystectomy, one of the most frequently performed abdominal surgeries, alters bile acid flow and enterohepatic circulation, which can modulate the gut microbial ecosystem and has been associated with increased risk of post-operative diarrhoea and even colorectal neoplasia. Despite these independent effects, the combined influence of cholecystectomy and PEG-based bowel preparation on the human gut microbiome has not been systematically investigated.

This is a prospective, parallel-group, observational cohort study conducted at Tongji Hospital, Huazhong University of Science and Technology. A total of approximately 20 participants will be recruited and allocated into two groups: the cholecystectomy group, consisting of individuals who have undergone cholecystectomy at least six months prior to enrolment and are scheduled for a screening or surveillance colonoscopy; and the control group, comprising individuals with an intact gallbladder who are matched for age and sex and are also scheduled for colonoscopy.

Eligible participants are aged 18 to 75 years, have no history of major organ disease, have not used antibiotics, probiotics, or prebiotics within six months before enrolment, and have no contraindications to colonoscopy or PEG intake. Exclusion criteria include pregnancy, severe cardiopulmonary insufficiency, mental disorders, and inability to comply with stool collection procedures.

All participants will undergo standard bowel preparation using 2-4 L of PEG-4000/3350 solution according to routine clinical practice. No experimental intervention is administered.

Stool specimens of approximately 10 g each will be self-collected by participants at home using provided collection kits at five predefined time points: within three days before bowel preparation (baseline), at the first non-watery stool after colonoscopy, and at one month, three months, and six months after colonoscopy, each with a permitted window of ±7 days for the one-month visit and ±14 days for the three- and six-month visits. Samples will be immediately frozen at -80 °C until DNA extraction. Faecal genomic DNA will be subjected to shotgun metagenomic sequencing on the DNBSEQ-T7 platform. Raw reads will be filtered using fastp to remove adapter contamination and low-quality sequences. High-quality reads will be aligned to the human genome to exclude host DNA, and the remaining reads will be used for taxonomic profiling with MetaPhlAn or Kraken2/Bracken, and functional profiling with HUMAnN3 against the UniRef90 and KEGG databases.

The primary outcome is the change in gut microbial α-diversity, as measured by Shannon, Simpson, and Chao1 indices, and β-diversity, assessed by Bray-Curtis dissimilarity and UniFrac distances, both within and between groups across the five time points.

Secondary outcomes include the temporal dynamics of specific taxa, such as the phylum Firmicutes/Bacteroidetes ratio and butyrate-producing genera; differential abundance of species and functional pathways, including KEGG modules and MetaCyc reactions; and the correlation between microbial compositional shifts and clinical parameters, for example time since cholecystectomy and bowel movement frequency.

All statistical analyses will be performed using R. Within-group comparisons over time will be assessed by the Friedman test or repeated-measures ANOVA, or non-parametric equivalents, followed by post-hoc pairwise Wilcoxon signed-rank tests with false discovery rate correction. Between-group comparisons at each time point will be analysed using the Mann-Whitney U test or linear mixed-effects models adjusting for potential confounders. Principal coordinate analysis based on Bray-Curtis distances will visualise community separation, and permutational multivariate analysis of variance will test for significant clustering. Linear discriminant analysis effect size will identify taxa with differential abundance between groups, using an LDA score threshold of >2.0 and p <0.05.

The study protocol has been approved by the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent will be obtained from all participants prior to enrolment. Results will be disseminated through peer-reviewed publications and scientific conferences.

By longitudinally profiling the gut microbiota of cholecystectomised and non-cholecystectomised individuals exposed to the same bowel preparation regimen, this study will delineate whether prior gallbladder removal predisposes patients to a more severe or prolonged PEG-induced dysbiosis. The findings may provide a microbiological rationale for tailored bowel preparation protocols or microbiota-targeted interventions, such as probiotics or post-colonoscopy dietary guidance, in this growing patient population.

Inclusion Criteria:

• Scheduled to undergo colonoscopy with polyethylene glycol (PEG)-based bowel preparation.
• For the gallbladder removal group: prior cholecystectomy performed at least 6 months before enrolment.
• For the normal group: intact gallbladder (no history of cholecystectomy).
• Willing and able to provide written informed consent.
• Willing to collect and mail stool samples at five specified time points (baseline, first non-watery stool after colonoscopy, 1 month, 3 months, 6 months).

Exclusion Criteria:

• Contraindications to colonoscopy (e.g., severe cardiopulmonary insufficiency, acute gastrointestinal bleeding, severe coagulopathy).
• Known intolerance or allergy to polyethylene glycol (PEG).
• Use of antibiotics, probiotics, or prebiotics within 6 months prior to enrolment.
• Pregnancy or breastfeeding.
• Mental disorders or any condition that may compromise the ability to comply with study procedures.
• Major organ diseases (e.g., liver cirrhosis, chronic kidney disease stage 4-5, uncontrolled diabetes mellitus).
• Participation in another interventional clinical trial within the last 3 months.

The study population consists of adult patients scheduled for colonoscopy at the Department of Gastroenterology, Tongji Hospital, Wuhan, China. Participants are recruited consecutively from the outpatient clinic and voluntarily enrolled after providing written informed consent. Two cohorts are defined based on history of cholecystectomy: individuals with prior cholecystectomy (gallbladder removal group) and individuals with an intact gallbladder (normal group). All participants undergo routine bowel preparation with PEG-based laxatives and provide stool samples longitudinally over a 6-month period. No experimental interventions are administered.

De-identified individual participant data collected during this study, including:

• Demographic information (age, sex)
• Clinical characteristics (time since cholecystectomy, bowel movement frequency)
• Metagenomic sequencing data (raw FASTQ files, taxonomic abundance tables, functional pathway profiles)
• Stool sample collection time points
• Laboratory processing and quality control data