Biliary stents reshape the bile microbiome in the absence of cholangitis

Abstract

Background and study aims

Biliary stents are widely used in endoscopic retrograde cholangiopancreatography (ERCP), yet their impact on the native bile microbiome under non-infectious conditions remains unclear. We aimed to characterize stent-associated alterations in the biliary microbiome using 16S rRNA gene sequencing.

Patients and methods

We analyzed bile samples collected during ERCP from 35 patients without clinical or laboratory evidence of acute cholangitis. Patients were categorized into a control group (n = 25; naïve papillae) and an endoscopic biliary stenting (EBS) group (n = 10; previously stented). Microbial composition was assessed using high-throughput 16S rRNA sequencing after propensity score matching to balance background characteristics.

Results

Beta diversity differed significantly between groups (PERMANOVA, P < 0.01), despite no significant differences in alpha diversity. The EBS group demonstrated increased relative abundance of Firmicutes and Fusobacteriota, and depletion of Proteobacteria. Notably, Enterococcus was significantly enriched in the EBS group (log fold change 6.74; q < 0.01), whereas Sphingomonas was reduced.

Conclusions

Endoscopic biliary stenting is associated with distinct bile microbiome alterations, characterized by enrichment of Enterococcus species in clinically stable patients. These findings suggest that stents may predispose to opportunistic colonization, providing a potential mechanistic link to future cholangitis. Recognizing such preclinical dysbiosis may inform tailored antimicrobial strategies and future stent design.

Keywords

Pancreatobiliary (ERCP/PTCD) - Quality and logistical aspects - Hygiene - ERC topics

Introduction

Endoscopic biliary stenting is a cornerstone of therapeutic endoscopic retrograde cholangiopancreatography (ERCP), widely used in management of both benign and malignant biliary obstruction [1]. Although stents improve biliary drainage and alleviate symptoms, they are also known to be factors that have significant impact on the causative pathogens in cases of cholangitis [2].

Human bile is not sterile. Recent advances in sequencing technologies, particularly 16S rRNA gene sequencing, have revealed a complex and diverse biliary microbiome in health and disease [3]. However, the effects of biliary stent placement on this microbial community in absent overt infection remain poorly understood. Previous studies have primarily focused on infected bile or stent occlusion [4], limiting our understanding of how stents may drive subclinical microbial shifts. Such dysbiosis may serve as a precursor to stent-related infections, influence antibiotic susceptibility, or impact stent patency.

In this study, we aimed to characterize the impact of prior biliary stent placement on composition of the bile microbiome in patients undergoing ERCP without clinical evidence of cholangitis. We hypothesized that stent presence, even in clinically stable patients, leads to specific and detectable alterations in the bile microbiota.

Patients and methods

Study design and setting

This study was a post hoc analysis using a biliary microbiome database collected at Keio University Hospital between August 2020 and February 2023. It was approved by the Institutional Review Board (IRB No. 20190055) and adhered to the ethical principles outlined in the Declaration of Helsinki. All participants provided written informed consent prior to study enrollment.

Study participants

The study included patients who required ERCP clinically and met the following inclusion criteria: 1 no clinical signs or symptoms of acute cholangitis defined as Tokyo guideline [5]; 2) not fitting the diagnostic criteria for primary sclerosing cholangitis due to the previous report that showed characteristic microbiome was thought to be associated [6]; 3) absence of recent antibiotic use within the last 30 days prior to ERCP; 4) no external or internal biliary drainage; and 5) no biliary duct operation.

Patients were divided into two groups: a control group with naïve papillae who underwent ERCP for the first time and an EBS group with prior endoscopic biliary stenting who were undergoing ERCP.

Sample collection

During the ERCP procedure, bile samples were collected directly from the bile duct using a sterile catheter immediately after accessing the duct without use of prophylactic antibiotics. The collected bile was then transferred into sterile containers and immediately frozen at -80°C until further analysis.

16S rRNA sequencing and microbial profiling

Microbial profiling was performed using 16S rRNA gene sequencing targeting the V3-V4 hypervariable regions. DNA was extracted from bile samples using the reported protocol [7]. Amplification of the V3-V4 regions of the bacterial 16S rRNA gene was carried out using two-step tailed polymerase chain reaction method. Sequencing was conducted on an Illumina MiSeq platform (Reagent Kit v3), generating paired-end reads of 300 bp. After sequencing, raw reads were quality-checked and filtered using FASTX-Toolkit (ver. 0.0.14) and Sickle (ver. 1.33), trimming bases with quality less than 20 and outputting paired-end reads with lengths greater than 130 bp. Operational taxonomic units (OTUs) were assigned after removing chimeric and noise sequences with DADA2 plugin of Qiime2 (ver. 2024.10) and outputting representative sequences and amplicon sequence variant (ASV) tables, and taxonomic classification was performed with the SILVA (ver. 138) database at a 99%.

Statistical analysis

Continuous variables were expressed as median and interquartile range (IQR) and compared using the Wilcoxon rank sum test. Categorical variables were expressed as proportions (%) and compared using Fisher's exact test. Propensity score was obtained by logistic regression model including following covariates: age, sex, disease, total bilirubin, status of gallbladder, taking ursodeoxycholic acid, and taking proton pomp inhibitors. Covariates were selected based on their potential clinical relevance and previously reported associations with bile microbiome composition. Propensity score matching (PSM) was performed with setting of 1:1 nearest-neighbor matching without replacement. To assess the balance of covariates, absolute standardized mean differences (ASMDs) were calculated and ASMDs < 0.1 were considered well balanced. Beta diversity was compared using permutational multivariate analysis of variance (PERMANOVA). Differences in microbial composition were assessed using the analysis of composition of microbiomes with bias correction (ANCOM-BC) method, with adjustments for multiple comparisons using the Holm method (showed as q value). P < 0.05 and q < 0.001 were considered statistically significant. All statistical analyses were performed using Qiime2 (ver. 2024.5) and R (ver. 4.41).

Results

Patient characteristics

A total of 35 patients were included in the study, with 25 in the control group and 10 in the EBS group. Ten paired cohort were obtained after PSM and almost all of variables were well balanced except for sex (ASMD = 0.2) ([Table 1]).

Table 1 Baseline characteristics after propensity score matching.

Characteristic	Control N = 10	EBS N = 10	P value	SMD
Data are presented as n (%) for categorical variables and median (interquartile range) for continuous variables. *Because it was not used as a covariate in propensity score matching, the SMD was not calculated. EBS, endoscopic biliary stenting; SMD, standardized mean difference.
Age (years)	73 (70, 80)	65 (56, 88)	0.8	0.09
Female	4 (40%)	5 (50%)	> 0.9	0.2
Total bilirubin (mg/dL)	0.70 (0.60, 0.80)	0.75 (0.50, 0.90)	0.8	0.03
Direct bilirubin (mg/dL)	0.10 (0.10, 0.10)	0.10 (0.10, 0.10)	0.2	-*
Presence of gallbladder	9 (90%)	9 (90%)	> 0.9	0
Ursodeoxycholic acid	2 (20%)	2 (20%)	> 0.9	0
Proton pump inhibitors	6 (60%)	6 (60%)	> 0.9	0
Disease			> 0.9	0.2
Gallstones related disease	5 (50%)	6 (60%)
Non-biliary disease	5 (50%)	4 (40%)

In the EBS group, all patients had plastic stents placed across the papilla. Median duration of stent placement before sampling was 77.5 days. Among these patients, four of 10 (40%) presented with cholangitis at time of initial ERCP when the biliary stent was placed. Three patients underwent stent exchange during this period, but none developed cholangitis.

Alteration of diversity

Alpha diversity did not differ between the two groups, as measured by the observed features, Chao1 index, Shannon index, and Pielou’s evenness ([Fig. 1]). Principal coordinates analysis (PCoA) plot of the Bray-Curtis dissimilarity revealed distinct clustering of microbial communities between the two groups ([Fig. 2]). There was a significant difference in it, as assessed by PERMANOVA (P < 0.01).

Fig. 1 Alpha diversity of the bile microbiome in the control and EBS groups.

Fig. 2 Beta diversity of the bile microbiome between the control and EBS groups.

Taxonomic composition

At the phylum level, both groups were dominated by Firmicutes and Proteobacteria, but the relative abundance of specific taxa varied between the groups. In the EBS group, mean proportions of Firmicutes and Fusobacteriota were higher compared with the control group (40.0% vs. 58.4% and 0.5% vs. 7.4%, respectively) ([Fig. 3]). Conversely, mean proportions of Proteobacteria, Bacteroidota, and Actinobacteriota were lower in the EBS group compared with the control group (48.4% vs. 29.9%, 7.6% vs. 2.1%, and 2.1% vs. 1.1%, respectively). ANCOM-BC analysis at the genus level revealed significant dysbiosis in the bile microbiome of the EBS group. Compared with the control group, the EBS group exhibited a significantly higher abundance of Enterococcus (6.74 log fold change; q < 0.01) and significantly lower abundance of Sphingomonas (-4.82 log fold change; q < 0.01) ([Fig. 4]).

Fig. 3 Relative abundance of dominant bacterial phyla in both groups.

Fig. 4 Differentially abundant genera identified by ANCOM-BC analysis.

Discussion

In this study, we demonstrated that prior biliary stent placement is associated with distinct changes in the bile microbiome, even in the absence of acute cholangitis. Specifically, patients with previously placed stents exhibited significant enrichment of Enterococcus and reduction in Proteobacteria, including Sphingomonas. These shifts occurred despite no clinical or laboratory evidence of infection at the time of bile sampling.

Our findings expand on prior studies that have focused on bile cultures during stent occlusion or cholangitis. Unlike those studies, our cohort was limited to patients without active infection, allowing us to isolate the effects of stents themselves on microbial composition. Enrichment of Enterococcus, which is a genus frequently implicated in stent occlusion and antibiotic resistance [8], may represent a predisposing factor for future infection.

Mechanistically, biliary stents may alter bile flow, introduce foreign surfaces that promote biofilm formation [9], or facilitate retrograde microbial migration from the duodenum [10]. These changes could select for specific taxa such as Enterococcus, which are capable of surviving in harsh, antimicrobial-rich environments and forming biofilms.

In contrast, reduction in Sphingomonas was observed. We note that Sphingomonas spp. are common environmental organisms in the biliary microbiome [11] and their depletion may reflect displacement by biofilm-forming taxa such as Enterococcus under altered bile flow caused by stents.

Limitations include the relatively small sample size, which may reduce power to detect subtler microbiome shifts, and the lack of longitudinal follow-up to determine the clinical consequences of these microbial alterations. To reduce bias, we employed PSM to reduce confounding due to clinical differences between groups. However, because there might be some unknown potentially relevant factors, the possibility of residual confounding due to unmeasured variables cannot be completely ruled out. There is also a possibility that stent-related factors such as stent type, placement method, and placement duration may influence the biliary tract microbial community; however, this was not examined in the present study. Furthermore, in addition to the small sample size, absence of functional metagenomic analysis limits our ability to determine whether the observed taxonomic shifts translate into functional alterations or contribute causally to infection risk.

Conclusions

In conclusion, biliary stents significantly reshape the bile duct microbiome, promoting expansion of Enterococcus species even absent infection. Recognizing these preclinical microbial shifts may inform future strategies for infection prevention, stent design, and antimicrobial stewardship in patients undergoing biliary interventions.

Contributorsʼ Statement

Atsuto Kayashima: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Seihiro Fukuhara: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation. Kentaro Miyamoto: Data curation, Formal analysis, Investigation, Methodology, Software, Supervision. Eisuke Iwasaki: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing - review & editing. Motohiko Kato: Funding acquisition, Methodology, Resources, Supervision. Tomohisa Sujino: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing.

Conflict of Interest

Kentaro Miyamoto is an employee of Miyarisan Pharmaceutical Co. The remaining authors declare that they have is no conflicts of interest.

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Publication History

Received: 27 June 2025

Accepted after revision: 22 October 2025

Accepted Manuscript online:
27 October 2025

Article published online:
11 November 2025