Changes in biliary microbiota before and after drainage of malignant biliary obstruction

Changes in biliary microbiota before and after drainage of malignant biliary obstruction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Changes in biliary microbiota before and after drainage of malignant biliary obstruction Jin-Liang Wu, Yan Chen, Zhi-Hua Ni, Tong-Lei Fang, Qing-Hua Tian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6538543/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background he biliary system stores bile for fat digestion and nutrient absorption. Surgery or other changes can disrupt this system, altering the microbiome and potentially leading to complications. Such disruptions may impact patient outcomes, highlighting the need for careful monitoring during biliary drainage procedures. Aims To investigate changes in biliary microbiota before and after MBO drainage and assess the impact on microbiota. Methods In this retrospective study, 42 MBO patients underwent percutaneous transhepatic catheter drainage (PTCD) from January 2020 to December 2022. The cohort included patients with cholangiocarcinoma, pancreatic cancer, hepatocellular carcinoma, and hilar lymph node metastasis. Bile samples were collected before and 7 days after drainage, analyzed using culture and next-generation sequencing (NGS). Data on acute cholangitis and antibiotic use were also collected. Results Prior to drainage, common bacteria included Burkholderia , Acinetobacter , Pseudomonas , and Staphylococcus , similar to normal biliary microbiota. Post-drainage, there was a notable increase in Staphylococcus , Klebsiella , and other pathogens, along with a decrease in microbial diversity and evenness. Conclusion In MBO patients, the biliary microbiota resembles that of non-diseased ducts but significantly changes post-drainage. There is increased pathogenic bacteria and reduced diversity, explaining the higher infection risk. Malignant biliary obstruction Digital subtraction angiography Percutaneous transhepatic catheter drainage Microbial community Figures Figure 1 Figure 2 Figure 3 Core Tip This study examines changes in the biliary microbiota before and after drainage in patients with malignant biliary obstruction (MBO). It was found that post-drainage, the abundance of bacteria like Staphylococcus and Klebsiella increased significantly, while overall microbial diversity and evenness decreased. This disruption in the microbial community could increase the risk of biliary tract infections, indicating the importance of monitoring and managing these microbial changes during and after drainage procedures to improve patient outcomes. Introduction The biliary system refers to a series of organs and structures involved in the generation, storage, and excretion of bile. Its primary function is to store bile produced by the liver and release it into the digestive system when needed, which is crucial for the digestion and absorption of fats and fat-soluble vitamins. For a long time, the normal biliary tract has always been considered sterile[ 1 , 2 ], as it has several mechanisms to inhibit bacterial growth. The Oddi sphincter resists reflux, preventing bacteria from the duodenum from entering the bile ducts. The gallbladder releases bile into the duodenum, preventing bacterial colonization in the bile ducts. Additionally, bile contains a large amount of bile acids with antibacterial properties, which can inhibit bacterial growth[ 3 ] . With the advancement of research on the human microbiota, the presence of a microbiota in the normal biliary system has become a topic of debate[ 4 ]. In 1995, with the emergence and wide application of 16S ribosomal RNA sequencing technology, it was confirmed that there were microorganisms in bile samples that "could not cultivate bacteria", leading to the proposal of the concept of "biliary microbiota"[ 5 ]. In 2019, Molinero et al[ 6 ] reported for the first time that the presence of a microbiota in the gallbladder bile of 13 liver transplantation donors without hepatobiliary and pancreatic diseases. The discovery of biliary microbiota has fundamentally changed our understanding of the development of biliary infectious diseases[ 7 ]. Research on the diversity, stability, and correlation of microbiota in the biliary system with physiological and pathological conditions has gradually been conducted. This study aims to investigate the changes in the bile microbiota of the bile duct after percutaneous transhepatic catheter drainage (PTCD) for malignant biliary obstruction. Using bile samples from liver transplant donors as the control group, and bile samples before and after obstruction and drainage as the experimental group, we aim to study the impact of biliary obstruction and drainage interventions on the bile microbiota from a microecological perspective. This research provides a factual and theoretical basis for further exploration of the pathogenesis of acute cholangitis and the improvement of drainage interventions. Materials and Methods Diagnostic criteria for malignant biliary obstruction The criteria for diagnosing malignant biliary obstruction were based on previous studies, and patients were required to meet all of the following three conditions: 1) Clinical manifestations: yellow staining of skin and sclera and epigastrium discomfort. 2) Imaging findings (at least one of the following): (1) Direct signs: presence of space-occupying lesions in the hilar or surrounding bile ducts and pancreatic ampullary lesions; and (2) Indirect signs: significant dilation of the intrahepatic and extrahepatic bile ducts, with a maximum diameter > 15mm and a "soft-rattan-like" appearance; abrupt cessation of biliary dilation, resulting in a "residual root shape"; presence of a "double duct sign" involving the common bile duct and pancreatic duct. 3) Laboratory tests or pathological results: elevation of tumor indicators (CA199, AFP, CEA, etc.) or biopsy findings indicating malignant tumors. Inclusion criteria 1) Patients who meet the above diagnostic criteria and are aged ≥ 18 years old; 2) Ability to provide informed consent form; 3) Indications for PTCD (at least one of the following): (1) Palliative biliary drainage for malignant obstruction caused by advanced tumors; (2) Preoperative preparation for patients with malignant biliary obstruction; and (3) Emergency biliary decompression and drainage for malignant biliary obstruction combined with acute biliary infection. 4) Adequate coagulation function, defined as an international normalized ratio (INR) ≤ 2.0. Exclusion criteria 1) Refusal to provide informed consent; 2) Contraindications for biliary drainage or inability to cooperate with the procedure; 3) Use of antibiotics or other medications within the past two months that may affect the study results; 4) Expected survival period of less than three months; 5) Pregnant or lactation; 6) Inability to provide complete clinical data. Patients who have not undergone biliary drainage are classified into the malignant biliary obstruction group (MBO group), while those who have undergone biliary drainage are classified into the biliary drainage group (BD group). The control group consisted of bile samples collected from liver donors at our hospital during the same period. This study strictly adheres to the ethical requirements of relevant medical research. Research subjects This retrospective study comprised 42 patients with malignant biliary obstruction who underwent PTCD at the department of radiology interventional in our hospital between January 2020 and December 2022. Comprehensive patient information was recorded and organized, including underlying disease, clinical symptoms, medical history, results of laboratory and imaging examination results, as well as bile culture and NGS technology detection results. The presence and severity of acute cholangitis were assessed and graded according to the 2018 Tokyo guidelines[ 8 ], and the use of antibiotics for treatment was also documented. The current study was approved by the Ethics Committee of Shanghai Sixth People's Hospital, and implemented under the guidance of the clinical operation. PTCD procedure and bile sample collection PTCD procedure: The patient was positioned flat on the bed, and the right upper abdomen was prepared and draped in a sterile fashion. After identifying the puncture site, 2% lidocaine was used to infiltrate and anesthetize the liver capsule. A 22G puncture needle was inserted into the bile duct, and bile overflow was confirmed either by seeing bile emerge from the needle or by using an empty needle to extract bile. A small amount of diluted contrast agent was injected to perform cholangiography, visualizing the degree of intrahepatic bile duct dilation, extent of obstruction, and stenosis. A 0.018-inch guide wire was advanced deep into the bile duct, and the puncture needle was removed. A vascular sheath was then introduced. After removing the guide wire, 15–20 ml of sterile bile samples were collected. Subsequently, a 0.035-inch guide wire was inserted through the narrow segment into the duodenum. Finally, an external or internal drainage tube was implanted: the distal end of the internal/external drainage tube was positioned outside the obstructive lesion (usually the duodenum), and the proximal drainage hole was placed in front of the obstructive lesion. The drainage tube, external tee, and drainage bag were secured to the skin surface. Bile sample collection: Bile was collected twice using a drainage tube, once during the PTCD procedure and once 7 days after the procedure. Sterile containers were used for collection, and each sample was divided into two parts. One part was immediately sent for bile culture and testing, while the other part was stored in a -80℃ refrigerator for subsequent NGS testing. Bile culture After the bile samples were collected, they were inoculated onto standard chocolate agar plates and blood agar plates as soon as possible. The blood agar plates were incubated in a conventional incubator at 35–37 ℃, while the chocolate agar plates were incubated in a 5% -10% carbon dioxide incubator. For anaerobic bacteria, the plates were observed 1–2 times every 24 hours according to standard anaerobic bacterial cultivation requirements, with results typically observed after 3 days of incubation. In cases where cultures yielded negative results but there was a high suspicion of infection, the observation period could be extended, but should not exceed 5 days. DNA processing and sequencing The Nextseq 550DX sequencing platform from Microgene Company (Shanghai) was used for this study. Total DNA was extracted from 5-10ml of bile samples using QIAgen's QIAamp ® UCP Pathogen DNA Kit following the manufacturer’s instructions. Human DNA was removed using QIAGEN's Benzonase and Sigma's Tween20[ 9 ]. The DNA sample library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina, USA)[ 10 ]. The quality of the library was evaluated using the Qubit dsDNA HS Detection Kit and the highly sensitive DNA kit on the Agilent 2100 Bioanalyzer. The library pool was loaded onto the Illumina Nextseq 550Dx sequencer, and 75 single-ended sequencing runs were performed, generating approximately 20 million reads per library. For negative controls, the same protocol was used to extract Hela cell samples at a concentration of 10 5 cells/mL from each batch, and sterile deionized water was extracted alongside the samples as a non-template control[ 10 ]. Bioinformatics analysis Low-quality reads, adapter contamination, duplicate reads, and reads less than 50 bp were removed. The Khendedy tool was used with default parameters to filter out low complexity reads[ 11 ]. Alignment software was employed to identify and exclude reads mapping to the human reference genome (hg38). The pathogen list was curated based on criteria from the Johns Hopkins University ABX Guide ( https://www.hopkinsguides.com/hopkins/index/Johns_Hopkins_ABX_Guide/Pathogens ), and the Clinical Microbiology Handbook, resulting in a final database comprising approximately 13,000 genomes. Microbial reads were aligned using the nucleotide alignment program SNAP v1.0beta 18. In order to mitigate cross-species imbalance among closely related microorganisms, if microorganisms sharing a genus or family name were identified, the reads per million (RPM) of species or genera sharing a genus or family name is reduced, with a 5% reduction applied to the species[ 12 ]. The bacterial species count and relative abundance table were input into the R language software (V.4.1.0 version) for statistical analysis. The Vegan package (2.5.7 version) in R was utilized to evaluate the alpha diversity of each subject's microflora at different levels of data, including the Shannon index, Chao1 index, Simpson index, Richness index, ACE index and Evenness index. To assess overall differences in microbial community structure among different groups, the Bray Curtis distance, principal component analysis (PCA), and principal coordinate analysis (PCoA) were used to cluster the samples, achieving visualization of sample clustering based on their genus or species level composition spectra. The results of principal coordinate analysis sorting were represented by the functionality of dudi.pco in the Ade4 package (version 1.7.18) in R. Linear discriminant analysis (LDA Effect Size, LEfSe) was used to determine the association between specific microbial species or genera and groups. Statistical Analysis The results of measurement data conforming to normal distribution were expressed as mean ± standard deviation ( ± s), and the intergroup differences were compared with independent sample t-tests. Non-normally distributed data were compared using the Wilcoxon test. For counting data, percentages were used for description and intergroup differences were analyzed and compared using the chi-square test. Species diversity analysis was considered statistically significant when P value was < 0.01. Results Clinical characteristics of subjects There were 42 patients and 10 healthy controls in the total retrospective study dataset.This study collected 10 normal bile samples from liver donors in the operating room: 5 samples were collected from the gallbladder, and 5 samples were collected from the common bile duct in a non-ischemic state, with bile continuously secreted by the liver for NGS analysis only. The control group was set up to exclude the effect of malignant bile duct obstruction on the microbiota. Among 42 patients with malignant biliary obstruction, there were 20 cases of cholangiocarcinoma, 13 cases of pancreatic cancer, 3 cases of hepatocellular carcinoma, and 6 cases of hilar lymph node metastasis (gastrointestinal tumor). The positive rate of bile culture in the MBO group was 14.28% (6/42), while in the BD group it was 76.19% (32/42), indicating a statistically significant difference in the positive rate of bile culture between the two groups. Among the 29 patients who received internal/external drainage, 28 cases had positive bile culture, whereas among the 13 patients who received external drainage, only 4 cases had positive bile culture. The postoperative image of PTCD is shown in Fig. 1 . Biliary drainage and stent implantation are the main methods for relieving biliary obstruction caused by malignant tumors in interventional radiology. There are two types of biliary drainage methods: postoperative of internal and external drainage ( Fig. 1 A, B ) , and postoperative of external drainage ( Fig. 1 C, D ) . Bile duct microbiota without liver and gallbladder lesions After culture, sequencing and bioinformatics analysis of samples from different groups, we obtained the microflora distribution of each group, as shown in Fig. 2 . Compared with each other, it could be found that the bacteria belonging to the Acinetobacter and Burkholderia genera were commonly present in the microbial communities of all bile samples and not affected by biliary obstruction. In the control group, the relative abundance of Mycobacterium and Nocardia was higher, while these genera decreased significantly in the MBO and BD groups ( Fig. 2 C, Fig. 2 D ) . A diversity index is a quantitative measure that reflects how many different types (such as species) there are in a dataset, and simultaneously takes into account how evenly the basic entities (such as individuals) are distributed among those types. The value of a diversity index increases both when the number of types increases and when evenness increases. For a given number of types, the value of a diversity index is maximized when all types are equally abundant. There are three types of diversity indices, namely alpha, beta, and gamma indices. Alpha diversity is a measurement used to measure the number of species within a community and the relative abundance between species. It reflects the coexistence results of species within a community through competition for resources or utilization of the same habitat, and can be divided into three categories: species richness index, species evenness index, and species diversity index. Beta diversity is a comparative analysis of the microbial community composition of different samples/samples between different groups. Alpha diversity analysis indicated that the Shannon index of the control group was 5.31 (SD = 1.00), followed by 3.98 (SD = 1.55) in the MBO group and 2.01 (SD = 1.44) in the BD group. Beta (inter sample) diversity analysis showed that the Bray Curtis distance within the control group was the smallest at 0.69 (SD = 0.07), followed by 0.75 (SD = 0.07) in the MBO group and 0.81 (SD = 0.08) in the BD group. These results suggested that the species diversity of the bile microbiota was high in the control group, with little change in the composition and abundance of the bile microbiota in each sample. Microbial community of malignant obstructive biliary tract Compared with the control group, the relative abundance of Burkholderia, Acinetobacter, Pseudomonas and Staphylococcus in the bile microbiota of MBO group increased, while the relative abundance of Mycobacterium and Nocardia decreased (Fig. 2 D). Alpha diversity analysis showed that only the Richness index of the MBO group exhibited statistical differences compared to the control group, while other indicators showed no statistical differences, indicating a decrease in the abundance of bile microbiota in the MBO group (Fig. 2 ). PCoA analysis indicated no significant difference in the composition of bile microbiota between the MBO group and the control group (Fig. 3 A). The findings suggested that the microorganisms in the malignant obstructive biliary tract were similar to those in the disease-free biliary tract, with a reduction in the number of some bacteria (such as Mycobacterium and Nocardia), leading to an increase in the relative abundance of Burkholderia, Acinetobacter and other bacteria. LEfSe analysis was used to identify characteristic bacterial species. In this study, the default cutoff value for the LDA score was set at 3.0, indicating that species with LDA scores greater than 3 are differentiated species and could be considered as biological markers with statistical differences between groups (Fig. 3 B). LEfSe analysis revealed that Burkholderia, Chlamydococcus, Methylbacillus, Bacillus, Escherichia coli, Multibacilli and Micrococcaceae were the characteristic bacteria of the MBO group. The symbolic bacteria of the control group and MBO group were found to be positively correlated with each other (Fig. 3 C). Biliary microbiota after biliary drainage The Shannon index of the BD group was 2.01 (SD = 1.44), significantly lower than that of the control group 5.31 (SD = 1.00) and the MBO group 3.98 (SD = 1.55). These differences were statistically significant. The Evenness index which evaluates species evenness, also showed significant statistical differences between the BD group and the other two groups (Fig. 2 A). The results indicated that the species diversity and evenness of the microbial community in bile samples decreased in the BD group, suggesting the emergence of dominant species in the biliary tract colony after biliary drainage, which inhibited the growth of other bacteria. Compared with the MBO group, the relative abundance of Acinetobacter, Burkholderia and Pseudomonas in the BD group decreased significantly, while that of Nocardia, Bacillus and Streptococcus increased significantly. This suggested that the abundance of Bacillus species in the biliary tract increased significantly after biliary drainage, while the relative abundance of most other bacteria decreased, leading to a decline of species diversity of the microbial community. The beta diversity (inter sample) results showed that the Bray Curtis value of the microbial community in the bile samples of the BD group was 0.81 (SD = 0.08), the highest among groups. This indicated a significant difference in species composition among the various samples in the BD group, with an increase in heterogeneity of the biliary microbial community after biliary drainage. PCoA analysis revealed differences in the microbial structure of bile in the BD group compared to the control group and MBO group. LEfSe analysis identified Staphylococcus, Klebsiella, Enterobacteriaceae, Aeromonas, Paracoccus, Anaerococcus, Diplococcus, Campylobacter, and Megabacteriumwere as the bacteria with significant identification characteristics in the BD group (Fig. 3 B). Spearman correlation analysis showed a negative correlation between the characteristic strains in the bile samples of the BD group and the characteristic strains in the MBO group (Fig. 3 C), suggesting a competitive relationship between the characteristic strains of the two groups. Discussion The microbiota exists in the normal bile duct The concept of a microbiota existing in healthy bile ducts has historically been underappreciated, partly due to the belief that the Oddi sphincter, bile flushing, and bile salts prevent bacterial invasion and maintain sterility in the biliary tract[ 1 , 2 ]. The discovery of a complex microbial community in healthy biliary systems in 1995 challenged this view. Several factors contribute to the limited understanding of bile microbiota: (1) Traditional cultures underestimate bacterial diversity[ 13 ]; (2) Invasive procedures like ERCP, PTCD, or surgery, typically used for diseased bile ducts, limit sample availability; (3) Ethical concerns prevent obtaining bile from healthy individuals; and (4) Traditional methods lack sensitivity for low microbial loads. In 2019, Molinero et al[ 6 ] used qPCR and 16S rRNA sequencing to identify microbial communities in gallbladder bile from liver transplant donors without biliary diseases, showing the presence of microbiota in a previously assumed sterile environment. Our study expands on this by analyzing both gallbladder and common bile duct bile, showing minimal differences in microbiota composition between these sites, and greater species diversity, suggesting a stable microbiota presence throughout the biliary system. This finding challenges current understandings of biliary infectious diseases and suggests that the biliary microbiota may play a significant role in biliary health and disease. Changes in biliary microbiota of malignant biliary obstruction The relationship between biliary microbiota and malignant tumors often focuses on the link between specific bacteria and biliary cancers. For instance, the bile culture positivity rate in gallbladder cancer patients (65–81%) is significantly higher than in patients with gallstones or controls[ 14 ]. In distal cholangiocarcinoma, the bile shows an increased presence of Escherichia coli/Shigella, Staphylococcus, and Klebsiella compared to bile duct stones[ 15 ]. Aviles Jimenez et al[ 16 ] found that Proteobacteria dominate the biliary microbiota in extrahepatic cholangiocarcinoma, along with Helicobacter and Campylobacter . They also detected Helicobacter pylori virulence genes like CagA and VacA , suggesting a potential carcinogenic role. Our study showed differences in the biliary microbiota between cholangiocarcinoma and benign lesions. We noted a decrease in microbiota abundance but not in biodiversity, consistent with reference[ 16 ], indicating an inhibitory effect in the obstructed biliary system. Our bile cultures had a lower positive rate (14.28%) in malignant cases compared to benign ones, supporting previous findings[ 17 ]. Our study found an increased abundance of Burkholderia, Acinetobacter, Pseudomonas , and Staphylococcus in malignant obstructive biliary tracts. Additionally, Burkholderia, Chlamycoccus, Methylbacillus, Bacillus, Escherichia coli, Multibacilli , and Micrococcaceae are identified as characteristic bacteria in these cases, consistent with existing literature[ 16 ]. These bacteria, along with Chlamycoccus and Methylbacillus, are difficult to culture and may survive under extreme conditions. Differences from previous studies[ 14 – 16 ] could stem from: (1) Different patient groups, as our study included various cancer types; (2) Different sampling methods, as we minimized contamination by using bile collected before PTCD; and (3) Variations in ethnicity and diet, with our study focusing on a Chinese population. Changes in biliary tract microbiota caused by biliary drainage Severe malignant biliary obstruction can lead to liver and other organ damage, often requiring ERCP or PTCD drainage to improve patient conditions for further treatment. These procedures introduce bacteria into the bile duct, resulting in an infection rate exceeding 60%[ 18 , 19 ]. Tabibian et al[ 17 ] reported bacterial growth in the bile of 18.6% of patients with periampullary cancer post-surgery, with the rate rising to 97% in those who had ERCP. This highlights the impact of sphincter and stent implantation on bile bacterial colonization. Post-PTCD complications include acute cholangitis, with some patients developing bacteremia and an 8.2% mortality rate[ 19 ]. Biliary stent implantation has been shown to induce significant bile microbiota metastasis and is associated with a higher risk of postoperative surgical site infection[ 20 ]. All patients with malignant obstruction and cholangitis have a history of stent implantation[ 21 ]. In our study, bile culture positivity in the BD group was 76.19%, rising to 96.55% post-drainage, indicating increased bacterial contamination. Understanding biliary drainage’s impact on microbiota can improve knowledge of biliary microbiota dynamics. Certain bacteria introduced during drainage can alter the microbiota composition, potentially affecting bile’s tumor-suppressive effects[ 22 ]. We found that while Bacillus increased in abundance post-drainage, the overall microbial diversity decreased. Significant differences in microbial composition were observed between the BD, MBO, and normal groups, indicating dysbiosis after drainage. A retrospective study of 528 cases showed different bile microbiota compositions in ampullary cancer patients with and without pre-surgery biliary drainage[ 23 ]. In our study, the BD group had an increased presence of common pathogens like Staphylococcus, Klebsiella, Enterobacteriaceae , and Aeromonas , mostly intestinal flora. These pathogens dominated, suppressing other bacteria and leading to microbiota dysbiosis. This imbalance may persist, facilitating further infections. Thus, ongoing infection in patients warrants repeated bile cultures and adjusted clinical treatment. Conclusion Bile samples obtained from three different groups of bile ducts–normal bile duct, malignant obstructive bile duct, and those post-biliary drainage–exhibited distinct microbial compositions. The presence of a stable microbial community within the normal biliary system was indicated by the abundance and diversity of bacteria in disease-free bile ducts. The microbial community composition of malignant obstructive bile ducts resembled that of normal bile ducts. However, the composition of biliary microbiota after drainage significantly differed from that before drainage and from the control group. The characteristic bacteria in the BD group included Bacillus, Streptococcus, Staphylococcus and Klebsiella. These dominant bacteria suppressed the growth of other original bacteria in the biliary tract, leading to reduced species diversity of the microbial community. This imbalance in biliary microbiota can explain the clinical phenomenon of patients being more prone to biliary tract infections after biliary drainage. Declarations Funding This work was supported by Science and Technology Innovation Special Fund Project of Shanghai Baoshan District Science and Technology Committee (2023-E-17), Excellent Youth Program of Shanghai Baoshan District Health Committee (BSWSYC-2023-04),the National Key R&D Program of the Ministry of Science and Technology (No. 2017YFC0109204),Shanghai Key Clinical Specialty(No. shslczdzk03203). Ethics approval documentation This study was conducted in accordance with the Declaration of Helsinki and approved by the [Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University] Ethics Committee (Approval No. 2019-0066). Written informed consent was obtained from all individual participants prior to their inclusion in the study. For participants under 16 years old, consent was provided by their parent or legal guardian. Availability of Data and Materials Data is provided within the manuscript or supplementary information files. Author contributions: Wu JL collected the data and guided the study; Chen Y, Ni ZH, Fang TL, Tian QH, and Wu JL contributed to the discussion and design of the manuscript, and literature search; Gu LR contributed to the discussion and design of the manuscript; Yang K designed the overall concept; Huang XL guided the study and wrote the manuscript. Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article. References Sung JY , Costerton JW, Shaffer EA. Defense system in the biliary tract against bacterial infection. Dig Dis Sci. 1992; 37 (5):689-96. doi: 10.1007/BF01296423. PMID: 1563308 Costello EK , Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R. Bacterial community variation in human body habitats across space and time. Science. 2009; 326 (5960):1694-7. doi: 10.1126/science.1177486. PMID: 19892944 Yamashita H , Nishiyama M, Ohbuchi K, Kanno H, Tsuchiya K, Yamaguchi J, et al. 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Role of bile bacteria in gallbladder carcinoma. Hepatogastroenterology. 2007; 54 (78):1622-5. https://www.ncbi.nlm.nih.gov/pubmed/18019679 Additional Declarations No competing interests reported. Supplementary Files EthicsapprovaldocumentationAppendix1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 30 May, 2025 Editor assigned by journal 26 May, 2025 Editor invited by journal 12 May, 2025 Submission checks completed at journal 09 May, 2025 First submitted to journal 09 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6538543","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464507193,"identity":"07530252-6884-4818-87ac-b39279f28f0a","order_by":0,"name":"Jin-Liang Wu","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jin-Liang","middleName":"","lastName":"Wu","suffix":""},{"id":464507194,"identity":"cfa7562c-d4cc-4be2-a621-3a814e116fc9","order_by":1,"name":"Yan Chen","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Chen","suffix":""},{"id":464507195,"identity":"43e55ec7-43fd-4de5-a9af-45e1a82a13be","order_by":2,"name":"Zhi-Hua Ni","email":"","orcid":"","institution":"Baoshan Hospital of Traditional Chinese Medicine and Western Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Hua","middleName":"","lastName":"Ni","suffix":""},{"id":464507196,"identity":"f0c55959-2b7a-404b-a7ab-59f99c398a6c","order_by":3,"name":"Tong-Lei Fang","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Tong-Lei","middleName":"","lastName":"Fang","suffix":""},{"id":464507198,"identity":"bcd0071e-a7c4-4a14-9b84-bfe917c241eb","order_by":4,"name":"Qing-Hua Tian","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Qing-Hua","middleName":"","lastName":"Tian","suffix":""},{"id":464507199,"identity":"d3b4241a-afdf-4543-9866-86753c421f3d","order_by":5,"name":"Liang-Rui Gu","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Liang-Rui","middleName":"","lastName":"Gu","suffix":""},{"id":464507204,"identity":"5c345157-c9ad-435f-8a9f-1584b54b7cb0","order_by":6,"name":"Kai Yang","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Yang","suffix":""},{"id":464507205,"identity":"b16ac67a-d76f-4178-ab80-38486046962f","order_by":7,"name":"Xiao-Lei Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACCQYGNjCDjb+x4cAHAxs74rXwSxw+eHBGQVoy8VokG9KSD/N8OMTYQEiL5Iwcs8c8NXfsNhw4Y3DYxuAAMwP74aMb8GmRlsgxN+Y59ix5w+Eeg8M5Bnf4GHjS0m7g0yInkWMmzcN2ONkAZEuOwTNmBgkeMyK0/ANpyTE4bGFwmLGBkBZpkBbetsN2QO8nHGYgRotkz7Myybl9hxOAgXzgYI9BWjIbIb9IHE/eJvHm22F7YFQ2f/jxx8aOn/3wMbxaGAQSwFRiA0yADa9yEOA/AKbsCSocBaNgFIyCkQsAAXxPr3W+vycAAAAASUVORK5CYII=","orcid":"","institution":"Baoshan Hospital of Traditional Chinese Medicine and Western Medicine","correspondingAuthor":true,"prefix":"","firstName":"Xiao-Lei","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-04-27 06:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6538543/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6538543/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83814759,"identity":"af146fd4-a717-492a-a895-0838e11ef95a","added_by":"auto","created_at":"2025-06-03 07:31:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":434689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe postoperative images of PTCD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Internal/external drainage for cholangiocarcinoma; B: Internal/external drainage for pancreatic cancer; C: Internal drainage for pancreatic cancer; D: Internal/external drainage for hilar lymph node metastasis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6538543/v1/1b7d29f4140c214709ec8b7f.png"},{"id":83814756,"identity":"819a9a79-1f29-4cd6-a7e5-fa355d6a0899","added_by":"auto","created_at":"2025-06-03 07:31:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":676914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of microbial community structures in three sets of bile samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Histogram of the microbial community structure of the top 15 dominant species at the genus level in the sample, and different colors represent different microbial species, the vertical axis represents the relative abundance of the species, and the horizontal axis represents the sample number; B: Cluster distribution map of the top 30 dominant species at the genus level among different groups, and each row represents a species, each column represents a sample. The color depth represents the abundance of bacteria, the brown area represents species enrichment, and the blue area represents species loss; C: Composition diagram of different groups of microorganisms, and different colors represent different microbial species, the vertical axis represents the relative abundance of the species, and the horizontal axis represents different groups; D: Bar chart of relative abundance of top 10 dominant species at the genus level of microbial communities in different groups, and color represents different groups, and the horizontal axis represents different dominant species.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6538543/v1/e61924a5fb14c967a8e0e21f.png"},{"id":83814755,"identity":"0897c430-acc5-42d2-8f69-1dcb914d97d6","added_by":"auto","created_at":"2025-06-03 07:31:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":708129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetagenomics analysis of the bile microbiota for different groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: αDiversity Index Box Chart; The horizontal axis represents different groups, and the vertical axis represents the Alpha index; There was a significant difference (P\u0026lt;0.001) between the BD group and the other two groups in terms of Shannon, Simpson, and Evenness, while there was no statistically significant difference between the control group and the MBO group. The Richness index of the MBO group showed statistical differences compared to the control group and BD group (P\u0026lt;0.01); B: Bile microbiota β PCoA analysis of diversity for three groups; Each point represents a sample, and the distance between the two points represents the difference in community composition; the shorter the distance, the smaller the difference. Different colors represent different groups; C: LEfSe analysis of characteristic bacterial species (LDA score\u0026gt;3.0); Different colors represent different groups, with the name of the bacterial species on the left; D: Distribution heatmap of biomarker bacterial species; The coordinates of the horizontal and vertical axes represent characteristic species, and different colors represent different groups. The color in the heat map represents the Spearman correlation coefficient between microbial species. Brown represents positive correlation, blue represents negative correlation, and color depth represents the size of the correlation coefficient.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6538543/v1/d9df7edc17d0bcd0cc9ebde5.png"},{"id":83816158,"identity":"c1bf8b3d-a01a-45cd-90c6-60fde75c86ce","added_by":"auto","created_at":"2025-06-03 07:47:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2825281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6538543/v1/dbe971dd-86c3-4d3d-aaf8-79adfd686734.pdf"},{"id":83815778,"identity":"46324575-d531-42f6-9342-56981a6ba058","added_by":"auto","created_at":"2025-06-03 07:39:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":502929,"visible":true,"origin":"","legend":"","description":"","filename":"EthicsapprovaldocumentationAppendix1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6538543/v1/3f18ce7d86f59f974da1d598.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in biliary microbiota before and after drainage of malignant biliary obstruction","fulltext":[{"header":"Core Tip","content":"\u003cp\u003eThis study examines changes in the biliary microbiota before and after drainage in patients with malignant biliary obstruction (MBO). It was found that post-drainage, the abundance of bacteria like Staphylococcus and Klebsiella increased significantly, while overall microbial diversity and evenness decreased. This disruption in the microbial community could increase the risk of biliary tract infections, indicating the importance of monitoring and managing these microbial changes during and after drainage procedures to improve patient outcomes.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe biliary system refers to a series of organs and structures involved in the generation, storage, and excretion of bile. Its primary function is to store bile produced by the liver and release it into the digestive system when needed, which is crucial for the digestion and absorption of fats and fat-soluble vitamins. For a long time, the normal biliary tract has always been considered sterile[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], as it has several mechanisms to inhibit bacterial growth. The Oddi sphincter resists reflux, preventing bacteria from the duodenum from entering the bile ducts. The gallbladder releases bile into the duodenum, preventing bacterial colonization in the bile ducts. Additionally, bile contains a large amount of bile acids with antibacterial properties, which can inhibit bacterial growth[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eWith the advancement of research on the human microbiota, the presence of a microbiota in the normal biliary system has become a topic of debate[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In 1995, with the emergence and wide application of 16S ribosomal RNA sequencing technology, it was confirmed that there were microorganisms in bile samples that \"could not cultivate bacteria\", leading to the proposal of the concept of \"biliary microbiota\"[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In 2019, Molinero et al[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] reported for the first time that the presence of a microbiota in the gallbladder bile of 13 liver transplantation donors without hepatobiliary and pancreatic diseases. The discovery of biliary microbiota has fundamentally changed our understanding of the development of biliary infectious diseases[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Research on the diversity, stability, and correlation of microbiota in the biliary system with physiological and pathological conditions has gradually been conducted.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the changes in the bile microbiota of the bile duct after percutaneous transhepatic catheter drainage (PTCD) for malignant biliary obstruction. Using bile samples from liver transplant donors as the control group, and bile samples before and after obstruction and drainage as the experimental group, we aim to study the impact of biliary obstruction and drainage interventions on the bile microbiota from a microecological perspective. This research provides a factual and theoretical basis for further exploration of the pathogenesis of acute cholangitis and the improvement of drainage interventions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDiagnostic criteria for malignant biliary obstruction\u003c/h2\u003e \u003cp\u003eThe criteria for diagnosing malignant biliary obstruction were based on previous studies, and patients were required to meet all of the following three conditions:\u003c/p\u003e \u003cp\u003e1) Clinical manifestations: yellow staining of skin and sclera and epigastrium discomfort.\u003c/p\u003e \u003cp\u003e2) Imaging findings (at least one of the following): (1) Direct signs: presence of space-occupying lesions in the hilar or surrounding bile ducts and pancreatic ampullary lesions; and (2) Indirect signs: significant dilation of the intrahepatic and extrahepatic bile ducts, with a maximum diameter\u0026thinsp;\u0026gt;\u0026thinsp;15mm and a \"soft-rattan-like\" appearance; abrupt cessation of biliary dilation, resulting in a \"residual root shape\"; presence of a \"double duct sign\" involving the common bile duct and pancreatic duct.\u003c/p\u003e \u003cp\u003e3) Laboratory tests or pathological results: elevation of tumor indicators (CA199, AFP, CEA, etc.) or biopsy findings indicating malignant tumors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInclusion criteria\u003c/b\u003e \u003c/p\u003e \u003cp\u003e1) Patients who meet the above diagnostic criteria and are aged\u0026thinsp;\u0026ge;\u0026thinsp;18 years old;\u003c/p\u003e \u003cp\u003e2) Ability to provide informed consent form;\u003c/p\u003e \u003cp\u003e3) Indications for PTCD (at least one of the following): (1) Palliative biliary drainage for malignant obstruction caused by advanced tumors; (2) Preoperative preparation for patients with malignant biliary obstruction; and (3) Emergency biliary decompression and drainage for malignant biliary obstruction combined with acute biliary infection.\u003c/p\u003e \u003cp\u003e4) Adequate coagulation function, defined as an international normalized ratio (INR)\u0026thinsp;\u0026le;\u0026thinsp;2.0.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExclusion criteria\u003c/b\u003e \u003c/p\u003e \u003cp\u003e1) Refusal to provide informed consent;\u003c/p\u003e \u003cp\u003e2) Contraindications for biliary drainage or inability to cooperate with the procedure;\u003c/p\u003e \u003cp\u003e3) Use of antibiotics or other medications within the past two months that may affect the study results;\u003c/p\u003e \u003cp\u003e4) Expected survival period of less than three months;\u003c/p\u003e \u003cp\u003e5) Pregnant or lactation;\u003c/p\u003e \u003cp\u003e6) Inability to provide complete clinical data.\u003c/p\u003e \u003cp\u003ePatients who have not undergone biliary drainage are classified into the malignant biliary obstruction group (MBO group), while those who have undergone biliary drainage are classified into the biliary drainage group (BD group). The control group consisted of bile samples collected from liver donors at our hospital during the same period. This study strictly adheres to the ethical requirements of relevant medical research.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eResearch subjects\u003c/h3\u003e\n\u003cp\u003eThis retrospective study comprised 42 patients with malignant biliary obstruction who underwent PTCD at the department of radiology interventional in our hospital between January 2020 and December 2022. Comprehensive patient information was recorded and organized, including underlying disease, clinical symptoms, medical history, results of laboratory and imaging examination results, as well as bile culture and NGS technology detection results. The presence and severity of acute cholangitis were assessed and graded according to the 2018 Tokyo guidelines[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the use of antibiotics for treatment was also documented. The current study was approved by the Ethics Committee of Shanghai Sixth People's Hospital, and implemented under the guidance of the clinical operation.\u003c/p\u003e\n\u003ch3\u003ePTCD procedure and bile sample collection\u003c/h3\u003e\n\u003cp\u003ePTCD procedure: The patient was positioned flat on the bed, and the right upper abdomen was prepared and draped in a sterile fashion. After identifying the puncture site, 2% lidocaine was used to infiltrate and anesthetize the liver capsule. A 22G puncture needle was inserted into the bile duct, and bile overflow was confirmed either by seeing bile emerge from the needle or by using an empty needle to extract bile. A small amount of diluted contrast agent was injected to perform cholangiography, visualizing the degree of intrahepatic bile duct dilation, extent of obstruction, and stenosis. A 0.018-inch guide wire was advanced deep into the bile duct, and the puncture needle was removed. A vascular sheath was then introduced. After removing the guide wire, 15\u0026ndash;20 ml of sterile bile samples were collected. Subsequently, a 0.035-inch guide wire was inserted through the narrow segment into the duodenum. Finally, an external or internal drainage tube was implanted: the distal end of the internal/external drainage tube was positioned outside the obstructive lesion (usually the duodenum), and the proximal drainage hole was placed in front of the obstructive lesion. The drainage tube, external tee, and drainage bag were secured to the skin surface.\u003c/p\u003e \u003cp\u003eBile sample collection: Bile was collected twice using a drainage tube, once during the PTCD procedure and once 7 days after the procedure. Sterile containers were used for collection, and each sample was divided into two parts. One part was immediately sent for bile culture and testing, while the other part was stored in a -80℃ refrigerator for subsequent NGS testing.\u003c/p\u003e\n\u003ch3\u003eBile culture\u003c/h3\u003e\n\u003cp\u003eAfter the bile samples were collected, they were inoculated onto standard chocolate agar plates and blood agar plates as soon as possible. The blood agar plates were incubated in a conventional incubator at 35\u0026ndash;37 ℃, while the chocolate agar plates were incubated in a 5% -10% carbon dioxide incubator. For anaerobic bacteria, the plates were observed 1\u0026ndash;2 times every 24 hours according to standard anaerobic bacterial cultivation requirements, with results typically observed after 3 days of incubation. In cases where cultures yielded negative results but there was a high suspicion of infection, the observation period could be extended, but should not exceed 5 days.\u003c/p\u003e\n\u003ch3\u003eDNA processing and sequencing\u003c/h3\u003e\n\u003cp\u003eThe Nextseq 550DX sequencing platform from Microgene Company (Shanghai) was used for this study. Total DNA was extracted from 5-10ml of bile samples using QIAgen's QIAamp \u0026reg; UCP Pathogen DNA Kit following the manufacturer\u0026rsquo;s instructions. Human DNA was removed using QIAGEN's Benzonase and Sigma's Tween20[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The DNA sample library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina, USA)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The quality of the library was evaluated using the Qubit dsDNA HS Detection Kit and the highly sensitive DNA kit on the Agilent 2100 Bioanalyzer. The library pool was loaded onto the Illumina Nextseq 550Dx sequencer, and 75 single-ended sequencing runs were performed, generating approximately 20\u0026nbsp;million reads per library. For negative controls, the same protocol was used to extract Hela cell samples at a concentration of 10\u003csup\u003e5\u003c/sup\u003e cells/mL from each batch, and sterile deionized water was extracted alongside the samples as a non-template control[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eLow-quality reads, adapter contamination, duplicate reads, and reads less than 50 bp were removed. The Khendedy tool was used with default parameters to filter out low complexity reads[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Alignment software was employed to identify and exclude reads mapping to the human reference genome (hg38). The pathogen list was curated based on criteria from the Johns Hopkins University ABX Guide (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.hopkinsguides.com/hopkins/index/Johns_Hopkins_ABX_Guide/Pathogens\u003c/span\u003e\u003cspan address=\"https://www.hopkinsguides.com/hopkins/index/Johns_Hopkins_ABX_Guide/Pathogens\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the Clinical Microbiology Handbook, resulting in a final database comprising approximately 13,000 genomes. Microbial reads were aligned using the nucleotide alignment program SNAP v1.0beta 18. In order to mitigate cross-species imbalance among closely related microorganisms, if microorganisms sharing a genus or family name were identified, the reads per million (RPM) of species or genera sharing a genus or family name is reduced, with a 5% reduction applied to the species[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe bacterial species count and relative abundance table were input into the R language software (V.4.1.0 version) for statistical analysis. The Vegan package (2.5.7 version) in R was utilized to evaluate the alpha diversity of each subject's microflora at different levels of data, including the Shannon index, Chao1 index, Simpson index, Richness index, ACE index and Evenness index. To assess overall differences in microbial community structure among different groups, the Bray Curtis distance, principal component analysis (PCA), and principal coordinate analysis (PCoA) were used to cluster the samples, achieving visualization of sample clustering based on their genus or species level composition spectra. The results of principal coordinate analysis sorting were represented by the functionality of dudi.pco in the Ade4 package (version 1.7.18) in R. Linear discriminant analysis (LDA Effect Size, LEfSe) was used to determine the association between specific microbial species or genera and groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe results of measurement data conforming to normal distribution were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u003cimg src=\"data:image/png;base64,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\" style=\"width: 15px; height: 19px;\" width=\"15\" height=\"19\"\u003e\u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003e\u0026plusmn;\u0026thinsp;s), and the intergroup differences were compared with independent sample t-tests. Non-normally distributed data were compared using the Wilcoxon test. For counting data, percentages were used for description and intergroup differences were analyzed and compared using the chi-square test. Species diversity analysis was considered statistically significant when P value was \u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics of subjects\u003c/h2\u003e \u003cp\u003eThere were 42 patients and 10 healthy controls in the total retrospective study dataset.This study collected 10 normal bile samples from liver donors in the operating room: 5 samples were collected from the gallbladder, and 5 samples were collected from the common bile duct in a non-ischemic state, with bile continuously secreted by the liver for NGS analysis only. The control group was set up to exclude the effect of malignant bile duct obstruction on the microbiota. Among 42 patients with malignant biliary obstruction, there were 20 cases of cholangiocarcinoma, 13 cases of pancreatic cancer, 3 cases of hepatocellular carcinoma, and 6 cases of hilar lymph node metastasis (gastrointestinal tumor). The positive rate of bile culture in the MBO group was 14.28% (6/42), while in the BD group it was 76.19% (32/42), indicating a statistically significant difference in the positive rate of bile culture between the two groups. Among the 29 patients who received internal/external drainage, 28 cases had positive bile culture, whereas among the 13 patients who received external drainage, only 4 cases had positive bile culture.\u003c/p\u003e \u003cp\u003eThe postoperative image of PTCD is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Biliary drainage and stent implantation are the main methods for relieving biliary obstruction caused by malignant tumors in interventional radiology. There are two types of biliary drainage methods: postoperative of internal and external drainage \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e, and postoperative of external drainage \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBile duct microbiota without liver and gallbladder lesions\u003c/h2\u003e \u003cp\u003eAfter culture, sequencing and bioinformatics analysis of samples from different groups, we obtained the microflora distribution of each group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Compared with each other, it could be found that the bacteria belonging to the Acinetobacter and Burkholderia genera were commonly present in the microbial communities of all bile samples and not affected by biliary obstruction. In the control group, the relative abundance of Mycobacterium and Nocardia was higher, while these genera decreased significantly in the MBO and BD groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. A diversity index is a quantitative measure that reflects how many different types (such as species) there are in a dataset, and simultaneously takes into account how evenly the basic entities (such as individuals) are distributed among those types. The value of a diversity index increases both when the number of types increases and when evenness increases. For a given number of types, the value of a diversity index is maximized when all types are equally abundant. There are three types of diversity indices, namely alpha, beta, and gamma indices. Alpha diversity is a measurement used to measure the number of species within a community and the relative abundance between species. It reflects the coexistence results of species within a community through competition for resources or utilization of the same habitat, and can be divided into three categories: species richness index, species evenness index, and species diversity index. Beta diversity is a comparative analysis of the microbial community composition of different samples/samples between different groups. Alpha diversity analysis indicated that the Shannon index of the control group was 5.31 (SD\u0026thinsp;=\u0026thinsp;1.00), followed by 3.98 (SD\u0026thinsp;=\u0026thinsp;1.55) in the MBO group and 2.01 (SD\u0026thinsp;=\u0026thinsp;1.44) in the BD group. Beta (inter sample) diversity analysis showed that the Bray Curtis distance within the control group was the smallest at 0.69 (SD\u0026thinsp;=\u0026thinsp;0.07), followed by 0.75 (SD\u0026thinsp;=\u0026thinsp;0.07) in the MBO group and 0.81 (SD\u0026thinsp;=\u0026thinsp;0.08) in the BD group. These results suggested that the species diversity of the bile microbiota was high in the control group, with little change in the composition and abundance of the bile microbiota in each sample.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial community of malignant obstructive biliary tract\u003c/h2\u003e \u003cp\u003eCompared with the control group, the relative abundance of \u003cem\u003eBurkholderia, Acinetobacter, Pseudomonas\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e in the bile microbiota of MBO group increased, while the relative abundance of Mycobacterium and Nocardia decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Alpha diversity analysis showed that only the Richness index of the MBO group exhibited statistical differences compared to the control group, while other indicators showed no statistical differences, indicating a decrease in the abundance of bile microbiota in the MBO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCoA analysis indicated no significant difference in the composition of bile microbiota between the MBO group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The findings suggested that the microorganisms in the malignant obstructive biliary tract were similar to those in the disease-free biliary tract, with a reduction in the number of some bacteria (such as Mycobacterium and Nocardia), leading to an increase in the relative abundance of Burkholderia, Acinetobacter and other bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLEfSe analysis was used to identify characteristic bacterial species. In this study, the default cutoff value for the LDA score was set at 3.0, indicating that species with LDA scores greater than 3 are differentiated species and could be considered as biological markers with statistical differences between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). LEfSe analysis revealed that Burkholderia, Chlamydococcus, Methylbacillus, Bacillus, Escherichia coli, Multibacilli and Micrococcaceae were the characteristic bacteria of the MBO group. The symbolic bacteria of the control group and MBO group were found to be positively correlated with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBiliary microbiota after biliary drainage\u003c/h2\u003e \u003cp\u003eThe Shannon index of the BD group was 2.01 (SD\u0026thinsp;=\u0026thinsp;1.44), significantly lower than that of the control group 5.31 (SD\u0026thinsp;=\u0026thinsp;1.00) and the MBO group 3.98 (SD\u0026thinsp;=\u0026thinsp;1.55). These differences were statistically significant. The Evenness index which evaluates species evenness, also showed significant statistical differences between the BD group and the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results indicated that the species diversity and evenness of the microbial community in bile samples decreased in the BD group, suggesting the emergence of dominant species in the biliary tract colony after biliary drainage, which inhibited the growth of other bacteria.\u003c/p\u003e \u003cp\u003eCompared with the MBO group, the relative abundance of Acinetobacter, Burkholderia and Pseudomonas in the BD group decreased significantly, while that of Nocardia, Bacillus and Streptococcus increased significantly. This suggested that the abundance of Bacillus species in the biliary tract increased significantly after biliary drainage, while the relative abundance of most other bacteria decreased, leading to a decline of species diversity of the microbial community.\u003c/p\u003e \u003cp\u003eThe beta diversity (inter sample) results showed that the Bray Curtis value of the microbial community in the bile samples of the BD group was 0.81 (SD\u0026thinsp;=\u0026thinsp;0.08), the highest among groups. This indicated a significant difference in species composition among the various samples in the BD group, with an increase in heterogeneity of the biliary microbial community after biliary drainage. PCoA analysis revealed differences in the microbial structure of bile in the BD group compared to the control group and MBO group.\u003c/p\u003e \u003cp\u003eLEfSe analysis identified Staphylococcus, Klebsiella, Enterobacteriaceae, Aeromonas, Paracoccus, Anaerococcus, Diplococcus, Campylobacter, and Megabacteriumwere as the bacteria with significant identification characteristics in the BD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Spearman correlation analysis showed a negative correlation between the characteristic strains in the bile samples of the BD group and the characteristic strains in the MBO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting a competitive relationship between the characteristic strains of the two groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe microbiota exists in the normal bile duct\u003c/h2\u003e \u003cp\u003eThe concept of a microbiota existing in healthy bile ducts has historically been underappreciated, partly due to the belief that the Oddi sphincter, bile flushing, and bile salts prevent bacterial invasion and maintain sterility in the biliary tract[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The discovery of a complex microbial community in healthy biliary systems in 1995 challenged this view. Several factors contribute to the limited understanding of bile microbiota: (1) Traditional cultures underestimate bacterial diversity[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; (2) Invasive procedures like ERCP, PTCD, or surgery, typically used for diseased bile ducts, limit sample availability; (3) Ethical concerns prevent obtaining bile from healthy individuals; and (4) Traditional methods lack sensitivity for low microbial loads.\u003c/p\u003e \u003cp\u003eIn 2019, Molinero et al[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] used qPCR and 16S rRNA sequencing to identify microbial communities in gallbladder bile from liver transplant donors without biliary diseases, showing the presence of microbiota in a previously assumed sterile environment. Our study expands on this by analyzing both gallbladder and common bile duct bile, showing minimal differences in microbiota composition between these sites, and greater species diversity, suggesting a stable microbiota presence throughout the biliary system. This finding challenges current understandings of biliary infectious diseases and suggests that the biliary microbiota may play a significant role in biliary health and disease.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChanges in biliary microbiota of malignant biliary obstruction\u003c/h2\u003e \u003cp\u003eThe relationship between biliary microbiota and malignant tumors often focuses on the link between specific bacteria and biliary cancers. For instance, the bile culture positivity rate in gallbladder cancer patients (65\u0026ndash;81%) is significantly higher than in patients with gallstones or controls[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In distal cholangiocarcinoma, the bile shows an increased presence of Escherichia coli/Shigella, Staphylococcus, and Klebsiella compared to bile duct stones[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Aviles Jimenez et al[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] found that \u003cem\u003eProteobacteria\u003c/em\u003e dominate the biliary microbiota in extrahepatic cholangiocarcinoma, along with \u003cem\u003eHelicobacter\u003c/em\u003e and \u003cem\u003eCampylobacter\u003c/em\u003e. They also detected \u003cem\u003eHelicobacter pylori\u003c/em\u003e virulence genes like \u003cem\u003eCagA\u003c/em\u003e and \u003cem\u003eVacA\u003c/em\u003e, suggesting a potential carcinogenic role.\u003c/p\u003e \u003cp\u003eOur study showed differences in the biliary microbiota between cholangiocarcinoma and benign lesions. We noted a decrease in microbiota abundance but not in biodiversity, consistent with reference[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], indicating an inhibitory effect in the obstructed biliary system. Our bile cultures had a lower positive rate (14.28%) in malignant cases compared to benign ones, supporting previous findings[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study found an increased abundance of \u003cem\u003eBurkholderia, Acinetobacter, Pseudomonas\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e in malignant obstructive biliary tracts. Additionally, \u003cem\u003eBurkholderia, Chlamycoccus, Methylbacillus, Bacillus, Escherichia coli, Multibacilli\u003c/em\u003e, and \u003cem\u003eMicrococcaceae\u003c/em\u003e are identified as characteristic bacteria in these cases, consistent with existing literature[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These bacteria, along with Chlamycoccus and Methylbacillus, are difficult to culture and may survive under extreme conditions. Differences from previous studies[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] could stem from: (1) Different patient groups, as our study included various cancer types; (2) Different sampling methods, as we minimized contamination by using bile collected before PTCD; and (3) Variations in ethnicity and diet, with our study focusing on a Chinese population.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eChanges in biliary tract microbiota caused by biliary drainage\u003c/h2\u003e \u003cp\u003eSevere malignant biliary obstruction can lead to liver and other organ damage, often requiring ERCP or PTCD drainage to improve patient conditions for further treatment. These procedures introduce bacteria into the bile duct, resulting in an infection rate exceeding 60%[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Tabibian et al[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] reported bacterial growth in the bile of 18.6% of patients with periampullary cancer post-surgery, with the rate rising to 97% in those who had ERCP. This highlights the impact of sphincter and stent implantation on bile bacterial colonization. Post-PTCD complications include acute cholangitis, with some patients developing bacteremia and an 8.2% mortality rate[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Biliary stent implantation has been shown to induce significant bile microbiota metastasis and is associated with a higher risk of postoperative surgical site infection[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All patients with malignant obstruction and cholangitis have a history of stent implantation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In our study, bile culture positivity in the BD group was 76.19%, rising to 96.55% post-drainage, indicating increased bacterial contamination.\u003c/p\u003e \u003cp\u003eUnderstanding biliary drainage\u0026rsquo;s impact on microbiota can improve knowledge of biliary microbiota dynamics. Certain bacteria introduced during drainage can alter the microbiota composition, potentially affecting bile\u0026rsquo;s tumor-suppressive effects[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We found that while Bacillus increased in abundance post-drainage, the overall microbial diversity decreased. Significant differences in microbial composition were observed between the BD, MBO, and normal groups, indicating dysbiosis after drainage.\u003c/p\u003e \u003cp\u003eA retrospective study of 528 cases showed different bile microbiota compositions in ampullary cancer patients with and without pre-surgery biliary drainage[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In our study, the BD group had an increased presence of common pathogens like \u003cem\u003eStaphylococcus, Klebsiella, Enterobacteriaceae\u003c/em\u003e, and \u003cem\u003eAeromonas\u003c/em\u003e, mostly intestinal flora. These pathogens dominated, suppressing other bacteria and leading to microbiota dysbiosis. This imbalance may persist, facilitating further infections. Thus, ongoing infection in patients warrants repeated bile cultures and adjusted clinical treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBile samples obtained from three different groups of bile ducts\u0026ndash;normal bile duct, malignant obstructive bile duct, and those post-biliary drainage\u0026ndash;exhibited distinct microbial compositions. The presence of a stable microbial community within the normal biliary system was indicated by the abundance and diversity of bacteria in disease-free bile ducts. The microbial community composition of malignant obstructive bile ducts resembled that of normal bile ducts. However, the composition of biliary microbiota after drainage significantly differed from that before drainage and from the control group. The characteristic bacteria in the BD group included Bacillus, Streptococcus, Staphylococcus and Klebsiella. These dominant bacteria suppressed the growth of other original bacteria in the biliary tract, leading to reduced species diversity of the microbial community. This imbalance in biliary microbiota can explain the clinical phenomenon of patients being more prone to biliary tract infections after biliary drainage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Science and Technology Innovation Special Fund Project of Shanghai Baoshan District Science and Technology Committee (2023-E-17), Excellent Youth Program of Shanghai Baoshan District Health Committee (BSWSYC-2023-04),the National Key R\u0026amp;D Program of the Ministry of Science and Technology (No. 2017YFC0109204),Shanghai Key Clinical Specialty(No. shslczdzk03203).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval documentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and approved by the [Shanghai Sixth People\u0026apos;s Hospital Affiliated to Shanghai Jiaotong University] Ethics Committee (Approval No. 2019-0066). Written informed consent was obtained from all individual participants prior to their inclusion in the study. For participants under 16 years old, consent was provided by their parent or legal guardian.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWu JL collected the data and guided the study; Chen Y, Ni ZH, Fang TL, Tian QH, and Wu JL contributed to the discussion and design of the manuscript, and literature search; Gu LR contributed to the discussion and design of the manuscript; Yang K designed the overall concept; Huang XL guided the study and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict-of-interest statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors report no relevant conflicts of interest for this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eSung JY\u003c/strong\u003e, Costerton JW, Shaffer EA. 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Hepatogastroenterology. 2007;\u003cstrong\u003e54\u003c/strong\u003e(78):1622-5. https://www.ncbi.nlm.nih.gov/pubmed/18019679\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Malignant biliary obstruction, Digital subtraction angiography, Percutaneous transhepatic catheter drainage, Microbial community","lastPublishedDoi":"10.21203/rs.3.rs-6538543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6538543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehe biliary system stores bile for fat digestion and nutrient absorption. Surgery or other changes can disrupt this system, altering the microbiome and potentially leading to complications. Such disruptions may impact patient outcomes, highlighting the need for careful monitoring during biliary drainage procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAims\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate changes in biliary microbiota before and after MBO drainage and assess the impact on microbiota.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this retrospective study, 42 MBO patients underwent percutaneous transhepatic catheter drainage (PTCD) from January 2020 to December 2022. The cohort included patients with cholangiocarcinoma, pancreatic cancer, hepatocellular carcinoma, and hilar lymph node metastasis. Bile samples were collected before and 7 days after drainage, analyzed using culture and next-generation sequencing (NGS). Data on acute cholangitis and antibiotic use were also collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to drainage, common bacteria included \u003cem\u003eBurkholderia\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e, similar to normal biliary microbiota. Post-drainage, there was a notable increase in \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e, and other pathogens, along with a decrease in microbial diversity and evenness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn MBO patients, the biliary microbiota resembles that of non-diseased ducts but significantly changes post-drainage. There is increased pathogenic bacteria and reduced diversity, explaining the higher infection risk.\u003c/p\u003e","manuscriptTitle":"Changes in biliary microbiota before and after drainage of malignant biliary obstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 07:31:17","doi":"10.21203/rs.3.rs-6538543/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-05-30T14:56:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-26T08:36:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-12T05:03:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-09T13:44:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Gastroenterology","date":"2025-05-09T13:43:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-gastroenterology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmge","sideBox":"Learn more about [BMC Gastroenterology](http://bmcgastroenterol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmge/default.aspx","title":"BMC Gastroenterology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84a8015f-1016-4d1d-b8c7-c2545a5285cf","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T07:31:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 07:31:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6538543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6538543","identity":"rs-6538543","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}