Analysis of Biliary Microbiota in Hepatobiliopancreatic Diseases Compared to Healthy People [MICROBILIO]
Conditions: Pancreatic Diseases, Microbiota, Early Detection of Cancer, Dysbiosis, Biliary Tract Diseases
Sponsor: University of Sao Paulo

1 
 
 
 
Official study title: “Analysis of the biliary MICRObiota in hepatoBILIOpancreatic diseases 
compared to healthy people [MICROBILIO]” 
NCT number: NCT04391426 
Date of the document: 06/04/2020 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
University of São Paulo 
Faculty of Medicine 
 

2 
Universidade of São Paulo 
Faculty of Medicine 
Analysis of biliary MICRObiota in liverBILIOpancreatic diseases compared to 
healthy people [MICROBILIO] 
 
Researchers Involved 
Fernanda Sayuri do Nascimento1, Milena Oliveira Suzuki1, João Victor Taba1,Vitoria 
Carneiro de Mattos1, Leonardo Zumerkorn Pipek1, Eugênia Machado Carneiro 
D’Albuquerque2, João Renato Rebello Pinho3, Alberto Meyer3, Wellington Andraus3, 
Eduardo Guimarães Hourneaux de Moura3, Luiz Augusto Carneiro-D’Albuquerque3, João 
Carlos Setubal4 
1: Faculty of Medicine FMUSP, University of  São Paulo, São Paulo, SP, BR; 2 Medicine Academic  -Santa 
Marcelina Faculty , São Paulo, SP, Brazil; 3: Department of Gastroenterology, Hospital das Clínicas, HCFMUSP, São Paulo, 
Brazil; 4: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil. 
Abstract 
The performance of the microbiota is observed in all clinical and pathological stages of 
carcinogenesis, since its development, diagnosis and treatment, including prognosis and 
survival. However, it was found that there is a scarcity of studies on biliary microbiota and its 
relationship with hepatobiliopancreatic diseases. Therefore, further investigation is necessary, 
since reaching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, 
tests and therapies in hepatobiliopancreatic diseases. For this, bile samples will be collected in 
cases and controls patients to characterize the microbiota and its variations according to the 
disease. 
Keywords: Pancreatic Diseases; Microbiota; Early Detection of Cancer; Dysbiosis; 
Biliary Tract Diseases 
1. Introduction 
Microbioma is the set of microorganisms that occurs naturally in a particular site, such 
as the human gastrointestinal tract. Typically, it has trillions of microbes, including fungi, 
viruses and bacteria [1], which coexist with human cells. 

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Normally, the microbiome bacteria interact with the epithelial barrier, with immune 
cells modulating their response, in addition to influencing local metabolism through their own 
metabolites. This maintains homeostasis [2]. Thus, an imbalance of the microbiota, such as the 
use of antibiotics or due to bacterial translocation, can lead to the development of diseases. It 
does this through the proliferation of pathogenic bacteria, for example, which can greatly affect 
the host and have potential pathological implications. [2-4] 
Studies have shown a close relation between dysbiosis and the outbreak of infections or 
chronic diseases. In 2019, Saus et al. [5] gathered data about the relation between the intestinal 
microbiota and the development of colorectal carcinoma (CRC), the most studied since the 
1990s. Currently, it is known that in patients with this neoplasm there is a co-abundance of pro-
inflammatory factors, opportunistic pathogens and other microbes. This is associated with 
metabolic dysfunction and the depletion of butyrate-producing bacteria, an important factor in 
intestinal homeostasis. With these studies, interest was raised in investigating other sites, such 
as the biliopancreatic tract (BPT). In 2015, Mitsuhashi et al. showed the association of the oral 
microbiota with the pancreatic carcinogenesis process. In periodontal diseases, Fusobacterium 
can be translocated via lympho-hematogenous pathways, leading to pancreatic dysbiosis. This 
would be associated with malignancy in the progression of pancreatic adenocarcinoma and 
worse prognosis. [4] 
Thus, research was conducted in rats, finding an association between the components of 
the tumour microbiota and the speed of progression of  biliopancreatic disease [6]. In pancreases 
of rats and humans with pancreatic duct adenocarcinoma (PDAC), a greater abundance of 
Malassezia spp. was found compared to bowel or pancreas controls without the disease. Due to 
the presence of the fungus, there is greater activation of mannose-binding lectin (MBL) and, 
consequently, the complement cascade is activated, leading to greater inflammation in the 
pancreas, which accelerates tumour progression [6, 7]. 
Traditionally, BPT neoplasia are diagnosed at an advanced stage, despite the 
improvement in the quality of diagnostic imaging. For early diagnosis, Mendez et al. conducted 
an experiment with PDAC-mutated mice before they developed the disease. By DNA 
sequencing of the fecal microbiota bacteria, it was found that, with the progression of pancreatic 
carcinogenesis, there was a change in the bacterial composition. The metabolites of these 
bacteria associated with the tumour promote greater production of polyamines, which increases 
as the neoplasia develops. Then, the dosage of polyamines could be used as a biomarker to track 
the progression of adenocarcinoma [8]. The analysis of the fecal microbiota, therefore, would 

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be a possibility for early diagnosis of PDAC, prompting research in human patients at high risk 
for carcinogenesis. 
Among the therapies, the most commonly used treatments for neoplasia are 
chemotherapy and radiotherapy. In BPT carcinomas, however, these methods have low 
sensitivity [9], which can be attributed, according to studies, to tumor dysbiosis. As previously 
mentioned, unchanged symbiotic microbiota mediates the immune response. Thus its 
imbalance decreases the expression of genes related to inflammation, phagocytosis, antigen 
presentation and adaptive immune response. On the other hand, genes related to tissue 
development, cancer and metabolism are stimulated [10]. Thus, the use of chemotherapeutic 
drugs loses effectiveness due to this negative regulation of the immune system's anti-tumor 
capacity. An example of this is oxaliplatin, whose effect is to stimulate the production of 
reactive oxygen species to promote DNA damage and tumor cell apoptosis. In mice injected 
with colon carcinoma cells, their cytotoxic effect decreased after being treated with antibiotics. 
[9, 10]  
There have been several advances in the introduction of new chemical compounds that 
interfere in specific signalling pathways of carcinogenesis, also affected by the microbiome. 
Because of this association, microbial agents and their metabolites are being tested to develop 
treatments that can reduce the tumor and are potentially preventive [11]. This was observed in 
studies with species of Lactobacillus, which modulates the expression of some enzymes such 
as beta-glucuronidase. The action of the enzyme is reduced by bacilli. This acts in the 
disjunction of carcinogenic agents, converting pro-carcinogens into their active form [12]. 
Lenoir et al. also demonstrated that L.casei has anti-tumor properties by decreasing the T-reg 
response and increasing Th17, promoting a decrease in CRC in rats. Thus, the microbe proved 
to be a protector and a new therapeutic alternative to carcinoma. [11, 13] 
Even with current treatments, mortality in some groups of BPT malignancies remains 
high, with low survival rates. However, in recent studies in patients with pancreatic duct 
adenocarcinoma, it was found that the greater variety of the tumor microbiota and the 
predominance of specific bacterial genera are related to a longer survival time when treated 
surgically [6]. Considering this recent progress, improvement is expected for the coming 
decades. 
Thus, the performance of the microbiota is observed in all clinical and pathological 
stages of carcinogenesis, from its development, diagnosis and treatment, including prognosis 

5 
and survival. However, there is a lack of studies on biliary microbiota and its relation with 
hepatobiliopancreatic diseases. Therefore, further investigation is necessary, since researching 
the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, interventions and 
therapies in hepatobiliopancreatic diseases. 
2. Aim 
In this study, our aim will be to characterize the specific composition of the biliary 
microbiota in patients with hepatobiliopancreatic diseases compared to healthy controls, using 
16S ribosomal RNA (rRNA) pyrosequencing methods. 
3. Material and Methods 
3.1 Study design and patients 
This is a case-control study that will be carried out at the Department of 
Gastroenterology of Clinicas Hospital in the Faculty of Medicine of the University of São 
Paulo. 
Patients who will undergo endoscopic retrograde cholangiopancreatography (ERCP) 
and donors previously selected to interventional liver transplantation will be recruited for the 
collection of bile, configuring case and control groups, respectively. The project outline is 
shown in figure 1. 
Figure 1 

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3.2 External Validation 
All patients who will not be included, with a condition for which a procedure is planned, 
such as ERCP or hepatectomy for liver transplantation, will have their records noted and stored, 
including information on date, sex, age, Body Mass Index (BMI) and the reason for non-
inclusion or exclusion. 
3.3 Patients who will undergo ERCP (case group) 
3.3.1 Inclusion criteria 
• 
Patients over 18 years old  
• 
Patients previously scheduled for ERCP 
• 
Cannulation of the bile duct, via the transpapillary route, with the aid of a 
papillotome with an end kept sterile until contact with the papilla 
3.3.2 Exclusion Criteria 

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• 
Use of antibiotics during ERCP or in the last 2 months prior to the procedure 
• 
Emergency ERCP 
• 
Pregnancy 
• 
Uncorrected coagulopathy 
History of previous ERCP will not be considered an exclusion criterion. 
3.3.3 ERCP Technical Description  
The procedure will be performed under conscious sedation or general anesthesia, at the 
discretion of the medical team responsible for carrying out the procedure. 
Before the beginning of ERCP, the end of the papillotome will be covered with sterile 
surgical plastic (plastic cover for videolaparoscopy), in order to avoid its contamination during 
passage through the working channel of the duodenoscope and contact with the digestive tract, 
up to the greater duodenal papilla. 
 The duodenoscope will be introduced according to the usual technique and positioned 
in front of the greater duodenal papilla. The papillotome with a sterile end will then be passed 
through the working channel of the duodenoscope. Before attempting cannulation of the papilla, 
air will be injected through the papillotome injection channel to remove the sterile plastic from 
its end. 
When performing cannulation of the bile duct, with the aid of endoscopic and 
radioscopic vision, the route of the papillotome injection until bile return will be aspirated with 
a sterile 5 ml syringe, to confirm the correct positioning of the instruments in the bile duct. 
Then, 1 ml of bile will be aspirated, which will be sent for analysis of the microbiota. 
If there is contamination of the papillotome prior to contact with the papilla, the patient 
will be excluded from the protocol. 
3.4 Living liver transplantation donors (control group) 
The correct selection of donors for living donor liver transplantation is essential not only 
to decrease the risk of complications for donors, but also to increase graft and recipient survival. 
First, there must be ABO blood type compatibility. Then, the compatibility between the 
weight and height of the patient and the donor is analysed. Subsequently, the size of the liver 
to be donated is evaluated, and it is necessary to calculate the relation between the weight of 

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the liver (donor) with the recipient. Finally, the entire anatomy of the donor and recipient is 
evaluated, such as veins, arteries and bile ducts. 
3.4.1 Inclusion Criteria 
• 
Patients over 18 years old up to 55 years old 
• 
Previously selected patients with scheduled surgery 
• 
BMI: 18 kg/m² to 28 kg/m² 
• 
Blood typing identical to the recipient 
• 
Absence of significant medical, psychiatric problems or previous abdominal 
surgery 
• 
Normal laboratory tests: liver function tests, blood count, coagulogram, 
pregnancy test and serology for hepatitis B, C and HIV 
• 
Normal imaging exams: CT of the abdomen and pelvis with liver volume 
(remaining volume - 30-40% of the total liver volume), MRI with cholangioresonance 
3.4.2 Exclusion Criteria 
• 
Use of antibiotics in the last 2 months prior to the procedure 
• 
Pregnancy 
• 
Uncorrected coagulopathy 
3.4.3 Description of the interventional liver transplantation technique 
The hepatectomy procedure for related living donor liver transplantation, left lobe or 
right lobe will be performed under general anesthesia. 
Started by a Makuuchi incision (“J” incision), followed by positioning the retractor and 
cavity inventory, and dissecting the cystic artery and cystic duct. 
The cystic duct will be opened and its catheterization and aspiration of 1 ml of bile will 
be carried out, which will be sent for analysis of the microbiota. 
Subsequently, intraoperative cholangiography will be performed to study the intra and 
extrahepatic biliary anatomy, followed by anterograde cystic-funicular cholecystectomy and 
left or right hepatectomy as clinical indication. 
3.5 Processing of samples 

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Sample collections will be sent to the Molecular Biology Sector of the Central 
Laboratory Division (LIM-03), which will be responsible for DNA extraction, construction of 
16S libraries and sequencing of microbiomes. 
3.5.1 DNA Extraction 
            DNA extraction will be performed from 1 ml of the content of the e-swab, using the 
DNA Kit Zymobiomics MiniPrep, according to the manufacturer's recommendations. The 
DNA will be stored immediately at -20 ° C until use. 
3.5.2 Sequencing of microbiomes 
The sequencing of microbiomes will be performed using the 16S Metagenomic 
Sequencing Library protocol (Illumina, San Diego, USA). In parallel, the microbiomes will also 
be sequenced based on total DNA extracted from the samples, for a more complete assessment 
of the bacterial species found in each patient. Genomic DNA libraries will be built using 
Nextera® XT DNA Sample Preparation Kit (Illumina) from an input of 1 µg of DNA, according 
to the manufacturer's recommendations. The 16S and genomic DNA libraries will be sequenced 
in MiSeq (Illumina, San Diego, USA), using the MiSeq® Reagent Kit v3 (600 cycles; Illumina, 
San Diego, USA). 
3.5.3 Data analysis 
The data analyses will be performed at the Bioinformatics Laboratory (Setulab), located 
in the Biochemistry Department, at the Chemistry Institute of the University of São Paulo. 
Sequencing the 16S amplicons (bacteria) from the different samples will result in sets of 
reads. The data will be sent to Setulab servers, where the analyses will be carried out, which 
will mainly follow the steps and programs available in the QIIME 2 package [14]. Briefly, the 
steps include quality control, to remove short reads or medium grades below a minimum 
limit; determination of amplicon sequence variants (ASVs) using the deblur [15] and / or 
DADA2 [16] programs; taxonomic classification of ASVs; alpha and beta diversity analyses; 
rarefaction analysis; and several statistical analyses, seeking to show differences in the 
microbial composition between control and patient samples. In the latter case, in addition to 
the QIIME 2 packages, we will use the STAMP program [17]. The results will be made 
available mainly in the form of tables and graphs. 
The analysis of total DNA data will follow the following steps: 
1.    Quality control for the removal of short reads or average quality below a threshold. 

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2.    Quality control to separate DNA from the microbiota from any human DNA that 
has been sequenced. 
3.    Classification of reads through the centrifuge program [18]. 
4. Application of the metaWrap pipeline [19] to recover data genomes (generation of 
MAGs). The application will be separated by sample, and the results will be compared 
later. 
5.    Classification of MAGs using the GTDB-tk program [20]. 
6.    Comparison of the results of taxonomic classification of total DNA data with those 
obtained by 16S. 
The results obtained will also be compared with results from the literature, in particular 
through the eHOMD website [21]. 
3.5.4 Data management 
Once this study is completed, the biological material will receive a code and will be filed in the 
biorepository anonymously, and may be used for other academic studies, without commercial 
purpose, as long as approved by the Research Ethics Committee, in accordance with the 
guidelines of the national body that coordinates the principles of research in our country, the 
National Commission for Ethics in Research. 
 
3.5.5 Safety and risks regarding adverse events 
Bile collection, both in the case group and in the control group, carries minimal risk and 
discomfort. However, serious adverse events related to examination (ERCP) and / or 
hepatectomy surgery will be documented on a form throughout the course of the study 
and will be reported to the principal investigator within 24 hours of observation. If the 
event is considered to be related to the collection of bile by the principal investigator, 
he will send a report to the local ethics committee within 3 days. 
3.6 Timetable 
The research is estimated to last twenty-four months, according to the following 
schedule: 
Month 
1 2 3 4 5 6 7 8 9 10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
Bibliographic 
Survey 
X X X X X X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sample collection 
 
 
 
X X X X X X X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Sample processing 
 
 
 
 
 
 
 
X X X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 

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Data tabulation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
Results analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
Completion of 
work 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
4. Result parameters 
The primary purpose of the study is to characterize the specific composition of the 
biliary microbiota in situations of disease and health. For this, the microbiota of patients with 
hepatobiliopancreatic diseases will be compared with that of healthy controls. In addition, it 
seeks to demonstrate the existence of changes in the case of illness. 
Further investigation includes disease biomarkers, diagnosis, interventions and 
therapies in hepatobiliopancreatic diseases. 
5. Dissemination policy 
MICROBILIO results should be presented at international medical conferences on 
corresponding areas of interest, for example, gastroenterology. Written publications will be 
submitted to surgical or endoscopic scientific journals. The authorship of written publications 
must be confirmed unequivocally by all leading researchers. 
6. Ethics and registration committee 
The study protocol was approved by the Hospital Ethics Committee (Faculty of 
Medicine, University of São Paulo - FMUSP - CAAE: 29547920.9.0000.0068). The study was 
registered at ClincalTrials.gov (Identifier: NCT04391426). 
7. Protocol version 
This manuscript refers to the first version of the complete study protocol, made on June 
4th, 2020. Modifications to the protocol will be reported to all investigators, the local Research 
Ethics Committee, all participants and the journal. 
8. Discussion 
 
The composition and role of the microbiota of the gastrointestinal tract is an increasing 
focus of study. There is even evidence that these microbes are related to cancer and other 
chronic diseases [5]. Biliary dysbiosis, for example, has been shown to be a protagonist in the 

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pathogenesis of several diseases in this tract, as has been recently described. However, given 
the proximity and connection between the pancreatic, hepatic and biliary tracts, it may be that 
the influence of this microbiota is more extensive, reaching the entire hepatobiliopancreatic 
tract [22]. 
On the other hand, the bile microbiota is still little studied, being sequenced in healthy 
individuals only recently [23]. Little is known about its composition in malignant diseases [24], 
with biliary lithiasis being the most studied and best characterized disease [23, 25]. 
This study aims to consolidate existing knowledge and deepen it with additional 
information. Thus, we seek to broaden the panorama about the different compositions of the 
biliary microbiota and, possibly, to shed light on a new form of study for hepatobiliopancreatic 
diseases, as well as strategies on how to deal with such conditions. 
9. Research Status 
This project is suspended due to the COVID-19 pandemic, which makes it impossible 
to recruit and collect data, scheduled to resume in August 2020. 
10. Interest Conflict 
The authors declare that they have no conflict of interest. 
11. References 
1. Sender R, Fuchs S, Milo R. Revised Estimates for the Number of Human and Bacteria Cells 
in the Body. PLoS Biol. 2016;14(8):e1002533. 
2. Cresci GA, Bawden E. Gut Microbiome: What We Do and Don't Know. Nutr Clin Pract. 
2015;30(6):734‐746.  
3. Akshintala VS, Talukdar R, Singh VK, Goggins M. The Gut Microbiome in Pancreatic 
Disease. Clin Gastroenterol Hepatol. 2019;17(2):290‐295.  
4. Mitsuhashi K, Nosho K, Sukawa Y, et al. Association of Fusobacterium species in pancreatic 
cancer tissues with molecular features and prognosis. Oncotarget. 2015;6(9):7209‐7220.  
5. Saus E, Iraola-Guzmán S, Willis JR, Brunet-Vega A, Gabaldón T. Microbiome and colorectal 
cancer: Roles in carcinogenesis and clinical potential. Mol Aspects Med. 2019;69:93‐106.  
6. Riquelme E, Zhang Y, Zhang L, et al. Tumor Microbiome Diversity and Composition 
Influence Pancreatic Cancer Outcomes. Cell. 2019;178(4):795‐806.e12.  

13 
7. Aykut B, Pushalkar S, Chen R, et al. The fungal mycobiome promotes pancreatic 
oncogenesis via activation of MBL. Nature. 2019;574(7777):264‐267.  
8. Mendez R, Kesh K, Arora N, et al. Microbial dysbiosis and polyamine metabolism as 
predictive markers for early detection of pancreatic cancer [published online ahead of print, 
2019 Aug 1]. Carcinogenesis. 2019;bgz116.  
9. Mima K, Nakagawa S, Sawayama H, et al. The microbiome and hepatobiliary-pancreatic 
cancers. Cancer Lett. 2017;402:9‐15.  
10.  Iida N, Dzutsev A, Stewart CA, et al. Commensal bacteria control cancer response to 
therapy by modulating the tumor microenvironment. Science. 2013;342(6161):967‐970.  
11. Wong SH, Yu J. Gut microbiota in colorectal cancer: mechanisms of action and clinical 
applications. Nat Rev Gastroenterol Hepatol. 2019;16(11):690‐704.  
12. Desrouillères K, Millette M, Bagheri L, Maherani B, Jamshidian M, Lacroix M. The 
synergistic effect of cell wall extracted from probiotic biomass containing Lactobacillus 
acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 on the anticancer activity of 
cranberry juice-HPLC fractions. J Food Biochem. 2020;44(5):e13195.  
13. Lenoir M, Del Carmen S, Cortes-Perez NG, et al. Lactobacillus casei BL23 regulates Treg 
and Th17 T-cell populations and reduces DMH-associated colorectal cancer. J Gastroenterol. 
2016;51(9):862‐873.  
14. Bolyen E, Rideout JR, Dillon MR, et al. Reproducible, interactive, scalable and extensible 
microbiome data science using QIIME 2 [published correction appears in Nat Biotechnol. 2019 
Sep;37(9):1091]. Nat Biotechnol. 2019;37(8):852‐857.  
15. Amir A, McDonald D, Navas-Molina JA, et al. Deblur Rapidly Resolves Single-Nucleotide 
Community Sequence Patterns. mSystems. 2017;2(2):e00191-16.  
16. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-
resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581‐583.  
17. Parks DH, Tyson GW, Hugenholtz P, Beiko RG. STAMP: statistical analysis of taxonomic 
and functional profiles. Bioinformatics. 2014;30(21):3123‐3124.  
18. Kim D, Song L, Breitwieser FP, Salzberg SL. Centrifuge: rapid and sensitive classification 
of metagenomic sequences. Genome Res. 2016;26(12):1721‐1729.  

14 
19. Uritskiy GV, DiRuggiero J, Taylor J. MetaWRAP-a flexible pipeline for genome-resolved 
metagenomic data analysis. Microbiome. 2018;6(1):158.  
20. Parks DH, Chuvochina M, Waite DW, et al. A standardized bacterial taxonomy based on 
genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018;36(10):996‐1004.  
21. Escapa IF, Chen T, Huang Y, Gajare P, Dewhirst FE, Lemon KP. New Insights into Human 
Nostril Microbiome from the Expanded Human Oral Microbiome Database (eHOMD): a 
Resource for the Microbiome of the Human Aerodigestive Tract. mSystems. 2018;3(6):e00187-
18.  
22. Nicoletti A, Ponziani FR, Nardella E, Ianiro G, Gasbarrini A, Zileri Dal Verme L. Biliary 
tract microbiota: a new kid on the block of liver diseases?. Eur Rev Med Pharmacol Sci. 
2020;24(5):2750‐2775.  
23. Molinero N, Ruiz L, Milani C, et al. The human gallbladder microbiome is related to the 
physiological state and the biliary metabolic profile. Microbiome. 2019;7(1):100.  
24. Chen B, Fu SW, Lu L, Zhao H. A Preliminary Study of Biliary Microbiota in Patients with 
Bile Duct Stones or Distal Cholangiocarcinoma. Biomed Res Int. 2019;2019:1092563.  
25. Shen H, Ye F, Xie L, et al. Metagenomic sequencing of bile from gallstone patients to 
identify different microbial community patterns and novel biliary bacteria. Sci Rep. 
2015;5:17450.  
26. Gao Z, Guo B, Gao R, Zhu Q, Qin H. Microbiota disbiosis is associated with colorectal 
cancer. Front Microbiol. 2015;6:20.