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European Review for Medical and Pharmacological Sciences 2020; 24: 2750-2775 microbiota: a new kid on the block of ?

A. NICOLETTI1, F.R. PONZIANI2, E. NARDELLA1, G. IANIRO2, A. GASBARRINI1, L. ZILERI DAL VERME2

1Internal , and , Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy 2Internal Medicine, Gastroenterology and Hepatology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy

Abstract. – The microbiome plays a crucial man body1,2. Indeed, a resident microbiota has recent- role in maintaining the homeostasis of the or- ly been described in several human environments ganism. Recent evidence has provided novel previously described as devoid of microorganisms, insights for understanding the interaction be- such as the urinary tract and the stomach3-9. Even tween the microbiota and the host. However, the 10 vast majority of such studies have analyzed the healthy placenta hosts microbial communities . interactions taking place in the intestinal tract. has traditionally been considered sterile The biliary tree has traditionally been consid- under normal conditions11-14. ered sterile under normal conditions. However, The physical and chemical features of bile and the advent of metagenomic techniques has re- its antimicrobial activity were supposed to create vealed an unexpectedly rich bacterial communi- a hostile environment for . Moreover, the ty in the biliary tract. Associations between specific microbiolog- difficulty in collecting bile samples, coupled with ical patterns and inflammatory biliary diseases the lack of sensibility of culture techniques in and have been recently described. Hence, detecting microbes in low-charge samples, sus- biliary dysbiosis may be a primary trigger in the tained this hypothesis for a long time. pathogenesis of biliary diseases. In particular, In 1967, while studying the microbial flora of recent studies have suggested that microorgan- patients undergoing percutaneous cholangiog- isms could play a significant role in the develop- raphy, Flemma et al15 observed that a consistent ment of , pathogenesis of autoimmune cholangiopathies and biliary carcinogenesis. number of patients had a positive bile culture with- Moreover, the intimate connection between out having had any signs, symptoms or history of the biliary tract, liver and , could reveal cholangitis. Ahead of their time, they hypothesized hidden influences on the development of diseas- that bacteria could exist in bile without causing es of these organs. any symptoms attributable to their presence. They Further studies are needed to deepen the named this condition “asymptomatic bactibilia”15. comprehension of the influence of the biliary microbiota in human pathology. This knowl- About 40 years later, the advent of 16S ribo- edge could lead to the formulation of strategies somal RNA sequencing confirmed the presence for modulating the biliary microbiota in order to of microbes in bile samples otherwise consid- treat and prevent these pathological conditions. ered sterile with culture-based techniques16. This knowledge has introduced the concept of “biliary Key Words: microbiota”. Biliary microbiota, Gallstones, Cholelithiasis, Primary At any level, the interplay between the micro- sclerosing cholangitis, Primary biliary cholangitis, Bili- ary tract cancer, , car- biota and the host plays a pivotal role in the main- cinoma, Personalized medicine. tenance of homeostasis. However, quantitative or qualitative changes in the composition of the mi- crobial community can derange this equilibrium, 17 Introduction favoring the development of diseases . Recent evidence has revealed rich microbial An increasing number of studies about the human communities in the biliary tract of patients affect- microbiota have dismissed the classical postulate ed by biliary tract diseases. A remarkable associ- which states that there are sterile sites within the hu- ation has been observed between certain microbi-

2750 Corresponding Author: Alberto Nicoletti, MD; e-mail: [email protected] Biliary tract microbiota: a new kid on the block of liver diseases? al strains and each pathology. Thus, possible roles the bile-duct . Cholangiocytes, which for bacteria in such pathogenic processes have are highly heterogeneous in both structure and been hypothesized18-22. function 23,32,33, modify bile through a sequence The understanding of the interplay between of secretory and absorptive processes in order to the microbiota and the host at this level may fa- regulate its flow and alkalinity according to the cilitate the formulation of novel strategies for the physiological functions24. Along the biliary tree, prevention and treatment of such pathological glandular elements called peribiliary glands or conditions. accessory glands are also present34. Ductal se- cretion is regulated by a wide range of factors, Overview of the Biliary System: including gastrointestinal hormones and choliner- Anatomical and Cellular Determinants gic nerves35. The final secretory product is deliv- for the Production and Secretion of Bile ered to the gallbladder and then to the . The biliary system represents a complex net- Although the gallbladder is not essential for the work of ducts and organs that are involved in secretion of bile, it helps its storage to prepare for the production and transportation of bile23. Bile fat digestion30. During fasting, the gallbladder is production is a complex biological process that filled with bile31. Only about 50% of the hepatic begins in the bile canaliculi, which are formed by bile reaches the gallbladder for concentration and the apical membranes of two adjacent pericentral storage, while the remaining bile bypasses the linked by tight junctions24. The he- gallbladder to enter the duodenum and undergo patocyte apical membrane is provided with both continuous enterohepatic cycling36. During diges- bile salt-dependent and independent transport tion, cholecystokinin stimulates the contraction systems, which are series of adenosine triphos- of the gallbladder and the common and phate-binding cassette transport proteins that the relaxation of the SO, resulting in the discharge function as export pumps for bile salts and other of up to 80% of the gallbladder contents into the organic solutes25. These transport systems create duodenum37,38. osmotic gradients in the bile canaliculi, which give the driving force for the flow into the lumen The Mutual Interaction Between Bile and through aquaporins24. Tight junctions hold the the Microbiota hepatocytes together and form a physical barrier Bile is a vital aqueous solution composed of between the blood and canalicular lumen, facil- ∼95% water in which organic and inorganic sol- itating “paracellular permeability”24,26. Bile can- utes, including bile acids, , phospho- aliculi conduct the flow of bile countercurrent to lipids, and amino acids, are dissolved24. the direction of the portal blood and connect with Bile acids (BAs) are the most prevalent organic the initial branches of the biliary tree, i.e., the compounds in bile, constituting approximately canals of Hering27,28. These structures continue 50% of the organic components of bile. BAs are into ducts that progressively increase in diameter: 24-carbon water-soluble products of cholester- small bile ductules (diameter <15 μm), interlobu- ol metabolism24,39. There are two processes and lar ducts (15-100 μm), septal ducts (100-300 μm), anatomical sites for the biosynthesis of BAs: the area ducts (300-400 μm), segmental ducts (400- primary BAs are first synthesized de novo from 800 μm), and hepatic ducts (>800 μm) as original- cholesterol in the liver and then are modified ly defined by Ludwig23,29. The confluence of the by bacterial enzymes in the intestine38. The two right and left hepatic ducts at the hepatic hilum primary BAs synthesized in the liver are cholic forms the that is joined by acid (CA), a trihydroxylated bile salt, and cheno- the from the gallbladder to form the (CDCA), a dihydroxy bile salt39. . The common bile duct runs These salts can be conjugated at the side chain through the head of the pancreas and ends in the with taurine or glycine, a process that metaboliz- sphincter of Oddi (SO), while penetrating the du- es BAs into stronger acids limiting their passive odenal wall to form the ampulla of Vater, which reabsorption at the biliary tree24. Intestinal bac- connects it to the pancreatic duct30. SO is a seg- teria, a consortium of a small number of species ment of circular and longitudinal belonging to the class Clostridia40, produce “sec- that incorporates the distal common bile duct and ondary BAs” by removal of the hydroxyl group at , contained in the duodenal wall31. C7, transforming to deoxycholic acid Once bile is secreted into the biliary tree, it is (DCA) and CDCA to lithocholic acid (LCA)38,39,41. exposed to cholangiocytes that form the lining of During transit through the caecum and colon,

2751 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme conjugated BAs can also be “deconjugated” from crease in the production of inflammatory cyto- the link with glycine or taurine by enzymes kines and in innate immune cells phagocytosis, known as bile salt hydrolases (BSH), which are which is mediated by the inhibition of NFκB expressed by Gram-positive intestinal bacterial pathway68,69. species such as Lactobacillus42-46, Enterococ- However, the aforementioned evidence is ob- cus47,48, Bifidobacterium49-51, and Clostridium52. tained from studies on the , BSH activity has also been described in the com- while the interaction between the host and the mensal Gram-negative spp. and in microbiota in the biliary environment is still in- the Archaea domain, such as Methanobrevibacter completely studied and poorly understood. smithii and Methanosphaera stadtmanae53. More- Along with gastric acid secretion and pancreat- over, numerous enteric species (Clostridium, Pep- ic enzymes, bile is responsible for the increasing tostreptococcus, Bacteroides, Eubacterium, and gradient of abundance of the from ) can oxidize and epimerize the the duodenum to the colon rectum70. In fact, bile hydroxy groups of BAs, leading to the generation has important antimicrobial properties. The am- of isobile (β-hydroxy) salts54, such as ursodeoxy- phipathic nature of BAs exerts membrane-dam- cholic acid (UDCA), which are among the most aging effects by binding and dissolving mem- hydrophilic BAs. Most of these conjugated and brane lipids and determine cellular lysis71-73. This deconjugated BAs are reabsorbed in the distal in- emulsification process involves a detergent action testine, where they undergo enterohepatic circu- that is negatively correlated with the number of lation, thus maintaining the BA pool36. This pool hydroxyl groups in the molecule. Thus, primary varies from 2 to 4 g and recirculates 6-10 times a BAs (CDCA and CA) are more toxic than second- day. This “recycle” is a highly economic circuit ary ones (LCA and DCA)69. Once BAs enter the that exerts important regulatory effects on several bacterial cytoplasm, they elicit other cytotoxic hepatic, biliary and intestinal functions55. mechanisms, including the internal acidification Thus, the gut microbiota exerts a strong influ- of cytoplasm and the generation of toxic com- ence on bile. Specifically, the intestinal bacteria pounds such as (H2S), which is are able to alter the composition of the BA pool. produced by the cleavage of taurine-conjugated Since the transformation of primary BAs into bile salts69. Moreover, bile is able to cause DNA secondary ones depends on the action of bacteria, damage74, oxidative stress75 and osmotic effects76 modifications in the gut microbiota that express against bacteria. BSH and -inducible (BAI) enzymes af- Besides the physical and chemical antimicrobi- fect the functions and signaling properties of al properties, bile contributes to the immunologi- BAs56. Quantitative or qualitative perturbations cal defense of organism against enteric infections of the BA pool have been related to several hu- by secreting immunoglobulins A (IgA), antimi- man diseases, such as metabolic syndrome57,58, crobial peptides, inflammatory cytokines (e.g., cancer59,60, inflammatory bowel diseases (IBD)61 tumor necrosis factor (TNF)-α), leukotrienes and and the occurrence and recurrence of Clostridium their metabolites and stimulating the innate im- difficile colitis62,63. BAs are also involved in the mune system in the intestine24,77-79. In addition, pathogenesis of several biliary diseases; for ex- BAs activate the nuclear receptor FXRα, that me- ample, in autoimmune cholangiopathies BAs play diates antibacterial effects by the upregulation of a significant role in the initiation of , genes involved in mucosal defense80. development of liver damage and progression to Altogether, these effects limit bacterial growth, liver fibrosis64. The magnitude of these pathogenic particularly in the . mechanisms is highlighted by the fact that the use of obeticholic acid, a CDCA-derived farnesoid X Bacterial Colonization of the Biliary receptor (FXR) agonist, is an effective treatment Tract: Biliary Defensive Systems and for primary biliary cholangitis65. Microbial Tolerance Mechanisms Furthermore, the interaction occurring in the The biliary tract owns several defensive sys- gastrointestinal tract between the gut microbiota tems to protect bile and the biliary mucosa from and the immune system is crucial for the main- bacterial colonization and infection. tenance of human homeostasis66,67. BAs are im- Firstly, the aforementioned antimicrobial prop- portant signaling mediators in immunological erties of bile reduce the concentration of bacteria mechanisms. Indeed, the activation of bile acid in the duodenum70. Secondly, the SO acts as a receptors, such as FXR and TGR5, causes a de- mechanical barrier that separates the duodenum

2752 Biliary tract microbiota: a new kid on the block of liver diseases? from the biliary tree. Its basal tone at rest of 15- and certain species of Helicobacter87 possess an 18 mmHg higher than duodenal pressure prevents incredible tolerance to high concentrations of BAs. the massive passage of bacteria from the gastroin- Several Gram-positive , including Liste- testinal tract, which would otherwise result from ria spp.88, Enterococcus faecalis89 and Clostridia90, the increased intestinal pressure caused by peri- have also demonstrated an ability to colonize bile. stalsis. Moreover, its coordinated action with the Microbes can reach the biliary tract through gallbladder allows the bile flow, which is another different routes, of which the ascending route functional cleansing effect to eliminate pathogens through the SO has traditionally been considered and potentially harmful substances from the bili- the most frequent route of entry of bacteria into ary tract. In fact, about 800-1000 ml of bile flows the biliary system. The dysfunctions of the SO, through the bile ducts everyday81. such as SO laxity, affect the activity of this “gate- Even if some microorganisms manage to over- keeper”, resulting in an increase in the passage of come these systems, the biliary secreted bacteria by duodenal reflux91. by biliary epithelium prevents them from adher- Sphincterotomy, performed during either en- ing to the biliary tract mucosa81. Furthermore, the doscopic retrograde cholangiopancreatography higher concentration of BAs at this level exerts (ERCP) or surgery, causes the loss of function and higher toxicity toward the bacteria38. integrity of SO. Similarly, the positioning of bili- The integrity of the continuous monocellular ary in order to treat mechanical stenosis of epithelium represents another important mechan- the biliary tree favors a direct passage92-95. An in- ical element that prevents the translocation of termittent or incomplete obstruction to bile flow, bacteria into the liver or the systemic circulation. as observed in choledocholithiasis and Tight junctions seal the intercellular spaces, en- of the ampulla, is another risk factor for biliary suring the continuity of the barrier81. contamination and infection15,96. The biliary epithelium also shows a wide range Furthermore, bacteria can reach the biliary of innate immune receptors, such as toll-like-re- tract through two hematogenous routes: via the ceptor (TLR) 1 to TLR6 and TLR9, and surface portal venous system or systemic circulation81,97. and intracellular adaptors that mediate the signal- Indeed, the biliary epithelium is nourished by a ing pathways and the initiation of inflammatory network of capillaries called peribiliary vascular responses82,83. In addition, antimicrobial peptides plexus98. This plexus originates from the terminal including human β-defensin-1 and -2 are widely branches of the hepatic artery and has anastomot- expressed in the intrahepatic biliary tree84. ic connections with the portal vein vasculature98. Tissue macrophages and liver Kupffer cells, Hence, as a consequence of increased intestinal activated by proinflammatory cytokines, are re- permeability, bacterial translocation into the por- sponsible for microbial killing and antigen pre- tal circulation can lead viable bacteria inside the sentation to the T cells and plasma cells in mes- biliary system99-101. enteric lymph nodes or minor lymphoid glands Finally, during bacteremia, microorganisms adjacent to bile ducts. The activation of the adap- can be transported into the biliary tract97. Using tive response enhances the production of immu- this route, enterica reaches the gall- noglobulins that can be found in bile, mainly as bladder, which represents its reservoir in typhoid secretory IgA77. carriers. Indeed, after disrupting of the intestinal Microorganisms must possess tolerance mech- epithelium, the bacterium infects the intestinal anisms in order to resist bile action. Thus, in order macrophages that reach the intestinal lymph to survive in the environmental conditions pre- nodes and then the systemic circulation102,103. sented by bile, bacteria respond with adaptations to the pH and detergent effects of bile. In partic- The Biliary Microbiota in ular, they strengthen their membrane, by modify- The knowledge about the composition of the ing its lipid composition and upregulate the ex- biliary microbiota in health represents the first pression of efflux pumps, porins, transmembrane step in the understanding of the influence of the proteins and BSH. However, bile tolerance is microbiota on the development of biliary diseases. strain-specific and in vitro models do not always Jiménez et al104 analyzed the bile, gallbladder coincide with in vivo observations38. mucus and mucosal microbiome of healthy pigs In general, Gram-negative bacteria show a high- using culture-based as well as metagenomics er resistance to bile than Gram-positive ones38. In techniques. All the cultured samples harvested particular, Salmonella spp.85, Escherichia coli86 bacterial species (6/6, 100%) and the number of

2753 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme identified species ranged from 3 to 20 per sample. tion from the duodenum, could better explain the Bacteria isolated from cultures were broadly bal- occurrence of some biliary infectious diseases. anced among Firmicutes (34%), Actinobacteria For ethical reasons, the majority of the research (32%) and (32%) phyla. Bacteroi- on the human biliary microbiota has focused on detes accounted for a lesser part (2% of the iso- the study of pathological models. Emerging evi- lates), suggesting an inadequate adaptation to this dence has provided new insights into the biliary environment. At the genus level, Staphylococcus, microbiota and has improved the understanding Streptococcus, Kocuria, Rothia, Acinetobacter of the pathogenesis of biliary diseases, such as and Psychrobacter were isolated from different gallstones, autoimmune cholangiopathies and bil- samples, suggesting a possible role as members of iary tract . the core biliary microbiota of pigs104. The 16S ribosomal RNA metagenomic profiling The Biliary Microbiota in the identified Streptococcus alactolyticus, a common Pathogenesis of Gallstones commensal in the gastrointestinal tract of pigs105, Since the 1920s, it has been known that the as the largely dominant species (>90%) in two an- formation of gallstones occurs irrespective of the imals104. It was also observed to be the prevalent presence of bile infection107,108. The first evidence isolate from the bile of another animal in the cul- of the possible involvement of microbial products ture-based assessment, as well. A higher bacterial in the pathogenesis of gallstones was obtained in diversity with a lower prevalence of some other the 1960s. Based on the previous observations that species (Lactobacillus salivarius and Bacillus sp.) infection with Escherichia coli could be impli- was observed in the remaining samples. Interest- cated in the pathogenesis of formation, ingly, apart from bile, the microbiological analysis Maki et al109 demonstrated that the of of gallbladder mucus and mucosa, broadened the bacterial β-glucuronidase in bile could hydrolyse spectrum of bacteria that could possibly colonize the into bilirubin and glu- the mucus and cellular brush border104. curonic acid, which could precipitate in the pres- Knowledge about the human physiological bili- ence of to form calcium bilirubinate109,110. ary microbiota has been lacking for years. Indeed, Indeed, β-glucuronidase expressing bacteria bile sampling techniques, such as ERCP, percuta- have been frequently identified in the samples neous biliary drainage and intra-operatory sam- of patients with pigmented gallstones111-115. Other pling, are invasive procedures that can only be bacterial enzymes, such as phospholipases and performed when a biliary tract is already BA hydrolases have later been shown to be impli- present or suspected. cated with similar mechanisms in the formation More recently, Molinero et al106 analyzed the of pigmented gallstones116-119. biliary microbiota of 27 liver donors (13 without Moreover, a study using scanning electron mi- and 14 with cholelithiasis). The 16S ribosomal croscopy (SEM) has demonstrated the presence of RNA sequencing revealed a prevalence of Acti- bacterial microcolonies or bacterial casts within nobacteria, Firmicutes and Bacteroidetes in both the pigmented gallstones along with bile coloni- the bile samples and gallbladder tissues of sub- zation assessed with bile culture. Bacteria, adher- jects without gallstones. A significant increase in ing to the pigment solids via glycocalyx, could the abundance of the Propionibacteriaceae fam- alter the local physico-chemical characteristics of ily and Sphingomonas genus was also reported bile by means of their enzymes, thus favoring the compared with individuals with gallstones. formation of pigmented gallstones120-124. This study provided the first evidence of the Thus, the studies conducted during the 1980s human biliary microbiota in subjects unaffected have confirmed Maki’s hypothesis and the role of by hepatopancreatobiliary diseases. However, bacteria in the pathogenesis of pigmented gall- larger samples are needed to confirm these re- stones is widely accepted16,114,117,125-128. sults and evaluate the core biliary microbiota of Interestingly, in a study using SEM and bile healthy individuals. culture, most of the patients with evidence of Confirmation of the hypothesis of stable colo- bacteria in the gallstones did not show any clin- nization of the biliary tract by resident microbial ical signs of biliary infection117. Considering the communities may revolutionize our knowledge selection bias in the collection of gallstones from on the development of biliary infectious diseases. patients undergoing surgery, this result under- Indeed, from a microbiota-centric view, a focal lines that dysbiosis of the biliary microbiota is a dysbiotic process, rather than an ascending infec- frequent occurrence.

2754 Biliary tract microbiota: a new kid on the block of liver diseases?

The importance of bacterial enzymes in the knowledgement of resident microbiota occurred in pathogenesis of pigmented and mixed gallstones 1998. Indeed, the same authors, using quantitative has been further highlighted by genomic tech- PCR, demonstrated that a vast majority (71/91, niques. In a previous study using polymerase chain 78%) of culture-negative cholesterol gallstones reaction (PCR)-based amplification and sequenc- had low bacterial concentrations of 103 CFU/10 ing of bacterial genes encoding various enzymes, mg, while only few culture-negative stones the presence of a gene encoding β-glucuronidase (11/91, 12%) had concentrations comparable to was observed in most of the mixed cholesterol culture-positive ones. Only 9 of the 100 cholester- gallstones, while bacterial sequences of E. coli and ol gallstones analyzed showed no bacterial DNA Pseudomonas sp. were identified in all the pig- and all of them had an elevated mean percentage mented and mixed cholesterol gallstones129. of cholesterol content (93.9±2.8%), confirming Conversely, the formation of cholesterol gall- the previous observation. The genomic analysis stones has traditionally been considered to be of gallstones with positive bile cultures showed dependent on metabolic imbalance and genetic a predominance of the bacterial strains identified variances rather than a bacterial detrimental ef- by the culture, suggesting an ongoing infection. fect126. Culture-based techniques and electron Interestingly, the genomic pattern of culture-neg- microscopy have failed to identify bacteria in this ative gallstones with high concentrations of bac- type of stones in most cases. In fact, a positive teria revealed a combination of different bacterial bile culture was observed in 10-33% of the sam- sequences, with no predominance of one partic- ples120,130-132. However, since the identification of ular strain compared to the others. Similarly, on microorganisms depends on their viability and average, 3.6 sequences per stone were observed cultivability, cultured bacteria are not representa- in the cholesterol gallstones with low bacterial tive of the complete biliary microbiome. concentration. Finally, after 6-month storage at A significant progress in research on the bili- -20°C, gallstones with both positive and nega- ary microbial system was made with the advent of tive bile cultures, but with high concentrations of bacterial genomic techniques16 (Figure 1, Table I). bacteria determined by genomic analysis, showed In 1995, Swidsinski et al16 analyzed the cho- the appearance of new bacterial sequences, that lesterol gallstones from patients with negative accounted for up to 20% of the total. Most of the bile culture using PCR-based amplification and sequences belonged to bacterial strains, such as 16S ribosomal RNA sequencing and found bac- Bacillus, Alcaligenes, Carnobacterium and Burk- terial DNA in 16 out of 17 patients (94%) with holderia, that are difficult to cultivate but are able gallstones with cholesterol content ranging from to survive and grow under extreme conditions134. 70 to 90%. Pure cholesterol gallstones (>90% While on the one hand the high concentration cholesterol content) showed no bacterial DNA. of a single bacterial species is consistent with an Although a thorough genus level identification infection, on the other hand, the simultaneous pres- was not feasible at the time of the study, the au- ence of multiple bacterial species suggests constant thors subdivided the identified bacteria into three colonization rather than a biliary infection. groups: Propionibacteria-related, Clostridia-re- According to the evidence described above, lated and Enterobacteria-related, accounting for pure cholesterol gallstones did not appear to host 45%, 35% and 25% of the total isolated strains, bacteria. In fact, only 1 out of 7 pure cholesterol respectively16. gallstones (14%) was reported to contain bacterial In a similar study using nested primers PCR, sequences in the study by Lee et al129, while none bacterial DNA was obtained in the gallstones (0/3, 0%) in Swidsinski et al16. of 26 out of 30 patients (86.7%). Propionibacte- In 2002, Kawai et al135 found bacterial DNA in ria-related (26.7%) and E. coli-related (23.3%) 12 out of of 21 (57%) pure cholesterol gallstones were the most prevalent bacterial DNA sequences (100% cholesterol content). Surprisingly, all isolated, while DNA of Streptococcus pyogenes the bacteria identified Staphylococcus ( aureus, was identified at a lower percentage (6.7%). How- Streptococcus salivarius, Streptococcus angino- ever, multiple heterogeneous sequences were sus, Streptococcus gordonii and Enterococcus found in 23.3% of the cases as a result of multi- faecalis) were Gram-positive cocci. Nevertheless, ple infections or repeated colonization by E. coli, this evidence seems robust due to the fact that the Propionibacterium acnes and Streptococcus pyo- analyzed material came from the core of the gall- genes or other unidentifiable microorganisms133. stone and had very high homology with known A shift from the concept of infection to the ac- bacterial 16S rRNA sequences135.

2755 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme

BILIARY MICROBIOTA

Enterobacteriaceae, Ruminococcaceae, Clostridiales, Alistipes, Bacteroidales, Gallstones Anoxybacillus, Clostridium (C.), Thermus, Catabacteriaceae, Propionibacterium, (genera) Enterococcus, Acinetobacter, Staphylococcus, Caulobacter, Pseudomonas, Massilia, Brevibacillus, Lactococcus, Paludibacter, Weissella Primary Biliary Staphylococcus, Enterococcus, Streptococcus, Lactohacillus, , Cholangitis (PBC) Propionibacterium, Corynebacterium, Agrobacterium, Flavobacterium, Clostridium, (genera) Micrococcus Primary Sclerosing Streptococcus, Prevotella, Fusobacterium, Veillonella, Haemophylus, Neisseria, Cholangitis (PSC) Alloprevotella, Leptotrichia, Porphyromonas, Cronobacter (genera) Cancer Prevotella, Actinomyces, Streptococcus, Fusobacterium Novosphingobium, Helycobacter. (genera)

GUT MICROBIOTA Bacteroides, Lachnospiraceae, Faecalibacterium, Clostridium (L.), Lachnospira, Gallstones Roseburia, , Phascolarctobacterium, Blautia, Clostridium (C.), (genera) Epulopiscium Primary Biliary Cholangitis Pseudomonas, , Streptococcus, Oscillospira, Sutterella, Bacteroides, (PBC) (genera) Veillonella Primary Sclerosing Bacteroides, Faecalibacterium, Roseburia, Blautia, Coprococcus, Runinococcus, Cholangitis (PSC) Bifidobacterium, Prevotella, Dorea, Alistipes, Anaerostipes, Streptococcus, Collinsella (genera)

Cancer (families) Moraxellaceae, Burkhoideriacae, Comamonadaceae, Bradyrhizobiaceae

Figure 1. Gut and biliary microbiota in biliary diseases. Biliary microbiota: gallstones (Wu et al19, 2013), PBC (Hiramatsu et al20, 2000), PSC (Pereira et al21, 2017), cancer (Avilés-Jiménez et al22, 2016), Gut microbiota: gallstones (Wu et al19, 2013), PBC (Tang et al172), PSC (Sabino et al181, 2016), cancer (Chng et al238, 2016).

2756 Table I. Studies on biliary microbiota using 16S rRNA gene sequencing. Biological References Country Model Sampling Method Evidence Specimen

HEALTHY

Gallbladder was removed from the sacrificed The gallbladder ecosystem of healthy pigs is mainly populated by bacteria broad- Bile, mucus sows. Bile was extracted using a sterile syringe. ly balanced among Firmicutes (34%), Actinobacteria (32%) and Proteobacteria and biopsies Jimenez et al104 Spain Pig Once the gallbladder was completely emptied, (32%) phyla. Bacteroidetes accounted for a lesser part (2% of the isolates). At the of the superficial mucus layer coating was collect- genus level, Staphylococcus, Streptococcus, Kocuria, Rothia, Acinetobacter and gallbladder ed and three biopsies were cut. Psychrobacter were isolated from different samples.

Prevalence of Actinobacteria, Firmicutes and Bacteroidetes in both the bile sam- Bile and Sterile sampling during liver transplants from ples and gallbladder tissues of subjects without gallstones. A significant increase in Molinero et al106 Spain Human gallbladder liver donors who had suffered a brain accident the abundance of the Propionibacteriaceae family and Sphingomonas genus was tissue or stroke. also reported compared with individuals with gallstones.

Cholelithiasis Bacterial DNA was found in gallstones with cholesterol content 70%-90%, in those Swidsinski et al16 Germany Human Gallstones Surgery with cholesterol content >90% no. Three bacterial groups were identified:Propion - ibacteria (45%), Clostridia (35%) and Enterobacteria (25%). Bacterial DNA was obtained in the 86.7% gallstones. Propionibacteria-related (26.7%) and E. coli-related (23.3%) were the most frequent DNA sequences iso- lated; Streptococcus pyogenes DNA was 6.7%, multiple heterogeneous sequences Wu XT et al133 China Human Gallstones Surgery were found in 23.3% of the cases as a result of multiple infections/colonizations by E. coli, Propionibacterum acnes and Streptococcus pyogenes or other unidentifi- able microorganisms. 78% of negative culture cholesterol gallstones had low bacterial concentrations and only few negative culture stones (12%) had concentrations comparable to posi- tive culture ones. The genomic analysis of the gallstone with positive bile culture Swidsinski et al134 Germany Human Gallstones Surgery showed a predominance of the bacterial strains identified by the culture, suggesting an ongoing infection. Most of them belong to bacterial strains, such as Bacillus, Alcaligenes, Carnobacterium and Burkholderia. Bacterial DNA sequences are usually present in mixed cholesterol (to 95% choles- terol content), brown pigment, and common bile duct, but rarely in pure cholesterol During and endoscopic retro- Lee et al129 USA Human Gallstones gallstones. The presence of a gene encoding β-glucoronidase was found in most grade colangio-pancreatography (ERCP) mixed cholesterol gallstones and bacterial sequences of E. coli and Pseudomonas were identified in all the pigment and mixed cholesterol gallstones. During cholecystectomy, one stone was re- Gut microbiota dysbiosis was observed among gallstone patients compared to moved aseptically from the gallbladder and a Gallstones, healthy subjects. Within the gut of patients, there exists an overgrowth of Proteo- Wu T et al19 China Human bile sample was extracted using a sterile needle bile, bacteria, TM7, Tenericutes, Actinobacteria, Thermi, and Cyanobacteria and a de- tubing. Prior to the operation, feces from all pa- crease in the abundance of Bacteroidetes in the biliary tract. tients were also collected. Table continued

2757 Table I (Continued). Studies on biliary microbiota using 16S rRNA gene sequencing.

Biological References Country Model Sampling Method Evidence Specimen

Cholelithiasis

Opisthorchis felineus infection modified the biliary microbiome. Bile from partici- During cholecystectomy, 5-10 ml of bile was Russian pants with showed greater numbers of Synergistetes, Spirochaetes, Saltykova et al137 Human Bile aspirated from the gallbladder under sterile con- Federation Planctomycetes, TM7 and Verrucomicrobia. Numbers of > 20 phylotypes differed ditions in bile of the O. felineus-infected compared to non-infected participants.

Salivary samples were collected after the pa- All observed biliary bacteria were detectable in the upper digestive tract. The bili- Salivary, tients gargled with 20 mL of sterile saline water. ary microbiota had a comparatively higher similarity with the duodenal microbiota, gastric, Patients expectorated their mouthwash into ster- Ye et al138 China Human vs. those of the other regions, but with a reduced diversity. Enterobacteriaceae duodenal fluid ile sputum cups. The gastric fluid, duodenal flu- genera (Escherichia, Klebsiella, and an unclassified genus) and Pyramidobacter and bile id, and bile samples were collected using strictly were abundant in bile. sterile side-viewing endoscopes. Oral cavity and respiratory tract inhabitants were more prevalent in bile samples Shen et al141 China Human Bile ERCP than intestinal inhabitants. Thus, in addition to gut species, bacteria from the oral cavity/respiratory tract might be relevant to human biliary infection. In cholelithiasic patients dairy product intake was negatively associated with the proportions of Bacteroidaceae and Bacteroides, and several types of fiber, pheno- Gutiérrez-Díaz Spain Human Bile Surgery lic, and fatty acids were linked to the abundance of Bacteroidaceae, Chitinophaga- et al142 ceae, Propionibacteraceae, Bacteroides, and Escherichia-. These results support a link between , biliary microbiota, and cholelithiasis. In the analysed pigmented stones, genes involved in biofilm formation were mainly recovered from clinically pathogenic Klebsiella and Enterococcus while bile re- sistance genes were present also in Escherichia, Shigella, Serratia and Bacillus. Kose et al143 Australia Human Gallstones During cholecystectomy Klebsiella was also present in one of the cholesterol gallstones, while the remaining analysed cholesterol stones showed a predominance of Gram-positive bacteria that were not identified within the pigmented stones. PRIMARY BILIARY CHONAGITIS (PBC) Bile was then taken aseptically from the gall- In 75% of PBC were identified Gram-positive cocci while these cocci were positive Hiramatsu et al20 Japan Human Bile bladders at the time of liver transplantation, just in only 5% in cholecystolithiasis. before explantation. Table continued

2758 Table I (Continued). Studies on biliary microbiota using 16S rRNA gene sequencing. Biological References Country Model Sampling Method Evidence Specimen

PRIMARY SCLEROSING CHOLANGITIS (PSC)

Scandinavia, A significant increase in the abundance of Firmicutes and a parallel decrease of Germany, Proteobacteria was observed along with differences in the abundance of Bacteroi- Folseraas et al202 Human Bile ERCP Central detes, Actinobacteria, and Tenericutes among patients with FUT2 loss-of-function Europe, USA genotypes and non-secretors.

The bacterial communities of non-PSC subjects and early stage PSC patients were similar. Streptococcus abundance was also positively correlated with an increase in Pereira et al21 Finland Human Bile ERCP disease severity. These findings suggest that the aetiology of PSC is not associated with changes in bile microbial communities, but the genus Streptococcus may play a pathogenic role in the progression of the disease. CANCER Microbiota in extrahepatic cholangiocarcinoma showed significant changes in mi- Epithelial cells Avilés-Jiménez crobial composition. Phylum Proteobacteria dominated all samples. Nesterenkonia Mexico Human from the bili- Brushing ERCP et al22 decreased, whereas Methylophilaceae, Fusobacterium, Prevotella, Actinomyces, ary duct Novosphingobium and H. pylori increased in patients with cholangiocarcinoma. Systemic perturbation of the microbiome was noted in tumor samples vs. non-can- cer normal for several bacterial families, with a significant increase in Stenotro- phomonas species in tumors. Comparison of Opisthorchis viverrini associated Singapore, Hepatic tissue, vs. non-associated groups identified enrichment for specific enteric bacteria (Bi- Chng et al238 Thailandia, Human bile, gastric Repository fidobacteriaceae, Enterobacteriaceae and Enterococcaceae). Functional analysis Romania mucosa of cholangiocarcinoma microbiomes revealed higher potential for producing bile acids and ammonia in O. viverrini associated tissues, linking the altered microbiota to carcinogenesis. Microbial community analyses revealed that fluke infection perturbed the gastro- intestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Bile from the gallbladder and colorectal con- Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, Plieskatt et al240 Thailandia Hamsters Feces, bile tents were collected from each hamster sacri- and Eubacteriaceae. Opisthorchiasis has a robust inflammatory phenotype with ficed at 6 weeks after infection byO. viverrini. conspicuously elevated IL-6. The inflammation of the biliary system leads to peri- ductal fibrosis, which is a precursor of cholangiocarcinoma. Table continued

2759 Table I (Continued). Studies on biliary microbiota using 16S rRNA gene sequencing. Biological References Country Model Sampling Method Evidence Specimen

CANCER

There are fundamental differences in the biliary microbiome of patients with periamp- ullary cancer who undergo preoperative biliary drainage (PBD) and those who do not. Scheufele et al93 Munich Human Bile Intraoperative PBD induces a shift of the biliary microbiome towards a more aggressive and resistant spectrum, which requires a differentiated perioperative antibiotic treatment strategy. Salmonella typhi and Helicobacter sp. were not detected in bile from any patients with gallbladder carcinoma (GBC). As the predominant species, Fusobacterium Bolivia, nucleatum, Escherichia coli, and Enetrobacter sp. were detected in bile from GBC Tsuchiya et al234 Human Bile Cholecystectomy Chile patients. Those in bile from patients with cholelithiasis were Escherichia coli, Salmo- nella sp., and Enerococcus gallinarum. Escherichia coli was detected in bile samples from both GBC and cholelithiasis patients. In patients with distal cholangiocarcinoma, the abundance of Gemmatimonadetes, Nitrospirae, Chloroflexi, Latescibacteria, Unclassified_Bacteria, and Planctomyce- Chen et al233 China Human Bile ERCP tes was increased compared with patients with choledocolithiasis. At the genus level, Escherichia/Shigella, Staphylococcus, Klebsiella, Unclassified_Enterobacteriaceae, and Faecalibacterium showed the highest abundance. , CHOLANGITIS AND OTHER BILIARY INFECTIOUS DISEASES

E. coli was the main biliary pathogenic microorganism, among others such as Klebsi- Faecal samples were collected in sterile tubes at ella spp., Clostridium perfringens, , and Enterobactercloacae in the hospitals. Bile samples were obtained during Liu et al244 China , bile the bile of the patients. Additionally, the amount of bile endotoxin significantly cor- percutaneous transhepatic cholangial drainage related with the number of Enterobacteriaceae, especially E. coli. Enterobacteriace- or gallbladder drainage. ae might play essential role in the pathogenesis and/or progress of acute cholecystitis.

Bile of patients with laparoscopic cholecystectomy may contain microorganisms, partic- ularly elderly patients, those with symptoms, and those who undergo preoperative ERCP. Yun et al245 Korea Human Bile Cholecystectomy Escherichia coli and Klebsiella were common in gram-negative bacteria. Enterococcus was the most common in gram-positive bacteria. Less than 5% resistance was observed against carbapenem, beta-lactam antibiotics, glycopeptide antibiotics, and linezolid. A bile duct microenvironment with more severe bacterial infection and stronger litho- Bile samples were extracted from the su- genicity was found in patients with sphincter of Oddi laxity (SOL). Proteobacteria praduo-denal segment of the common bile duct and Firmicutes were the most widespread phylotypes, especially Enterobacteriaceae. Liang et al246 China Human Bile with a 5-mL germ-free injector before any in- Patients with SOL possessed more varied microbiota. In the SOL group, pathobionts, vasive manipulation on the bile duct occurred. such as Bilophila and Shewanella algae had richer communities, and harmless bac- teria were reduced. For necropsy, hamsters were anesthetized with The identities of bacteria cultured for enrichment suggested that chronic O. viverri- Hamsters Itthitaetrakool ether. Liver tissue at the hilar region and ni infection changes the liver microbiome and promotes Helicobacter spp. growth. Thailand and Liver tissue et al241 containing a large bile duct was immediately There may be synergy between O. viverrini and the liver microbiome in enhancing worms collected. immune response-mediated hepatobiliary diseases. 2760 Table continued Table I (Continued). Studies on biliary microbiota using 16S rRNA gene sequencing.

Biological References Country Model Sampling Method Evidence Specimen

BILIARY STENTING The most common bacteria identified were Pseudomonas, Citrobacter, Klebsiella, Staphylococcus, Serratia, Escherichia coli, Streptococcus, Enterococcus, Aero- monas, Proteus and Enterobacter. The protein concentration of the biofilms was Vaishnavi et al247 India Human Stents Stents were retrieved endoscopically found to be significantly higher in stents placed in patients with cholangitis than those without cholangitis and those with smaller diameter stents. Longer indwelling dura- tion had more biofilm formation. LIVER TRASPLANTATION Bile of liver transplant recipients is frequently colonized with microorganisms. Of isolated bile samples, 64.2%were Gram-positive, 22.2% were Gram-negative, and Bile was collected via percutaneous biliary Bile and 13.6% revealed albicans. Most detectable Gram-positive bacteria were En- Kabar et al248 Germany Human drainage and during ERCP, after liver transplan- feces terococcus faecium. Most detectable Gram-negative bacteria were E. coli and Kleb- tation siella . There was high correlation between microorganisms found in bile and those isolated from stool. Firmicutes and Proteobacteria were the predominant phyla. Enterococcus, Rhizobi- Liu et al249 China Human BIle Collection from T-tube after sterilization um, Nevskia, Lactococcus, Bacillus were the most common genera.

2761 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme

However, genomic techniques confirm only the Notably, in some of these studies, the Shannon presence of microorganisms within the gallstone diversity index and richness of bacterial commu- and not their vitality. The evidence that viable nities were significantly higher in the gallstone bacteria are present inside the gallstone core un- and some bacteria identified in the gallstones derlines the relevance of bacterial metabolism in were not found in the bile139. This evidence sug- the development of gallstones136. gests that the gallstone may represent a protective In 2013, the core biliary microbiota in patients environment within which the microorganism with cholesterol gallstones was described. Indeed, can create a separate niche that is resistant to the Wu et al19, through 16S rDNA pyrosequencing, antimicrobial effect of bile and has favorable con- identified 106 bacterial species belonging to 6 phy- ditions for its growth. la both in the gallstones as well as in bile. Impor- The use of advanced PCR techniques, such tantly, a higher microbial diversity was observed in as PCR-denaturing gradient gel electrophoresis the biliary tract compared to the gut microbiota of (DGGE) and whole-metagenome shotgun (WMS) the same patients. At the phylum level, increased sequencing, has further increased taxonomic reso- levels of Proteobacteria, TM7, Tenericutes, Ac- lution, facilitating the identification of new biliary tinobacteria, Thermi, and Cyanobacteria and a bacterial genera in the stones (Brucella, Citro- decrease in the abundance of Bacteroidetes were bacter, Shinella, Aurantimonas, Lachnospiraceae reported in the biliary tract. The dominant phyla and Lactobacillus) as well as in the bile of patients of the biliary microbiota in patients with gallstones with cholelithiasis (Bacillus, Enterobacter and have been later confirmed by other studies137,138. Acinetobacter)139,141. Furthermore, metagenomic As expected, some of these phyla possess a higher techniques have improved our understanding of resistance to extreme environmental conditions, the complex interactions between the environment, such as those present in the biliary tract. Notably, individual habits and microbiota. The interplay in- the phylum Proteobacteria includes genera such as fluences the host metabolism, which in turn influ- Escherichia, Salmonella, Vibrio, and Helicobacter, ences the development of gallstones141-143. all of which have been associated with several These studies have demonstrated an unexpect- gastrointestinal diseases7. At the taxon level, a sig- edly rich bacterial community in a hostile envi- nificant increase was observed in the abundance ronment. This evidence collectively suggests that of Enterobacteriaceae, Ruminococcaceae, Clos- bile colonization is common and may play a piv- tridiales, Bacteroidales, Acinetobacter, Staphy- otal role in the formation of gallstones. lococcus, Caulobacter, Pseudomonas, Massilia, Brevibacillus and Lactococcus in the biliary tract. The Biliary Microbiota and Autoimmune Several previously undescribed bacterial species as Cholangiopathies Primary Biliary well as a high interpersonal variation were reported Cholangitis (PBC) in this study, suggesting a correlation with dietary, PBC is a chronic autoimmune disease affecting environmental and genetic factors. Furthermore, the small bile ducts. Currently, the most widely over 85% of the bacterial operational taxonomic accepted hypothesis proposes that, in genetically units (OTUs) were observed in the bile as well as predisposed individuals, an exaggerated immune in gallstones. The biliary tract shared about 70% response is produced against self-antigens ex- of the OTUs of the patients’ gut microbiota, while pressed in the biliary tract. It has been proposed this percentage dropped to 40% when the gut mi- that molecular mimicry between host antigens crobiota from healthy individuals was compared and microbes may act as a possible trigger144. An- with the biliary microbiota of the patients19. In a timitochondrial antibodies, serological markers study comparing the biliary microbiota of patients of disease observed in about 95% of patients with having gallstones with salivary, gastric and duo- PBC, target the pyruvate dehydrogenase com- denal microbiota, all the bacterial genera found in plex E2 (PDC-E2) and other proteins that share the bile tract were observed in at least one other lipoic acid residues145. This enzymatic complex analyzed gastrointestinal site138. Similarly, Peng et expressed in the mitochondria of biliary epi- al139 reported the presence of common intestinal thelial cells shows cross-reactivity with several colonizers in the bile of patients with cholelithiasis. bacterial proteins, such as pyruvate dehydroge- These findings support the hypothesis that the bil- nase complex146, ATP-dependent Clp protease147, iary microbiota originates from the gut, either by dihydrolipoamide acetyltransferase (E2p)148 and direct passage across the SO or by bacterial trans- other proteins of E. coli149-151, lipoyl domains of location81,99,140. Novosphingobium aromaticivorans152,153, heat

2762 Biliary tract microbiota: a new kid on the block of liver diseases? shock proteins of Mycobacterium gordonae154,155, position of bile in PBC or its consequences. Nev- pyruvate dehydrogenase complex of Mycoplasma ertheless, quantitative and/or qualitative modifica- pneumoniae156 and β-galactosidase of Lactoba- tions of bile have been observed in PBC, resulting cillus delbrueckii157. Hence, an immune reaction in an increase in the concentration of CA174-176. against one or more of these bacteria, combined These alterations of bile exert a profound impact on with a loss of immunotolerance to pyruvate dehy- the composition of the gut microbiota: CA, in fact, drogenase complex E2, could lead to the develop- possesses the lower anti-microbial activity among ment of PBC145,146. the BAs69. Moreover, immune dysregulation could Furthermore, PBC seems to occur more fre- imbalance the bacterial regulation by the secretion quently in patients with urinary tract infec- of anti-microbial peptides and immunoglobulins. tions158-162, particularly by E. coli163 or other in- Hence, gut dysbiosis may simply be a consequence fections by Mycobacteria164, Chlamydia165-167 and of the chemical composition and the impaired an- Helicobacter pylori168. Elevated antibodies titers ti-microbial activity of bile. against Enterobacteriaceae169 Toxoplasma gondii Interactions between the host and bacteria, that and Helicobacter pylori170 have also been reported. result in the activation of the immune system to- Over the past few years, advancement in the wards biliary epithelial cells, could directly take 16S RNA sequencing-based knowledge on the place in the biliary tract. Hence, the biliary mi- influence of the gut microbiota in human pathol- crobiota could may play a pivotal role in disease ogies has led to the study of its involvement in development. autoimmune cholangiopathies (Figure 1). Indeed, bacterial compounds from Streptococ- In a study by Lv et al171, the gut microbiota of pa- cus intermedius and Propionibacterium acnes tients with early stage PBC showed a higher abun- have been identified in the liver tissue of patients dance of potentially opportunistic pathogens, such with PBC177,178. Similarly, bacterial proteins have as the families Enterobacteriaceae, been found in the sera of the affected patients179. and Enterococcaceae and the genera Streptococ- So far, Hiramatsu et al20 investigated the bili- cus, Veillonella and Haemophilus parainfluenzae ary microbiota through 16S rRNA profiling. Bile compared to healthy controls. Simultaneously, a samples were collected from the gallbladder of 19 decreased abundance of health-promoting bacte- patients with PBC during liver transplantation. ria, such as Lachnospiraceae and some beneficial Bacterial sequences were found in 10 out of 15 Bacteroidetes was observed171. PBC patients. Staphylococcus aureus was the Tang et al172 reported a decrease in the richness most frequently detected microorganism (5/15 of the gut microbiota in PBC patients compared PBC patients, 33%; 40% of all PBC clones). En- to healthy controls. Similar to Lv et al171, the terococcus faecium, Lactobacillus plantarum, abundance of the genera Haemophilus, Veillonel- , Streptococcus pneumoniae la, Clostridium, Lactobacillus, Streptococcus, and other Streptococci were the other commonly Pseudomonas, Klebsiella and Enterobacteriace- found bacteria (Figure 1, Table I). Importantly, ae was significantly increased in patients with this study was limited by the analysis of only 10 PBC. Most of the bacteria included in these gen- clones that were selected from the total number of era are responsible for infectious diseases, such the amplified PCR products. Hence, the identified as urinary tract infections, which are associated bacteria should be considered as “major clones” with the development of PBC. According to these rather than the complete biliary microbiota20. findings, a microbiome signature, composed of 12 Further studies using next-generation metage- genera associated with the disease was described. nomic techniques should be carried out in order to Conversely, the abundance of Faecalibacteri- better understand the biliary microbiota in PBC and um, Bacteroides, Sutterella and Oscillospira its influence in the different phases of the disease. was decreased in PBC172. Among these bacteria, Faecalibacterium prausnitzii exerts a significant Primary Sclerosing Cholangitis (PSC) beneficial effect on the homeostasis of the gut mu- PSC is a chronic cholestatic autoimmune dis- cosa173. Interestingly, the alterations in some of the ease that affects the bile ducts causing biliary PBC-enriched genera as well as the PBC-depleted inflammation and fibrosis. Hereditary alterations ones were partially reversed after six months of of the genes that regulate immune response, par- therapy using ursodeoxycholic acid172. ticularly HLA class and IL-2 receptor genes, have It is presently under debate as to whether these been shown to confer higher susceptibility to the alterations are causes of the alteration in the com- development of the disease, following which the

2763 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme environmental factors may represent the final biliary tree that occurs during ERCP was later trigger. Considering the strong association with been confirmed by the same group201. IBD, it has been proposed that a primary intestinal Pereira et al21 studied the biliary microbiota of dysbiosis causing inflammation and consequent patients with PSC at different disease stages us- exposure of cholangiocytes to cytokines and mi- ing 16S rRNA profiling. Notably, they did not find crobial products could initiate the pathogenesis180. significant differences in the biliary microbiota of Therefore, several studies have recently ana- early stage PSC patients compared to controls. At lyzed the gut microbiome of PSC patients (Figure advanced disease stages, the abundance of Strep- 1). An increase in the abundance of potentially tococcus genus was significantly elevated. Lower harmful bacterial genera, including Veillonella, microbial diversity and a further increase in the Enterococcus and Escherichia, has been ob- abundance of Streptococcus spp. characterized served. Likewise, the bacterial genera Fusobacte- the biliary microbiota of the patients who devel- rium, Lactobacillus, Blautia, Barnesiella, Lach- oped dysplasia or cancer. nospiraceae and Megasphaera were reported to As observed from culture-based studies95,201, be associated with PSC compared to IBD patients the limitations of sampling during ERCP and se- and healthy controls181-185. A parallel decrease lection of patients with a history of ERCP could was reported in the abundance of some anaero- have affected the results. bic taxons, such as Clostridiales II, Bacteroides, Along with a genome-wide association study, Prevotella and Roseburia186. In particular, Rose- Folseraas et al202 studied the genotype-dependent buria exerts well-recognized beneficial effects changes in the biliary microbiota composition in 39 on the maintenance of intestinal homeostasis187. patients with PSC, considering the presence, het- It is known to produce butyrate, which exerts a erozygosity or absence of allele “G” of FUT2. This trophic effect toward enterocytes, thus maintain- gene has an effect on the expression of fucosylated ing the integrity of the gut barrier188. It has been glycan expression in the bile duct epithelium and demonstrated in germ-free murine models that was found to be associated with PSC. Interestingly, the protective effects of some bacterial strains a significant increase in the abundance of Firmic- could play an even more important role than the utes and a parallel decrease of Proteobacteria was detrimental effects of pathogenic species189. observed along with differences in the abundance According to these observations, several an- of Bacteroidetes, Actinobacteria, and Tenericutes tibiotics, including tetracycline190,191, vancomy- among patients with FUT2 loss-of-function geno- cin192-194, azithromycin195, metronidazole194,196, mi- types and non-secretors. nocycline197, rifaximin198, probiotics199 as well as These findings have laid the foundations for fecal microbiota transplantation have been tested further studies. Hopefully, a multiple “omics” in patients with PSC200. approach and an improved understanding of the In a culture-based study on a group of 36 PSC interaction between the host and the microbiome patients undergoing liver transplantation, the bile will unravel the complexity of the pathogenesis of or bile walls of 20 patients were culture-posi- autoimmune cholangiopathies. tive. α-haemolytic Streptococcus was the most frequently identified bacterial species (16/20 pa- Influence of Biliary Bacteria on the tients), while Enterococcus and Staphylococcus Development of Biliary Tract Cancer were isolated from five cultures. The authors Recent evidence has begun to clarify the com- attributed these results to possible bile contami- plex influence of the human microbiota on the nation and consequent colonization during previ- development and progression of cancer. Indeed, ously performed ERCP. Moreover, most of the pa- bacteria promote carcinogenesis by altering the tients who had not received antibiotic prophylaxis metabolism, proliferation and death of cells by before ERCP showed a higher number of isolates. dysregulating the immune response or by actively In addition, a positive correlation was observed inducing DNA damage via toxins97,203,204. Bacte- between the number of identified bacteria and the ria possess carcinogenetic potential as they can length of the period elapsed after the last ERCP. enhance the release of the mediators of inflam- About 50% of the patients had a history of bil- mation, such as TNF-α and IL-1. Moreover, they iary infection during the previous six months; are able to trigger the activation of NFκB, either thus they had either received or were undergoing directly or indirectly via proinflammatory cyto- antibiotic therapy at the time of liver transplanta- kines205. NF-κB activation further exacerbates the tion95. The relevance of the contamination of the inflammatory response and upregulates genes in-

2764 Biliary tract microbiota: a new kid on the block of liver diseases? volved in cell cycle control (cyclin D1, CDK2 ki- These findings suggest a significantly higher nase, c-myc) and apoptosis (p21, p53 and pRb)206. risk of developing cancer in patients with chronic Several bacterial toxins have possible roles in bile colonization, particularly for chronic typhoid the development and progression of cancer97. The carriers. study of the expression of specific bacterial toxins The genus Helicobacter has also been associ- in the bile could further clarify the importance of ated with biliary tract cancers87,227,228. However, in this mechanism. several studies using PCR primers, a large vari- Since gallstones represent the strongest risk ability in the detection rate in bile ranging from factor for developing biliary tract cancer207 and 0 to 82.8% has been found. Although the choice are associated with mortality208, other bacteria of primers may have influenced the results, an in- implicated in the formation of gallstones could creasing prevalence gradient has been observed also play a role in carcinogenesis. from Western to Eastern countries87. The most The term “biliary tract cancers” refers to ma- frequently identified species are Helicobacter lignant tumors of the bile duct, such as extrahe- bilis229 and H. hepaticus230. Details about the pos- patic cholangiocarcinoma, gallbladder and am- sible pathogenesis are still unknown. However, pulla of Vater. In Western countries, the overall Helicobacter is able to colonize the bile, interact incidence of these tumors is modest and ranges with BAs and cause inflammation and neoangio- between 0.5 and 5 per 100000 annually, mak- genesis231,232, mechanisms that are potentially in- ing them the sixth most common cancers of the volved in carcinogenesis. gastrointestinal system. Owing to dietary, envi- Avilés-Jiménez et al22 analyzed compared the ronmental and microbiological factors, their inci- biliary microbiota of 100 patients with extrahe- dence in Eastern countries is significantly higher patic cholangiocarcinoma to 100 patients with (up to 100/100000). Generally, they are associated benign biliary tumors, using 16S RNA sequenc- with low survival rates and poor prognosis, since ing (Figure 1, Table I). At the phylum level, a they are quite often diagnosed at late stages209. dominance of Proteobacteria (60.4% on average) In the culture-based microbial studies, patients was observed in all the samples. Methylophilace- with gallbladder carcinoma had a significantly ae, Fusobacterium, Prevotella, Helicobacter and higher frequency of positive bile cultures (65-81%) were the most frequently identi- compared to the patients with cholelithiasis and fied genera in patients with cholangiocarcinoma. controls210,211. In another study, bacterial growth The authors detected Helicobacter pylori-associ- was observed in the bile of 22 out of 118 patients ated virulence genes, such as CagA and VacA, in (18.6%) with periampullary cancer undergoing most samples from both groups, indicating a pos- surgery. In patients who underwent preoperato- sible carcinogenic role in the biliary tract. With the ry ERCP, the percentage of culture-positive bile exclusion of four OTUs that were considered as samples rose to 97%, underlining the significant potential contaminations, 21 OTUs showed a con- impact of sphincterotomy and biliary stenting on siderable modification in the cholangiocarcinoma bile colonization93. group:. In particular, 12 increased (Novosphingo- Several studies212-217 have reported an associ- bium, Prevotella, Streptococcus, Dialister, Fuso- ation between typhoid carriage and the develop- bacterium, two Actinomyces, two genera belong- ment of hepatobiliary cancer. Caygill et al218 re- ing to Methylophilaceae, one to Sinobacteriaceae ported that typhoid carriers possessed a lifetime and one to Neisseriaceae families, one to class risk of 6% of developing . In ), while 9 (Rothia, two Nest- several studies211,219-222 the relative risk of devel- erenkonia, three Mesorhizobium, one unclassified oping biliary tract cancer ranged from 2.1 in low genus belonging to Micrococcaceae and one to prevalence infection areas to 22.8 in endemic ar- Phyllobacteriaceae families, one to Rhizobiales eas. Both direct DNA damage via toxins, such as order) decreased in abundance. Importantly, the cytolethal distending toxin223,224, and an indirect analysis revealed distinct clusters between chol- detrimental modification of the bile composition angiocarcinoma and controls22. via bacterial enzymes225,226 have been suggested In another recent investigation, patients with to be potential carcinogenic mechanisms. distal cholangiocarcinoma had a prevalence of Interestingly, Nath et al222 demonstrated us- Gemmatimonadetes, Nitrospirae, Chloroflexi, ing nested PCR that specific Salmonella typhi Latescibacteria, Unclassified_Bacteria, and Planc- sequences were found in the bile of 35 out of 52 tomycetes compared with patients with choledoco- patients (67%) with gallbladder carcinoma. lithiasis. At the genus level, Escherichia/Shigella,

2765 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme

Staphylococcus, Klebsiella, unclassified_Entero- understanding of the relationship between the gut bacteriaceae, and Faecalibacterium showed the and the biliary microbiota could be derived by highest abundance233. studying the modifications of the biliary microbi- In a study using Next Generation Sequencing ota in patients on treatment with probiotics. (NGS)-PCR, Fusobacterium nucleatum, E. coli Furthermore, alterations of the bile composi- and Enterobacter sp. were the predominant bac- tion have been associated with the development of teria in the bile of patients with gallbladder carci- several other gastrointestinal diseases243. Hence, noma234. Interestingly, these bacterial strains have biliary dysbiosis could represent a primary patho- been linked to the development of colon cancer235 genic step in the development and progression of and thus could possess an intrinsic carcinogenic these pathological conditions. A detailed compre- potential irrespective of the site colonized by them. hension of the impact of the biliary microbiota on Furthermore, considering how important the bile composition may facilitate the development microenvironment is in tumorigenesis and how the of strategies for modulating the microbiota in or- microbiota is involved in shaping it236,237, Chng et der to prevent the occurrence of such diseases. al238 for the first time described the tissue micro- Therefore, the modulation of the biliary microbi- biome of Opisthorchis viverrini associated chol- al community should be considered for the preven- angiocarcinoma. Indeed, patients affected by liver tion of biliary and other gastrointestinal diseases. fluke are well-recognized models of biliary tract Finally, the biliary tree is intimately connect- carcinogenesis239 and the parasite is able to modify ed with the pancreas and liver. Hence, the study the microbiome of infested individuals137,240-242. An of the biliary microbiota could reveal a profound increase in the abundance of Bifidobacteriaceae influence of the biliary microbiota on the patho- and Enterobacteriaceae abundance was observed genesis of illnesses of these organs. in the tissue microbiome of the Opisthorchis In summary, recent evidence has paved the group, while an interesting prevalence of Stenotro- way for a better understanding of a crucial site phomonas was found in non-affected patients238. in the development of gastrointestinal diseases. Future studies are needed to explore the influence of the biliary microbiota in human pathology. Conclusions This knowledge could lead to the formulation of strategies for modulating the biliary microbiota in An unexpectedly rich bacterial community has order to treat and prevent several gastrointestinal recently been discovered in an environment that diseases. was previously considered to be hostile to bacte- rial growth. However, the stages and factors that favor the colonization of the biliary tract are in- Conflict of Interests completely understood. The Authors declare that they have no conflict of interests. As for the methodology, the standardization of the sampling methods should be considered. Several techniques have been used to perform bile sampling, but some of them have witnessed Author contributions a possible risk of contamination. Separate assess- AN and EN reviewed the literature, prepared the initial ments of the performance of each technique and manuscript and produced tables and illustrations. FRP and GI revised the manuscript critically for important intellec- sampling standards are currently lacking. tual content. AG and LZDV conceived the topic and revised Since studies on healthy human biliary micro- the manuscript critically for important intellectual content. biota are not feasible for ethical reasons, compar- All authors approved the final version. ative studies on the biliary microbiota of patients with different biliary illnesses could identify a microbial fingerprint of each disease. References Moreover, an understanding of the modifi- cations of the biliary microbiota after treatment 1) Costello EK, Lauber CL, Hamady M, Fierer N, Gor- with the available therapies could provide new don JI, Knight R. Bacterial community variation insights on the impact of bacterial communities in human body habitats across space and time. in the pathogenic mechanisms of biliary diseases. Science 2009; 326: 1694-1697. In particular, probiotic therapy modifies the 2) Zhou Y, Gao H, Mihindukulasuriya KA, La Rosa PS, composition of the gut microbiota. An improved Wylie KM, Vishnivetskaya T, Podar M, Warner B, Tarr

2766 Biliary tract microbiota: a new kid on the block of liver diseases?

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