Bacterialflorainboth Gut And The Biliary Tract Associated With Cholelithiasis

Objective:The aims of this study are:1) to reveal the variation of bacterial community and composition in both gut and biliary tract (gallstones and bile) in patients with gallstones;2) clarify whether there exist gut bacterial dysbiosis in patients with gallstones;3) to identify the biliary tract bacteria associated with the formation of cholesterol gallstones, as well as to elucidate the relationship between the biliary tract bacteria and cholesterol gallstones formation out of four potential bacterial factors contributing to cholesterol gallstones. The study will provide foundationalfindings for further studying bacteriological mechanism of the cholesterol stone formation, which will be helpful for both preventions and therapeutics for cholelithiasis.Methods:We collected samples of stone, bile and feces from29patients with gallbladder stones, and also fecal samplesfrom38normalindividuals, and finally obtained a total of120samples. The content of cholesterol in gallstones was analyzed for the classification of gallstone disease. The bacterial16S rRNA gene was amplified by PCR methods using bacterial DNAs extracted from all120samples and was used to perform454-barcoded pyrosequencing. Bacterial16S rDNA sequences were filtered and used to identify bacterial OTUs (equal to bacterial species) according to97%sequence identity. Final OTUs were used to clarify thecommunity structure and composition of bacteria in both gut and biliary tractwith cholelithiasis. Based on these results, a series of analyses were further performed, including heat-map based abundance, network analysis, and functional annotation depending on similar species’ genomes.Results:1. Cholesterol stone proportion is92.55%of all sampled stones, which represents the main type of gallstone disease.2. Bacterial DNAs were detected from all samples.3.We obtained a dataset consisting of299,217high-quality and classifiable16S rRNA gene sequences. From the dataset, we identified a total of4637operational taxonomic units (OTUs). These identified bacterial OTUs can be classified into20bacterial phyla.4. Comparison of gutbacteria community (normal individuals and patients with gallbladder stone) was conducted. Within the gut, the relative abundance of the bacterial phylum Proteobacteria among patientswere significantly (P<0.001) higher than that in normal. The relative abundances of three bacterial genera (Faecalibacterium, Lachnospira,andRoseburia) in normal subjects were significantly (P<0.001) lower in those in patients.5. Gut bacteria were compared with biliary bacteria in the gallstone patients.(1) This study found that Bacteroidetes phylum in biliary tract was significantly lower than that in the gut (P<0.001).The abundances of six bacterial phyla in the biliary tract, includingProteobacteria, TM7, Tenericutes, Actinobacteria, Thermi,and Cyanobacteriawere significantly higher than those in the gut (P<0.001).(2) This study also found that, in the biliarytract, there was a significant (P<0.001) decreasing of two bacterial genera and increasing of16otherbacterial genera (P<0.001). Bacteriadiversity in the biliary tract was significantly higher than that in the gut (P<0.001).6. This study showed that over85%of bacterial OTUs were shared by both bile and gallstones, but only60%ofgut bacterial OTUs were shared by patients and healthysubjects. Around70%of gut bacterial OTUs from patients and40%OTUs from healthy subjects were found in the biliary tract, respectively.7. Based on the dataset described above, this study identified biliary tract core microbiome out of gallstone patients, which includes106bacterial OTUs belonging to six bacterial phyla. Only33.96%(36/106) of the members of the biliary tract core microbiomecould be matched to known bacterial species. About15of the36-matched bacterial species havepublicly available genomes. Based on reference genomesof those15bacterial species, this studyfurther analyzed the presence or absence of four genes potentially associated withthe formation of gallstones, includingMDR, BSH, bG,andphL.The results found each of15speciesat least contained one of the four genes.Conclusions:1. This is the first study to use a454-basedpyrosequencing of bacterial16S rDNAs to clarify the structure and composition of bacteria in both gut and biliary tract with cholelithiasis.2. To the best of our knowledge, this is the first study todiscover a potential association of the gut microbiotadysbiosis that is present among gallstone patients.Our results discovered significant changes of gut microbial components between gallstone patients andhealthy subjects. This raises a possibility that biliary tractmicrobiota could be partially originated from the gut. Furtherevidence is obviously needed to explore this possibility,but nonetheless it is an intriguing prospect. 3. This study supports mounting evidence that cultureindependent454-based16S rDNA sequencing is necessaryas a diagnostic tool in gallstone-associated bacterial infection, because it provides more detailed information thancurrently employed and sequence analysis of clonedmicrobial16S rDNA.4. This study reveals the core of biliary tract bacteria and identified four types of bacteria associated with stone formation factors (MDR efflux pump protein, BSH, bG and PhL). The biliary core bacteria are closely related to the formation of gallstones.5. These findings indicatedthat bacterial community assembly might be more important than single species in the formation of cholesterol gallstones.6. This study makes a comprehensive discussion of the relationship between the bacteria community assembly and cholesterol stone formation. These findings have numerous medical implications for both prevention and therapeutics for cholelithiasis or other relative GIT healthy risks, warranting further follow-up studies that are needed to verify these findings and move forward. The most poignant implication of this study is that prevention and management of bacterial infections in the biliary tract may be a target for lowering the risks of cholesterol gallstones. The study also provides some clues for further exploringthe mechanism of bacteriology leading to the cholesterol stone formation.