| Emerging evidences reveal that the human microbiome has close relationship with human health and disease. The microorganisms resident in the human body interact with the host through their metabolites and immunomodulatory effects, thus having profound impacts on energy intake, metabolism and immune system development. Human diseases like obesity, type 2 diabetes mellitus and inflammatory bowel disease have been found to be related to the human microbiota. In recent years, with the rapid development of next-generation sequencing technology, metagenomic sequencing technology has become a critical approach in investigations of microbial communities. This technology could avoid the shortcomings of conventional microorganism identification methods including microorganism cultivation method, polymerase chain reaction(PCR) amplification method and phylogenetic microarrays. Compared to conventional microorganism identification methods, metagenomic sequencing technology usually could handle multiple samples in parallel and has a higher sequencing throughput and resolution. Unlike above approaches, metagenomic sequencing technology can provide a comprehensive picture of microbial communities. Metagenomic sequencing technology has characterized the microbial diversities of human body sites containing the human gastrointestinal tract, oral cavity, respiratory tract, skin, and vagina, which has laid solid foundations for further characterization of associations between the human microbiome and human health and disease. However, due to issues about sample collection and data analysis, there are few investigations on the microbiota of specific human diseases, leading to a unclear understanding of the composition and function of the microbiome and the roles of the microbiome in disease pathogenesis.By using metagenomic sequencing technology, the first two parts of this study mainly focused on the poorly explored biliary microbiota of patients with common bile duct stones(choledocholithiasis), including the composition of the microbial community, the identification of novel biliary bacteria and microbial genes, and the relationship between the biliary microbiota and the microbiota of the upper digestive tract. In the third part, as the complement of the human microbiome, the gastrointestinal microbiome of mice with Alzheimer?s disease(AD) would be investigated.Current studies on the biliary microbiota of patients with choledocholithiasis are mainly based on microorganism cultivation method and PCR amplification method. There is so far no systematic investigation of the biliary microbiota of choledocholithiasis patients. We performed whole-metagenome shotgun(WMS) sequencing and parallel 16 S ribosomal RNA(rRNA) amplicon sequencing(referred as “16S sequencing”) of bile samples from 15 Chinese choledocholithiasis patients. This study is the first biliary WMS survey of common bile duct bile samples. Each sample obtained an average of 7.29 Gbp WMS reads. After quality control and alignment analysis, we found that the ratios of the host reads relative to the raw reads were all above 98%, and the numbers of reads aligned to bacterial reference genomes ranged from 593 to 1,242,270. Based on composition analysis and clustering analysis, we found that the microbial communities of most individuals(13/15) were clustered into two types, according to the relative enrichment of different intestinal bacterial species. Cluster I was composed of species from the gut and oral cavity/respiratory tract, in which the bacterial read ratios ranged from 0.62% to 1.66%. And Cluster II was mainly composed of species from the oral cavity/respiratory tract, in which the bacterial read ratios ranged from 0.0008% to 0.056%. However, this cluster only contained two intestinal bacterial species. Thus, in bile samples, bacteria from the oral cavity/respiratory tract were more prevalent than gut bacteria. Combining with the metadata of these samples with the microbial structure, we found that the microbial community patterns were not associated with fever status or surgical history. We also identified many patient-specific species in only one(n = 77) or two individuals(n = 43), revealing heterogeneity among individuals. WMS and 16 S sequencing results both showed that the biliary microbiota had lower within-sample diversities than those of the intestinal microbiota from healthy adults. With whole-metagenome shotgun sequencing, 13 novel biliary bacteria were detected, including Pyramidobacter piscolens and Cellulosimicrobium cellulans. These novel bacteria had a genome coverage ranging from 1% to 33.13%, and most of them were oral and environmental bacteria. Moreover, we identified genes encoding putative proteins related to gallstone formation and bile resistance, like β-glucuronidase, phospholipase and multidrug efflux pump proteins. We reconstructed the metabolic KEGG pathways of the biliary microbiota and compared them with those of stool samples from healthy adults. The biliary microbiota was enriched in pathways of inflammation, oxidative stress and flagellar assembly, whereas carbohydrate metabolic pathways showed varying behaviours.In order to further study the origin of the biliary microbiota, we then presented an investigation on bacterial communities of the human biliary tract, duodenum, stomach, and oral cavity from six gallstone patients. Employing 16 S rRNA amplicon sequencing, we obtained an average of 12,299 reads with high quality from each sample and detected a total of 1,398 operational taxonomic units(OTUs). We found that all biliary bacteria could be identified in the upper digestive tract. At the phylum level, the seven biliary phyla could be identified in at least one sampling site of the upper digestive tract in the same patient. At the genus level, 53 high abundant genera in bile samples were also contained in the microbiota of other sampling sites. Based on the correlation analysis and microbial diversity index, we found that the biliary microbiota resembled, to some extent, the duodenal microbiota. The diverse changes of the biliary microbiota relative to those of the rest sampling sites were also observed. Although the relative abundances of most bacteria, including Veillonella, Prevotella, Porphyromonas and Rothia, were greatly diminished in bile samples, three Enterobacteriaceae genera(Escherichia, Klebsiella, and an unclassified genus) and Pyramidobacter were abundant in bile. Based on the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States tool which could predict functional contents from 16 S sequencing data, we predicted the relative abundances of Kyoto Encyclopedia of Genes and Genomes pathways for every sampling site. Comparisons between the biliary tract and other sites indicated relatively elevated abundances of environmental information processing-related and cell motility-related pathways in bile samples. Moreover, differential representation of pathways related to energy and nutrient sources between bile samples and other digestive tract sites were also observed. Hence, with this study, we provide evidence for the potential source of biliary bacteria and depict the potential selective pressure imposed on the microbiota by the biliary system.To investigate the microbiota associated with Alzheimer?s disease, we finally performed metagenomic analysis on the gastrointestinal microbiome of AD mice by 16 S rRNA amplicon sequencing and validated the impacts of a Chinese Traditional Medicine LW-AFC on the cognitive abilities and the gastrointestinal microbiota of AD mice. The cognitive experiments, including Morris-water maze test, shuttle-box test and novel object recognition test, indicated that the SAMP8 mice, a model of AD, had defects in cognitive abilities like spatial learning and memory ability, active avoidance response and object recognition memory capability and the treatment of LW-AFC could improve cognitive impairments of SAMP8 mice. Based on 16 S sequencing, each sample obtained an average of 160,684 16 S reads. Among these samples, a total of 13,727 OTUs were detected. And the gut microbiota of SAMP8 mice treated with LW-AFC were more similar to those of the control SAMR1 mice. At the genus level, SAMP8 mice had 8 significantly increased and 12 decreased OTUs when compared to the control SAMR1 mice. Compared to SAMP8 mice, SAMP8 mice treated with LW-AFC also had 20 significantly abundant OTUs, including 15 increased OTUs and 5 decreased ones. Among all the differentially abundant OTUs, the abundances of 13 OTUs were reversed by LW-AFC. Moreover, correlation analysis revealed that at the genus level, a total of 18 genera were significantly correlated with at least one of the three tested cognitive abilities. There were 7 genera associated with all the three cognitive abilities. It should be noted that the relative abundances of the 7 genera were all reversed by LW-AFC. In all, relative to the control mice, SAMP8 mice had significant alterations in their gut microbiota, and LW-AFC might have the ability to restore the imbalance in the gut microbiota.In general, by using metagenomic sequencing technology, we delineated the microbial structure and function of the biliary microbiome and the gut microbiome under specific disease conditions, and laid foundations for investigating the roles of the microbiota in disease development. This study has firstly made a systematic description of the microbial composition of the common bile duct bile. It has dectected 13 unreported biliary bacteria and identified two cluster patterns among the biliary microbiota. Moreover, functional genes of the biliary microbiota were predicted and the metabolic pathway network was reconstructed. The whole study is novel and has never been done before. Meanwhile, our study firstly compared the biliary microbiota with the microbiota from three upper digestive tract sites and provided evidence for the potential source of the biliary bacteria and the impacts of the biliary system on the biliary microbiota. Furthermore, the gut microbiota of AD mice revealed imbalance when compared to the control mice. And LW-AFC might have the ability to restore such imbalance, which provided clues for the potential targets of LW-AFC. Our studies also revealed that metagenomic sequencing technology would be widely applied in microbiome studies and clinical studies. |
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