Effect of periampullary diverticulum on biliary flora and the formation of common bile duct stone

Background: Bile duct stone is closely related to periampullary diverticulum, but it is not clear whether the formation of it was affected by the diverticulum through the biliary flora. To explore the diversity and correlation of biliary and intestinal flora in the patients with choledocholithiasis and the effects of periampullary diverticulum on the flora and bile duct stone. Methods: Bile and intestinal fluid were collected from patients with primary common bile duct stones, and then divided into diverticulum group and none- diverticulum group according to the presence or absence of paravertebral diverticula, DNA of these samples was extracted and a bacterial gene library was constructed, and related bioinformatics analysis was performed after high-throughput sequencing to obtain the bacterial components and community structure of the sample. Result: A total of 3001,613 valid sequences were obtained, with an average of 136436.95±3696.842 sequences, which were classified into 6021 ASV/OUT. Alpha diversity analysis showed that the species richness and diversity in the diverticulum group were lower than those in the non-diverticulum group. According to the species annotation results, the advantage bacterium group of the bile is Proteobacteria (BG 80.41%, Bg 70.95%), and advantage bacterium group of the intestinal fluid is Firmicutes and Proteobacteria (BG 89.39%, Bg 74.11%). A large proportion of Enterobacter was found in bile. Escherichia coli, Klebsiella, Streptococcus and other bacteria closely related to stone formation have been found. The proportion of E. coli in the diverticulum group was increased and due to the existence of the diverticulum Enterobacteria in the bile were increased and more complex. The bacteria that produce Beta-glucuronidase are found to be increased in bile. Due to the influence of the periampullary diverticulum, the intestinal flora will be changed and then the biliary flora will also change. Conclusion: The existence of periampullary diverticulum will affect the biliary tract flora and lead to the increase of bacteria related to stone formation, which will affect the formation of choledocholithiasis and make it easier for choledocholithiasis to form.


Background
There are thousands of species and trillions of bacteria in the human gut, which some scholars regard as the body's "hidden organs". The research found that human health is not only related to its genetic genes but also has a subtle connection with intestinal microorganism [1], gene sequencing analysis has revealed the intestinal bacteria that play an incredible role, they have important effects on health, especially the gut microbes are associated with a variety of diseases, such as cardiovascular disease, intestinal disease, autoimmune disease, metabolic disease, biliary tract disease, depression, Alzheimer's, cancer [2][3][4][5][6][7][8][9], etc. Moreover, the duodenal flora has been shown to affect the overgrowth of intestinal bacteria, irritable bowel syndrome, and celiac disease [10], and these association studies have redefined the concept of the human body as a superorganism composed of its cells and symbiotic microorganisms.
Current studies have found that intestinal microbial changes can occur in patients with primary cholangitis, but the application of ursodeoxycholic acid treatment can partially recover [11].
Common bile duct stones are a common and frequently-occurring disease, and the primary common bile duct stones are mostly pigmented stones. Increasing evidence suggests that bacteria play an important role in the pathogenesis and formation of pigmented stones [12][13][14]. However, only 1% of bacteria in complex samples can be cultured, and studies have shown that even if bacteria are not cultured in patients' bile, bacterial biofilms may still exist on the surface of stone [15]. This provides us with a biased view of the relative abundance of existing species.
Periampullary diverticulum refers to the saclike process formed within a radius of about 3cm around the large duodenal nipple. At present, the existence of periampullary diverticulum and choledocholithiasis, cholangitis, and choledochal dilatation is considered to be significantly related [16]. Other studies have shown that patients with common bile duct stones are more likely to be found combined with periampullary diverticulum and that the periampullary diverticulum is only related to common bile duct stones but not to gallbladder stones [17]. The existence of parapapillary diverticulum will not only cause cholestasis but also make duodenal fluid retrograde into the bile duct so that some bacteria that can produce β-glucuronidase can be infected [18]. It has been reported that even the presence of parapapillary diverticulum can lead to recurrent bacterial cholangitis [19]. Previous studies have shown that the existence of periampullary diverticulum is associated with an increased incidence of choledocholithiasis. Whether the periampullary diverticulum makes the formation of stones easier by affecting the flora is an unsolved problem at present. This study takes the flora as the starting point to explore the effect of periampullary diverticulum on choledocholithiasis.
With the emergence of a new generation of sequencing technology, people can deeply study the microbial communities in different parts of the body, including those that are difficult to cultivate, and predict the metabolic function of the microbial community to find its impact on human health [20]. However, there is no related research that will be periampullary diverticulum associated with bacteria to analyze its influence on common bile duct stone, has yet to someone use the gene sequencing method for further study.

Subject investigated
The subjects were selected from the patients who were admitted to the

DNA extraction
The E.Z.N.A.® DNA extraction kit was used to extract DNA according to the instructions, and then DNA was quantified by Nanodrop.
Finally, the extraction quality of DNA was detected by 1.2% agarose gel electrophoresis.

16S rRNA amplicon sequencing
The 16SrRNA gene V3V4 variable region was amplified by PCR, the target fragment length was 480bp, the sequencing strategy was NovaSeq-PE250, the upstream primer was 338F(ACTCCTACGGAGGCAGCA), and the downstream primer was806R(GGACTACHVGGTWTCTAAT).

Bioinformatics analysis process
The sequence denoising is performed according to the analysis process of QIIME2 DADA2 [21]. According to the distribution of ASV/OTU in different samples, evaluate the Alpha diversity of each sample and calculate the corresponding indexes, and corresponding graphs were drawn.
Species annotation was performed, and a histogram of species composition was then produced for all samples at the phylum and genus levels.
GraPhlAn [22] was used to draw the GraPhlAn evolutionary tree graph to show the evolution of each sample in different species and understand the overall situation. Use the Venn diagram for community analysis to study which species are common and which are unique among different samples.

Result Sample sequence and ASV/OTU
Sequence denoising is carried out by using the DADA2 method after sequencing. In the past, sequences with similarity higher than a certain threshold are usually merged into an OTU (OperationalTaxonomicUnit, operable taxon) [23], but the DADA2 method is no longer clustering with similarity, but only removes repetition, which is equivalent to clustering with 100% similarity. Each sequence that removes repetition is called ASV (amplicon sequence variants), or call it a feature sequence (corresponding to the OTU representative sequence). After quality control, denoising, splicing, and de-chimerism of the original sequence, we finally obtained 3501497 original sequences and 3002685 high-quality sequences. Finally, after removing the ASV sequence with a total number of only 1 in all samples by default, 3001613 valid sequences were obtained, with an average of 136436.95 ±3696.842 (Fig 1A and 1B ).

Alpha diversity analysis
Alpha diversity is a comprehensive index, which can not only reflect the species diversity in the sample area, but also describe the richness and evenness Alpha diversity is evaluated by calculating the corresponding diversity index, the richness is characterized by Chao1 index and Observedspecies index, the higher the community richness is, the larger the index is, and the higher the index value is, the higher the community diversity is. The coverage is characterized by the Goodscoverage index.
The higher the index, the less the proportion of undetected species in the sample. The specific results are shown in Table 1.

Rarefaction curves and Rank abundance curve
Rarefaction curve can be used to evaluate the diversity and abundance of sample species, so we constructed the Observed species index rarefaction curve and Shannon index rarefaction curve (Fig 2, 3).  The rank abundance curve can be used to reflect the ASV/OUT abundance distribution of each sample (Fig 4). Different from the rarefaction curve, the rank abundance curve can reflect species diversity and the richness and uniformity of sample species. The richer the species composition, the wider the curve. The width of the curve reflected the abundance of the species.
The smoother the curve, the more uniform is the species distribution.
Similarly, the curve of the non-diverticular group was wider and smoother than that of the diverticular group.    The general composition of the genus can reflect the change of habitat in different parts of the human body (Fig 8, 9). Genus is the lowest taxon except for species in the bacterial taxon. However, due to the wide variety of microorganisms and the lack of accurate species information in the current database, the microbial sequences have not been completely covered by the sequencing sequence or the reference sequence lacks accurate species information, leading to the inability to identify unclassified species among some specific genera. Therefore, some characteristic sequences may not be able to obtain genera-level annotation information in the analysis process.

Fig 8. TOP20 species distribution at genus level of all bile samples
From S8 Fig, it can be seen that Enterobacter is the most different between the two groups, there is no Enterobacter in the BG group. This is due to technical limitations not able to obtain genera-level annotation information in the analysis process. But its Enterobacteriaceae do occupy a relatively high proportion, indicating that the proportion of Enterobacter in the bile of the diverticulum group is higher and more complicated, and its analysis needs further exploration. It can also be seen from Figure 9 that there is a certain proportion of Escherichia in the bile of the diverticulum group, which is not found in the bile of the non-diverticulum group. Secondly, Veillonella also has a higher share in BG than in the Bg group. Proportion, the rest of the genus can be seen from the chart that the two groups are not much different. and in the BG group, Weirongococcus is also found to occupy a certain proportion and is higher than the Bg group. It can also be seen from the figure that the DG group is Cray The genus Pleurotus has a high proportion (12.75%), while it is almost absent in the Dg group (0.03%). Its proportion in the BG group is 0.26%, but it is also almost absent in the Bg group (0.01%). The proportion of the DG group (5.56%) is also much higher than that of the Dg group (0.19%). This is no accident, indicating that the existence of diverticulum not only changes its flora but also affects the biliary flora. Propionibacterium is a typical bacterium in the formation of cholelithiasis. Currently, there are few reports on the isolation of Propionibacterium from bile, which is only isolated and found in a single study. However, Propionibacterium is found in all the samples in this study, BG 0.55%, Bg0.84%, DG0.08%, Dg0.39%.

Species Difference Analysis and Marker Species
There are 626 ASV/OTUs shared by the BG and Bg group, and the species abundance composition of their consensus characteristic sequences at the phylum and genus levels is shown in S10 Fig. BG   Thus it can be seen that the bacteria in bile may be more derived from the intestinal tract, more specifically from around the duodenal papilla.
It is well known that bile is a natural antibacterial compound and essential for digestion and nutritional absorption, but some enteropathogens have not only evolved to resist the bactericidal conditions of bile, but these bacteria also use bile as a signal to enhance virulence regulation and thus effectively infect, and E. coli is one of them [29]. At present, it is believed that the mechanism of stone formation caused by bacteria is mainly related to several factors or enzymes produced by bacteria, such as β-glucuronidase, phospholipase, and bacterial hydrolase.
This study also found that the existence of diverticulum can increase the production of β-glucuronidase species in bile. β-glucuronidase is a key enzyme that regulates the release of free bilirubin and glucuronic acid from Lactobacillus is a kind of probiotics, but it is easy to retrograde into the bile duct because of its over-reproduction due to the relatively closed environment formed by diverticulum. In this study, we found that

Conclusion
The existence of periampullary diverticulum will affect the biliary tract flora and lead to the increase of bacteria related to stone formation, which will affect the formation of choledocholithiasis and make it easier for choledocholithiasis to form.

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