Association of Microbial Dysbiosis with Gallbladder Diseases Identified by Bile Microbiome Profiling

Abstract

Background: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully. We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis.

Methods: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants.

Results: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions.

Conclusion: We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Keywords: Bile; Chronic Cholecystitis; Gallbladder Diseases; Gallbladder Neoplasms; Microbiota.

Fig. 1. Flowchart of 25 participants. 10 Healthy controls and 15 patients with gallbladder diseases were enrolled. 10 Healthy controls were living-donors for the liver transplant and underwent right hepatectomy and cholecystectomy. Among 15 patients, 10 patients with chronic cholecystitis underwent laparoscopic cholecystectomy and 5 patients with gallbladder cancer underwent extended cholecystectomy. Bile samples were collected aseptically and analyzed.

Fig. 2. The bacterial composition of the bile of the healthy individuals and patients with chronic cholecystitis and gallbladder cancer. The number of sequences profiled at the genus level was normalized using the CLR method. The normalized values under 0 were colored white. The maximum normalized value of pseudo-count was −0.3072.

CLR = centered log-ratio, N = normal control, CC = chronic cholecystitis, GBC = gallbladder cancer.

Fig. 3. The bacterial species identified in patients with GBC and CC. The y-axis of each boxplot indicates CPM reads calculated from the result of read mapping in CC4 (A), GBC1 (B), and GBC2 (C). Phylogenetic tree of the bacterial species in CC4 and Citrobacter species (D), in GBC1 and four Enterobacteriaceae species (E), and in GBC2 and Streptococcus species (F). The proteins obtained from the bile microbiomes are in bold.

GBC = gallbladder cancer, CC = chronic cholecystitis, CPM = counts per million.

Fig. 4. The distribution of the six major bacteria in three different clinical groups.

Genera that show significant difference between two different groups are marked with asterisk: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Normalized values lower than −0.3072 are all derived from pseudo-count. N = normal control, CC = chronic cholecystitis, GBC = gallbladder cancer.

Fig. 5. Radiologic and pathologic findings of CC patients with or without a dysbiotic microbiome. B-mode ultrasound (A) and coronal computed tomography scan (B) obtained on portal phase show the stone with the largest dimension (4.4 cm) in the gallbladder of a 77-year-old man. (C) CC with a gallbladder cancer-associated microbiome pattern reveals severe infiltration of lymphoplasmacytic cells and neutrophils and shallow mucosal erosion. On higher magnification, the epithelium shows enlarged, hyperchromatic, crowded nuclei with prominent nucleoli. However, CC with a normal microbiome pattern shows nonspecific chronic inflammation without epithelial atypia. (D) The overall histologic score of CC with dysbiosis is significantly higher than the normal, especially regarding the degree of activity and epithelial atypia.

CC = chronic cholecystitis.