Characterization of the bile and gall bladder microbiota of healthy pigs

Abstract

Bile is a biological fluid synthesized in the liver, stored and concentrated in the gall bladder (interdigestive), and released into the duodenum after food intake. The microbial populations of different parts of mammal's gastrointestinal tract (stomach, small and large intestine) have been extensively studied; however, the characterization of bile microbiota had not been tackled until now. We have studied, by culture-dependent techniques and a 16S rRNA gene-based analysis, the microbiota present in the bile, gall bladder mucus, and biopsies of healthy sows. Also, we have identified the most abundant bacterial proteins in the bile samples. Our data show that the gall bladder ecosystem is mainly populated by members of the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, fluorescent in situ hybridization (FISH) and transmission electron microscopy (TEM) allowed us to visualize the presence of individual bacteria of different morphological types, in close association with either the epithelium or the erythrocytes, or inside the epithelial cells. Our work has generated new knowledge of bile microbial profiles and functions and might provide the basis for future studies on the relationship between bile microbiota, gut microbiota, and health.

Keywords: Bile; gall bladder; microbiome; microbiota; proteome.

Figure 1

FISH in situ localization of bacteria in gall bladder biopsies. The nuclei of the gall bladder epithelial cells appeared blue due to DAPI staining while bacteria appeared green due to the hybridization with the EUB338 probe. Figure shows the same section observed by using (A) bright field microscopy and (B) a composition of two filters, one for DAPI blue detection and the other for detection of green FAM fluorescence. The figure suggests the presence of bacteria inside the host cells (Fig. 1C and D) and in the borders of the section (Fig. 1E).

Figure 2

TEM ultramicrophotographic images of bacteria within the gall bladder tissue. A and B show bacteria-like structures closely associated to erythrocytes, while C–F show potential gall bladder bacteria at a higher magnification. Magnification: (A) 6000×, (B) 5000×, (C) 25,000×, (D and E) 40,000×, and (F) 150,000×. B, bacteria-like structures; E, erythrocyte; C, collagen fibers.

Figure 3

Best hit comparison of bacterial phyla in the four bile (B) and two mucus (M) samples analyzed by pyrosequencing in this study. The percent of sequences of each phyla was assigned according to the Best Blast Hit paradigm.

Figure 4

Species for which DNA was detected in the four bile (B) and two mucus (M) samples analyzed by pyrosequencing in this study. Bacterial diversity was assessed using the Shannon–Weaver diversity index.

Figure 5

Bacterial phyla as deduced from the taxonomic analysis of biliary proteins.