Metagenomics of pigmented and cholesterol gallstones: the putative role of bacteria

Abstract

There is growing evidence for bacteria playing a role in the pathogenesis and formation of pigmented gallstones from humans. These studies mainly involved cultivation of gallstone-associated bacteria and 16S rRNA profiling, providing an indirect link between processes involved in gallstone formation by the bacteria in-situ. Here, we provide functional metagenomic evidence of a range of genes involved in bile stress response, biofilm formation, and anaerobic energy metabolism by Gram-negative Klebsiella in pigmented gallstones from a 76-year-old male patient. Klebsiella was also present in one cholesterol-type stone in a 30-year-old female patient who had additional cholesterol gallstones characterised by Gram-positive bacteria. Pigmented stones further revealed a predominance of genes involved in carbohydrate metabolism, whilst cholesterol stones indicated a profile dominanted by protein metabolism possibly reflecting known chemical differences between Gram-negative and Gram-positive biofilm matrices. Archaeal genes were not detected. Complementary carbon and hydrogen isotopic analyses of cholesterol within the patients' stones revealed homogeneity, suggesting a common diet or cholesterol biosynthesis pathway that has little influence on microbial composition. This pilot study provides a framework to study microbial processes that play a potential role in gallstone formation across markedly different types of stones and patient backgrounds.

Figure 1

Principle coordinate analysis (PCoA) ordination of Bray-Curtis similarity (square root transformed) between total bacterial genera in the four gallstones of patients PM1 and CF4. Shown is the first two principle coordinate axes, which combined explain 93.5% of the variation between the patients. Coloured ellipses signify the percentage of similarity between the patients’ native stones.

Figure 2

Relative abundance of bacteria, archaea, eukaryotes and viruses recovered from metagenomes in patients PM1 (left) and CF4 (right).

Figure 3

Heatmap with the major bacterial genera identified in the gallstones (n = 4) of patients PM1 (pigmented) and CF4 (cholesterol). The color key shows the relative abundance of the genera in the gallstones. The dendrograms illustrate the relationship between samples showing that the distribution of genera is relatively similar between replicate stones, but greatly differ between the two patients.

Figure 4

Heatmap with the major functional gene categories (acquired from the Subsystems, SEED Database) identified in the gallstones (n = 4) of patients PM1 (pigmented) and CF4 (cholesterol). The color key shows the relative abundance of the gene categories in the gallstones. The dendrograms illustrate the relationship between samples showing that the distribution of genes is relatively similar between replicate stones, but greatly differ between the two patients. For example, bacterial genes involved in the carbohydrate vs. protein metabolism were more abundant in gallstones from PM1 vs. CF4.