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

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