Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing.

Recent advances in metagenomics have expanded our ability to detect low-abundance microbial communities. While the gut remains the most densely populated microbial habitat, emerging evidence has proposed that microorganisms might also inhabit anatomical sites once considered sterile, such as the biliary system. We apply next-generation DNA sequencing to characterize the bacterial community of bile in obese and non-obese patients with symptomatic gallstones. Bile samples were collected from 64 patients (32 obese, 32 non-obese) undergoing elective cholecystectomy. We incorporated negative (sterile tubes) and positive (mock microbial community standard) controls to evaluate contamination risks. We applied both 16S rRNA gene amplicon and shotgun metagenomic sequencing. Both sequencing methods detected extremely low bacterial biomass in bile. Specifically, shotgun metagenomic sequencing identified bacterial DNA traces in only eight samples, displaying minimal community similarity. In the positive controls, our measurements confirmed the expected microbial community composition, and in the negative controls, no bacterial DNA was detected. In contrast, 16S rRNA gene sequencing showed bacterial DNA in all bile samples as well as in negative controls, suggesting a higher susceptibility to contamination. Our findings suggest that bile may not be consistently colonized by bacterial communities in uncomplicated gallstone disease.