Metagenomic next-generation sequencing and conventional microbiology for microbial profiling in biliary tract infections: a comparative study with clinical stratification

Background Biliary tract infections (BTIs) are clinically important conditions characterized by complex heterogeneous microbial profiles. However, the complete spectrum of implicated microorganisms, particularly anaerobic species and polymicrobial signatures, remains insufficiently defined due to the inherent limitations of conventional microbiological methods. Methods In this retrospective study, bile samples obtained from patients with BTIs were analyzed using DNA- and RNA-based metagenomic next-generation sequencing (mNGS) alongside conventional microbiological techniques, including culture and smear microscopy. The breadth of microbial detection across these methods was systematically compared. Microbial profiles were further stratified according to clinical variables, including sex, infection site, and disease severity. Results Metagenomic sequencing revealed a broad array of microorganisms, including bacteria, fungi, viruses, and parasites, with bacteria constituting the predominant component. A considerable proportion of identified bacterial taxa were obligate or facultative anaerobes, and polymicrobial detection patterns were frequently observed. Gram-positive genera such as Streptococcus and Enterococcus , as well as Gram-negative genera including Klebsiella and Escherichia , were commonly detected. Male patients had higher detection rates of Enterococcus and Enterobacter , whereas Staphylococcus species were more frequently identified in gallbladder infections than in bile duct infections. In this cohort, DNA-based mNGS showed broader microbial detection coverage than RNA-based mNGS and conventional culture methods. Conclusion These results underscore the complexity of microbial detection in bile-associated infections and support the value of metagenomic sequencing for comprehensive microbial characterization in BTIs. The marked heterogeneity in microbial distribution indicates that bile samples from patients with BTIs commonly show polymicrobial profiles rather than a uniform single-organism profile.