Abstract
BACKGROUND: Despite advances and successes in precision oncology, pancreatic cancer (PC) remains a tumor with extremely low survival rates, and many of these cases experienced postoperative recurrence and metastasis. Alterations in the gut microbiota have been linked to the survival rates of PC patients. Nevertheless, the complexity of gut microbiota composition poses significant challenges in identifying definitive clinical biomarkers for PC. METHODS: Fecal samples were collected from PC patients, half of whom had metastasis, and their matched healthy controls (HCs). A metagenomic analysis was employed to further investigate the functional features of gut microbiota with both PC and metastatic PC. The clinical correlations, microbial metabolic pathways and antibiotic resistome were further assessed. In a follow-up validation, intraoperative tumor tissue and pancreatic fluid were sampled from PC patients and underwent comprehensive microbiological analysis, including bacterial culture, mass spectrometry-based identification, and third-generation whole-genome sequencing of Klebsiella pneumoniae isolates. RESULTS: We observed a significant alteration of the gut microbiota in PC patients, highlighted by an overall increase in microbial diversity compared to healthy controls (p < 0.05). Comparative abundance analysis identified 59 differentially abundant microbial species in non-metastatic pancreatic cancer (NMPC) (56 increased, 3 decreased) and 21 in metastatic pancreatic cancer (MPC) (19 increased, 2 decreased), alongside 18 significantly altered microbial metabolic pathways (FDR-adjusted p < 0.05). Notably, Klebsiella pneumoniae, Klebsiella oxytoca, and Akkermansia muciniphila were identified as prominent antibiotic resistance gene (ARG) carriers in the gut microbiota of PC patients, with 653 ARG subtypes detected across fecal samples, 38-47% of which were shared among groups. Strong co-occurrence patterns between ARGs (e.g., acrB, mdtC, cpxA, emr, pmrF) and the above species were observed predominantly in MPC samples (p < 0.05). Whole-genome sequencing of 14 isolates obtained from tumor tissue and pancreatic fluid revealed consistent ARG profiles and virulence genes, corroborating the metagenomic findings and supporting the hypothesis of gut-to-tumor translocation and potential intratumoral colonization. CONCLUSION: This study provides a comprehensive microbiome-based insight into PC and its metastatic subtypes. By integrating microbiome analysis with microbial culture, this study provides direct evidence of gut-derived multidrug-resistant (MDR) K. pneumoniae colonization in PC tissues.