PreTA-mediated metabolism of 5-fluorouracil by intratumoral Citrobacter freundii drives chemoresistance in pancreatic cancer

Pancreatic cancer is highly malignant, and while fluoropyrimidines (5-fluorouracil [5-FU] and capecitabine) are critical first-line treatments for metastatic cases, drug resistance remains a major challenge. In this study, we identified an association between Citrobacter enrichment in pancreatic tumors and poor overall survival in patients with pancreatic cancer, particularly in patients receiving fluoropyrimidine-based treatment. Co-culture of 5-FU with the Citrobacter freundii strain isolated from pancreatic cancer and intratumoral injection of this strain in xenograft models significantly reduce the antitumor efficacy of 5-FU. Subsequent analyses using mass spectrometry, bioinformatics, and gene knockout experiments revealed that C. freundii strain inactivates 5-FU via the PreTA, which is homologous to human dihydropyrimidine dehydrogenase (DPD). Gimeracil, a DPD inhibitor, preserves the efficacy of 5-FU by blocking PreTA activity. Other preTA-harboring bacteria also metabolized 5-FU, indicating broader bacteria-mediated 5-FU resistance. These findings reveal a potential microbiome-driven mechanism of chemotherapy failure and identify PreTA as a druggable target.