Intratumoral Enterobacter hormaechei drives gemcitabine resistance in pancreatic cancer via cdd L ‐mediated drug inactivation

Gemcitabine resistance poses a critical barrier to improving survival in pancreatic cancer, yet the microbial drivers remain elusive. By integrating 16S rRNA amplicon sequencing with large-scale culturomics across 114 clinical samples, we identified Enterobacter hormaechei as a key intratumoral pathogen. We demonstrate that E. hormaechei confers resistance by enzymatically converting the drug to its inactive metabolite dFdU via a unique long-isoform cytidine deaminase encoded by cddL. Kinetic analysis revealed exceptional catalytic efficiency (Km = 0.22 mM, kcat = 194.05 s−1), and genetic ablation of cddL fully restored drug sensitivity. In vivo, antibiotic co-treatment eliminated intratumoral bacteria and potentiated gemcitabine efficacy, enabling a 50% dosage reduction without comprising therapeutic outcome. Pan-cancer analysis further confirmed the broad prevalence of Enterobacter across multiple solid tumor types. These findings elucidate a cddL-mediated mechanism of chemoresistance and identify intratumoral E. hormaechei as a tractable therapeutic target for optimizing gemcitabine-based regimens and improving patient outcomes.