In the human gut, the growth of Clostridioides difficile is impacted by a complex web of inter-species interactions with members of human gut microbiota. We investigate the contribution of inter-species interactions on the antibiotic response of C. difficile to clinically relevant antibiotics using bottom-up assembly of human gut communities. We discover two classes of microbial interactions that alter C. difficile’s antibiotic susceptibility: infrequent increases in tolerance at high antibiotic concentrations and frequent growth enhancements at low antibiotic concentrations. Based on genome-wide transcriptional profiling data, we demonstrate that metal sequestration due to hydrogen sulfide production by the prevalent gut species Desulfovibrio piger increases metronidazole tolerance of C. difficile. Competition with species that display higher sensitivity to the antibiotic than C. difficile leads to enhanced growth of C. difficile at low antibiotic concentrations. A dynamic computational model identifies the ecological design principles driving this effect. Our results provide a deeper understanding of ecological and molecular principles shaping C. difficile’s response to antibiotics, which could inform therapeutic interventions.

RNA Seq data for metronidazole (MTZ) samples: C. difficile in fresh media (CD_ABB), C. difficile in D. piger spent media (CD_DPSM), and C. difficile in coculture with D. piger (CD_DP).