Clostridioides difficile
infection (CDI) is a leading cause of hospital-acquired diarrhea, which often stems from disruption of the gut microbiota by broad-spectrum antibiotics. The increasing prevalence of antibiotic-resistant
C. difficile
strains, combined with disappointing clinical trial results for recent antibiotic candidates, underscores the urgent need for novel CDI antibiotics. To this end, we investigated
C. difficile
enoyl ACP reductase (
Cd
FabK), a crucial enzyme in
de novo
fatty acid synthesis, as a drug target for microbiome-sparing antibiotics. To test this concept, we evaluated the efficacy and
in vivo
spectrum of activity of the phenylimidazole analog 296, which is validated to inhibit intracellular
Cd
FabK. Against major CDI-associated ribotypes 296 had an Minimum inhibitory concentration (MIC
90
) of 2 µg/mL, which was comparable to vancomycin (1 µg/mL), a standard of care antibiotic. In addition, 296 achieved high colonic concentrations and displayed dosed-dependent efficacy in mice with colitis CDI. Mice that were given 296 retained colonization resistance to
C. difficile
and had microbiomes that resembled the untreated mice. Conversely, both vancomycin and fidaxomicin induced significant changes to mice microbiomes, in a manner consistent with prior reports.
Cd
FabK, therefore, represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.