The effects of novel oral preclinical PolC inhibitor antibiotic candidates on the gut microbiome in a murine thigh infection model

ABSTRACT Antibiotics can negatively affect gut microbiome composition and function. However, preclinical evaluations of antibiotic effects on the microbiome are rarely undertaken. The purpose of this study was to characterize pharmacokinetics and metagenomic profiles of PolC inhibitor compounds compared to linezolid in a neutropenic murine thigh infection model. Neutropenic mice thighs were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and dosed orally starting 1 h later with ACX-728, ACX-801, ACX-978, or linezolid. Untreated control mice were euthanized at 1 h for baseline data, and a second untreated group were euthanized at 24 h after the start of dosing, when all remaining mice were euthanized. At euthanasia, thigh muscles were excised for MRSA quantification, and fecal pellets, colon tissue, and plasma samples were collected for drug concentration and microbiome analyses. All PolC inhibitors were detectable in plasma and feces at 25 h. Compared with linezolid, PolC inhibitors maintained greater gut microbial richness and diversity in this acute murine infection model. PolC-treated samples preserved overall community structure and Bacteroidota dominance, whereas linezolid induced marked disruption, with secondary expansion of Pseudomonadota , primarily Escherichia coli . Microbial pathway analysis showed preservation of community-level metabolic function for PolC inhibitors but marked disruption by linezolid. Compared to controls, linezolid produced a 4.4 log reduction in MRSA colony-forming unit per gram, while the PolC inhibitors produced a mean log reduction of 0.4–2.5 per group. In these exploratory studies, PolC inhibitor compounds preserved anaerobic community structure and fermentative capacity, whereas linezolid induced depletion of healthy commensals and expansion of aerotolerant taxa. These findings highlight the importance of microbiome-informed antibiotic evaluation in preclinical studies. Larger preclinical and clinical studies are warranted to confirm these findings. IMPORTANCE This study evaluated the class effect of PolC inhibitors on the microbiome as part of the preclinical development plan for systemic PolC inhibitors. Similar to the non-absorbable antibiotic, ibezapolstat, a narrow effect on the microbiome was observed with these novel compounds.