Cinchonidine enhances intracellular erythromycin activity against Rhodococcus equi

Rhodococcus equi is a facultative intracellular pathogen that causes severe infections in foals and immunocompromised individuals. Its ability to survive within macrophages renders conventional antibiotics ineffective and promotes multidrug resistance. Novel therapeutic strategies are needed.

We screened a library of 3251 natural compounds to identify antibacterial candidates targeting R. equi. Strains included wild-type 103S⁺, an mrx-deficient triple mutant, and a biosensor-expressing strain carrying the Mrx1-roGFP2 probe. Minimal inhibitory concentrations, checkerboard synergy assays, and time-kill kinetics were performed. Intracellular efficacy was assessed in infected J774.A1 murine macrophages. Redox stress induction was quantified using confocal microscopy.

Thirty-eight compounds selectively inhibited the mrx-deficient mutant, suggesting redox-mediated mechanisms. While most natural compound combinations showed limited intracellular activity, the pairing of erythromycin (ERY) and cinchonidine (CIN) exhibited synergy, significantly reducing intracellular bacterial load. Time-kill assays revealed bactericidal activity at 8 × MIC. Biosensor analysis confirmed that ERY-CIN synergy correlated with elevated oxidative stress, supporting a redox-based mechanism.

Our findings highlight CIN as a redox-active adjuvant that potentiates erythromycin's intracellular efficacy against R. equi. This combinatorial approach targets redox homeostasis and enhances bacterial clearance, providing a promising strategy against multidrug-resistant intracellular pathogens.