Reassessment of extracellular and intracellular activity of macrolides, rifampicin, and doxycycline against Rhodococcus equi based on bacterial counts and microscopy

Rhodococcus equi is an opportunistic intracellular pathogen responsible for severe respiratory infections in foals, requiring prolonged antibiotic treatment. Despite its in vitro susceptibility to multiple antibiotics, the in vivo eradication of intracellular R. equi remains challenging due to its ability to persist and survive within alveolar macrophages. This study aimed to assess in vitro the extracellular and intracellular activity of rifampicin, doxycycline, and four macrolides against R. equi while assessing current methodologies used to differentiate intracellular and extracellular bacteria. We found that the removal of extracellular R. equi with short times of exposure to bactericidal drugs was challenging. We thus proposed an alternative approach with washing steps to assess intracellular antibiotic activity by combining bacterial counts and optical microscopy. Our results indicated that clarithromycin alone, as well as azithromycin or gamithromycin in combination with doxycycline, dramatically reduced both intracellular and extracellular R. equi. This study also highlighted the need for refined in vitro methodologies to assess antibiotic efficacy against R. equi, ultimately contributing to the development of optimized treatment strategies for R. equi infections in foals. Importance: Rhodococcus equi is a major cause of pneumonia in foals and represents a serious challenge for the equine sector due to the need for prolonged and sometimes ineffective antibiotic treatments. This study highlights the limitations of current in vitro methods used to evaluate antibiotic activity against R. equi, by demonstrating that widely used bactericidal antibiotics like gentamicin may not eliminate extracellular bacteria as effectively as previously assumed, and we emphasize the risk of overestimating intracellular bacterial survival in experimental models. We therefore propose a new method that allows more accurate assessment of antibiotic efficacy within host cells. Our findings help refine in vitro testing strategies and support the identification of antibiotic combinations with effective intracellular activity, offering new perspectives for improving treatment protocols against R. equi infections in foals.