Reassessment of extracellular and intracellular activity of macrolides, rifampicin, and doxycycline againstbased on bacterial counts and microscopy.

Abstract

UNLABELLED: is an opportunistic intracellular pathogen responsible for severe respiratory infections in foals, requiring prolonged antibiotic treatment. Despite itssusceptibility to multiple antibiotics, theeradication of intracellularremains challenging due to its ability to persist and survive within alveolar macrophages. This study aimed to assessthe extracellular and intracellular activity of rifampicin, doxycycline, and four macrolides againstwhile assessing current methodologies used to differentiate intracellular and extracellular bacteria. We found that the removal of extracellularwith 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. This study also highlighted the need for refinedmethodologies to assess antibiotic efficacy againstultimately contributing to the development of optimized treatment strategies forinfections in foals. IMPORTANCE: 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 currentmethods used to evaluate antibiotic activity against, 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 refinetesting strategies and support the identification of antibiotic combinations with effective intracellular activity, offering new perspectives for improving treatment protocols againstinfections in foals.