Extracellular and Intracellular Activity of Rifaximin α Against Bovine Staphylococcus aureus: Implications for Early Mastitis Therapy

Subclinical mastitis caused by Staphylococcus aureus remains a major challenge due to persistence, recurrence and antimicrobial use. Bacterial adaptation to mammary gland conditions and intracellular survival contribute to therapeutic failure. Rifaximin α (RIFα), a semisynthetic rifamycin widely used for intramammary therapy, exhibits strong antibacterial activity; however, an integrated pharmacodynamic evaluation considering both extracellular and intracellular compartments is limited.

In vitro pharmacodynamic studies were performed to characterize the extracellular and intracellular activity of RIFα against bovine S. aureus. Extracellular activity was assessed using minimum inhibitory concentration determination, post-antibiotic effect, and time–kill curves under mammary-simulated conditions, including different pH values (7.4, 6.5 and 5.0) and media supplemented with bovine serum or milk. Antibacterial activity was quantified using the antibacterial effect index (E). Intracellular activity was evaluated in polymorphonuclear cells (PMNs) isolated from bovine blood. PMNs were infected with S. aureus ATCC 29213, extracellular bacteria were eliminated, and infected cells were exposed to RIFα at 3×MIC and 10×MIC. Intracellular viable bacteria were quantified by colony-forming unit enumeration following cell lysis.

RIFα demonstrated potent extracellular activity, achieving stable bactericidal effects (E ≤ –3) at 4–8×MIC across pH conditions and in the presence of milk or serum, confirming high antibacterial efficacy under mammary-relevant conditions. In contrast, intracellular exposure resulted in only partial reductions in intracellular S. aureus, with decreases of approximately 1.5–1.7 log₁₀ CFU after 8 h, without reaching bactericidal thresholds.

From a PK/PD standpoint, the contrast between potent extracellular bactericidal activity and limited intracellular efficacy underscores the importance of early exposure-driven antimicrobial action. These findings emphasize the value of dynamic pharmacodynamic approaches that integrate both extracellular and intracellular compartments to better inform rational dosing strategies against persistent Staphylococcus aureus infections.