BackgroundStaphylococcus aureus survival inside phagocytes is considered to provide a reservoir of bacteria that are relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy.PurposeThe objective of this study was to develop a nanovesicle using exosomes loaded with linezolid to overcome intracellular infections by pathogenic bacteria.MethodsExosomes were collected from the culture supernatants of RAW 264.7 cells. Their size distribution and zeta potential were characterized by dynamic light scattering, their morphology was characterized by transmission electron microscopy, and their protein content (CD63 and Flotillin 1) was assessed by Western blotting. Linezolid was incorporated into exosomes by co-incubation at 37°C and it’s accumulation in RAW264.7 cells and release in vitro were determined by high performance liquid chromatography. The intracellular bactericidal effect was evaluated in methicillin-resistant S. aureus (MRSA)-infected macrophages in vitro and MRSA peritonitis model in vivo.ResultsWe prepared a nanoformulation of the antibiotic linezolid using exosomes harvested from mouse RAW264.7 macrophages. The exosomal formulation of linezolid was more effective against intracellular MRSA infections in vitro and in vivo than the free linezolid. Our data also showed no signs of cytotoxicity in macrophages.ConclusionExosomes provide an effective alternative for intracellular antibiotic delivery of antibiotic that is efficacious, cost-effective, and safe. This regimen can be viewed as a potential antimicrobial agent for use against intracellular infections.
Antibiotic activity can differ depending on whether the bacterial target is extracellular or intracellular. To determine extracellular and intracellular activities of sitafloxacin (STX) against Staphylococcus aureus in comparison with levofloxacin (LVX) and moxifloxacin (MXF) in vivo and in vitro, three S. aureus strains (ATCC25923, 29213, 43300) were evaluated. MIC, MBC and mutant prevention concentration (MPC) of the test quinolone for S. aureus were determined by microdilution in broth, and intracellular activity was determined in RAW264.7 cells after phagocytosis of bacteria. Cellular quinolone accumulation was determined by HPLC. The time-and concentration-kill relationships were examined in vitro (in broth and in RAW264.7 cells, respectively) and in vivo by use of a mouse peritonitis model. The results showed that the activity of STX in broth cultures, including the MIC, MBC, MPC and the time-and concentration-kill relationships, were greater for STX than those for LVX and MXF. In particular, STX exhibited the strongest activity against intramacrophage S. aureus. The intracellular effects could be ranked in the following order as the mean change in the log10 number of cfu ml À1 (log10 cfu ml À1 ) between treated and untreated mice: STX4LVX4MXF. It also showed that the dominant factor of intracellular activity in vivo was the frequency of doses. There was a poor correlation between the intracellular accumulation of the three different quinolones and the actual intracellular effect. The results of the intracellular and extracellular time-and concentration-kill relationships indicated that STX has the potential to display useful activity against extracellular and intracellular S. aureus.
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