Isolation of Extracellular Outer Membrane Vesicles (OMVs) from Escherichia coli Using EVscore47 Beads.

Outer membrane vesicles (OMVs) are attractive for biomedical applications based on their intrinsic properties in relation to bacteria and vesicles. However, their widespread use is hampered by low yields and purities. In this study, EVscore47 multifunctional chromatography microspheres were synthesized and used to efficiently isolate functional OMVs from Escherichia coli. Through this technology, OMV loss can be kept to a minimum, and OMVs can be harvested using EVscore47 at 11-fold higher yields and ~13-fold higher purity than those achieved by means of ultracentrifugation. Based on the results presented here, we propose a novel EVscore47-based isolation of OMVs that is fast and scalable.

Eradicating intracellular MRSA via targeted delivery of lysostaphin and vancomycin with mannose-modified exosomes.

Intracellular methicillin-resistant Staphylococcus aureus (MRSA) is extremely difficult to remove by common antibiotics, leading to infection recurrence and resistance. Herein we report a novel exosome-based antibiotic delivery platform for eradicating intracellular MRSA, where mannosylated exosome (MExos) is employed as the drug carrier and preferentially taken up by macrophages, delivering lysostaphin (MExoL) and vancomycin (MExoV) to intracellular pathogens. Combination of MExoL and MExoV eradicated intracellular quiescent MRSA. Moreover, MExos rapidly accumulated in mouse liver and spleen, the target organs of intracellular MRSA, after intravenous (IV) administration. Thus, the MExos antibiotic delivery platform is a promising strategy for combating intracellular infection.

Exosome-encapsulated antibiotic against intracellular infections of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus 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. PURPOSE: The objective of this study was to develop a nanovesicle using exosomes loaded with linezolid to overcome intracellular infections by pathogenic bacteria. METHODS: Exosomes 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. RESULTS: We 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. CONCLUSION: Exosomes 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.

Activity of sitafloxacin against extracellular and intracellular Staphylococcus aureus in vitro and in vivo: comparison with levofloxacin and moxifloxacin.

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(-1) (log10 cfu ml(-1)) between treated and untreated mice: STX>LVX>MXF. 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.