Antibiotic carry over is a confounding factor for cell-based antimicrobial research applications

Area of Science:

• Microbiology
• Cell Biology
• Biotechnology

Background:

• Chronic wounds frequently harbor Staphylococcus aureus, necessitating novel antimicrobial and wound healing strategies.
• Extracellular vesicles (EVs) are a promising area for therapeutic development, but their isolation relies on conditioned medium (CM).
• Standardization of EV isolation techniques is increasing, yet upstream tissue culture methods for CM collection are less scrutinized.

Purpose of the Study:

• To investigate the antimicrobial properties of conditioned medium (CM) used for extracellular vesicle (EV) enrichment.
• To determine the source of antimicrobial activity observed in CM.
• To assess the impact of antibiotic carry-over on CM's antimicrobial potential.

Main Methods:

• Culturing cells to collect conditioned medium (CM).
• Testing CM for bacteriostatic effects against penicillin-sensitive and penicillin-resistant Staphylococcus aureus strains.
• Analyzing CM composition to identify antimicrobial factors, including residual antibiotics.
• Evaluating the effect of pre-washing cells and reduced antibiotic concentrations on CM antimicrobial activity.

Main Results:

• CM demonstrated bacteriostatic effects against penicillin-sensitive S. aureus but not penicillin-resistant strains.
• The observed antimicrobial activity was attributed to residual penicillin retained and released from tissue culture plastic, not cell-secreted factors.
• Pre-washing cells and using lower antibiotic concentrations in basal medium mitigated the antibiotic carry-over effect.

Conclusions:

• Residual antibiotics in conditioned medium can be mistaken for inherent antimicrobial properties of cell-secreted factors or extracellular vesicles.
• Controlling antibiotic usage in tissue culture is vital to prevent misleading results in antimicrobial and therapeutic evaluations.
• Accurate determination of CM's antimicrobial mechanisms is essential for validating future cell-based therapeutic applications, including those involving EVs.