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

Elfar, Miran Yousri, Brown, Helen Louise, Clayton, Aled ORCID: https://orcid.org/0000-0002-3087-9226 and Stephens, Phil ORCID: https://orcid.org/0000-0002-0840-4996 2025. Antibiotic carry over is a confounding factor for cell-based antimicrobial research applications. Scientific Reports 15 (1) , 28310. 10.1038/s41598-025-14186-7

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Abstract

Chronic wounds often host pathogens like Staphylococcus aureus, prompting interest in developing new antimicrobial and wound healing strategies, including the utilisation of extracellular vesicles (EVs). Whilst there has been a recent emphasis within the EV community to ensure standardization of characterization and isolation techniques, there has been less focus placed on the upstream tissue culture methodologies used for collection of vesicle-containing conditioned medium (CM). Hence, this study investigated the antimicrobial properties of the CM used for EV enrichment. CM exhibited bacteriostatic effects against penicillin-sensitive S. aureus NCTC 6571, but not penicillin-resistant S. aureus 1061 A. Further analysis revealed that the antimicrobial activity was due to residual antibiotics rather than cell-secreted factors, specifically the retention and release of penicillin to tissue culture plastic surfaces. Pre-washing cells and minimizing antibiotic concentrations in basal medium reduced this carry-over effect. These findings emphasize the importance of controlling antibiotic use in tissue culture to avoid misleading conclusions about the antimicrobial potential of CM or EVs. Researchers should carefully consider medium selection and supplementation during method development as accurately determining the antimicrobial mechanisms of any CM is essential for validating future cell-based therapeutic applications.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Schools > Medicine
Schools > Biosciences
Schools > Dentistry
Publisher: Nature Research
ISSN: 2045-2322
Funders: Dunhil Medical Trust
Date of First Compliant Deposit: 5 August 2025
Date of Acceptance: 29 July 2025
Last Modified: 07 Aug 2025 11:02
URI: https://orca.cardiff.ac.uk/id/eprint/180278

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