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bioRxiv · 10.64898/2026.06.15.732343

Does media matter? Growth environment influence antimicrobial tolerance and expression of virulence and transmembrane ion transport-associated genes in MRSA

Orababa, O. Q.·Ayomikun, K.·Uzairue, L. I.
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Abstract

Clinically relevant pathogens are often tested for antimicrobial susceptibility using standard laboratory media that poorly reflect the in vivo environments in which they cause infections, leading to poor clinical outcomes. In this study, we aim to understand the impact of media on the global transcriptome, biofilm formation, and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus USA300 when cultivated in a physiologically relevant wound medium, such as simulated wound fluid (SWF), compared to cation-adjusted Mueller-Hinton broth (caMHB), a general-purpose medium. The transcriptomics analysis showed upregulation of 865 genes and downregulation of 792 in SWF compared to caMHB. Upregulated genes in SWF are associated with virulence, such as genes coding for fibronectin-binding proteins (fnaAB), serine proteases (splABCDE), as well as genes involved in antimicrobial resistance, such as multidrug efflux pump genes (norB, norC). Conversely, genes associated with transmembrane ion transport, including phosphate transport (pstSCAB, phoU) and potassium intake (kdpABCF), were significantly downregulated in SWF, as further confirmed by increased membrane disruption upon exposure to a membrane-potential-sensitive dye (DiSC3). Biofilm assay showed reduced surface attached biofilm but increased cell-to-cell attachement in SWF compared to caMHB. Antimicrobial susceptibility testing revealed a 2- to 4-fold increase in tolerance to clinically relevant antibiotics in SWF compared to caMHB. Overall, our findings revealed that media affects gene expression, membrane physiology, virulence, and antibiotic tolerance in MRSA, underscoring the need to use physiologically relevant media in routine antimicrobial susceptibility testing and the drug development pipelines.

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Orababa, O. Q., Ayomikun, K., Uzairue, L. I.. 2026-06-15. Does media matter? Growth environment influence antimicrobial tolerance and expression of virulence and transmembrane ion transport-associated genes in MRSA. https://doi.org/10.64898/2026.06.15.732343

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