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bioRxiv · 10.64898/2026.04.23.720151

A liquid handling platform for standardised quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes

Bergum, M.·Martin, B.·Sutton, J. M.·Moore, S. J.
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Abstract

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterising emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression (CFE) systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimising the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, while arr-3 increased the IC50 of rifampicin by approximately 20-fold compared to controls. Future work could extend this platform to characterise novel ARGs identified through genomic surveillance or rapidly profile activity of new or derivative antibiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/720151v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@50bbdcorg.highwire.dtl.DTLVardef@5d98e4org.highwire.dtl.DTLVardef@e0a3fforg.highwire.dtl.DTLVardef@2ef12a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Bergum, M., Martin, B., Sutton, J. M., Moore, S. J.. 2026-04-23. A liquid handling platform for standardised quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes. https://doi.org/10.64898/2026.04.23.720151

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