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A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance

Chengan, Kameshwari, Hind, Charlotte, Stanley, Maria, Wand, Matthew E., Nagappa, Lakshmeesha K., Howland, Kevin, Hanson, Tanith, Martín-Escolano, Rubén, Tsaousis, Anastasios D., Bengoechea, José A., and others. (2023) A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance. npj Antimicrobials and Resistance, 1 (1). Article Number 16. ISSN 2731-8745. (doi:10.1038/s44259-023-00018-z) (KAR id:104336)


Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.

Item Type: Article
DOI/Identification number: 10.1038/s44259-023-00018-z
Projects: Grant in Aid for Open Innovation in AMR (Project 111742)
Additional information: For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: University of Kent (
Royal Society (
Biochemical Society (
Biotechnology and Biological Sciences Research Council (
Medical Research Council (
SWORD Depositor: JISC Publications Router
Depositing User: JISC Publications Router
Date Deposited: 19 Feb 2024 11:48 UTC
Last Modified: 21 Feb 2024 04:19 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

Chengan, Kameshwari.

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Stanley, Maria.

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Howland, Kevin.

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Martín-Escolano, Rubén.

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Tsaousis, Anastasios D..

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Smales, Christopher Mark.

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