Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus

Hamidreza Ardalani, Syariful Anam, Kresten J.K. Kromphardt, Dan Staerk, Kenneth T. Kongstad*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC50 values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD600 measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.
Original languageEnglish
Article number1550
JournalMolecules
Volume26
Issue number6
Number of pages12
ISSN1420-3049
DOIs
Publication statusPublished - 2021

Keywords

  • Dye-free microplate assay
  • Antibacterial
  • High-resolution antibacterial profiling
  • Natural products
  • Staphylococcus aureus

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