4.6 Article

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

Journal

MOLECULES
Volume 26, Issue 6, Pages -

Publisher

MDPI
DOI: 10.3390/molecules26061550

Keywords

dye-free microplate assay; antibacterial; high-resolution antibacterial profiling; natural products; Staphylococcus aureus

Funding

  1. INEF (Iran's National Elite Foundation) [1286782590]
  2. Indonesia Endowment Fund for Education (LPDP) [PRJ-4250/LPDP.3/2016]
  3. Danish National Research Foundation [DNRF137]
  4. Carlsberg Foundation

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A novel dye-free antimicrobial assay in 96-well microplate format was developed and validated for expedited screening of crude extracts, enabling determination of IC50 values and high-resolution inhibition profiling.
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.

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