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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

Ardalani, Hamidreza LU ; Anam, Syariful ; Kromphardt, Kresten J. K. ; Staerk, Dan and Kongstad, Kenneth T. (2021) In Molecules (Basel, Switzerland) 26(6).
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... (More)

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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author
; ; ; and
publishing date
type
Contribution to journal
publication status
published
subject
in
Molecules (Basel, Switzerland)
volume
26
issue
6
article number
1550
pages
12 pages
publisher
MDPI AG
external identifiers
  • scopus:85103920102
  • pmid:33799878
ISSN
1420-3049
DOI
10.3390/molecules26061550
language
English
LU publication?
no
id
c92b5eb2-ab87-48a3-bcc5-af91d79588e2
date added to LUP
2021-05-13 14:08:24
date last changed
2024-09-07 19:15:43
@article{c92b5eb2-ab87-48a3-bcc5-af91d79588e2,
  abstract     = {{<p>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 IC<sub>50</sub> 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 OD<sub>600</sub> measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against <i>Staphylococcus aureus</i>, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution <i>S. aureus </i>growth inhibition profiling was evaluated on a crude extract of an inactive <i>Alternaria </i>sp., spiked with ciprofloxacin.</p>}},
  author       = {{Ardalani, Hamidreza and Anam, Syariful and Kromphardt, Kresten J. K. and Staerk, Dan and Kongstad, Kenneth T.}},
  issn         = {{1420-3049}},
  language     = {{eng}},
  month        = {{03}},
  number       = {{6}},
  publisher    = {{MDPI AG}},
  series       = {{Molecules (Basel, Switzerland)}},
  title        = {{Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts : Validation and Proof-of-Concept Study with <i>Staphylococcus aureus</i>}},
  url          = {{http://dx.doi.org/10.3390/molecules26061550}},
  doi          = {{10.3390/molecules26061550}},
  volume       = {{26}},
  year         = {{2021}},
}