Introduction
Staphylococcus aureus is a Gram-positive spherically shaped bacterium. It is a widespread pathogen and is known for its ability to become resistant to a wide range of antibiotics1. The urgent need for the development of novel antibiotics to combat the resistance, brings scientists to search for new potential candidates in previously untouched fields. Venoms include antimicrobial peptides (AMPs) which represent attractive candidates for the development of novel antibiotic therapeutics2.
In this approach, venom fractions from Naja nigricollis, Agkistrodon controtrix pictigaster, Poecilotheria and Pandinus cavimanus were screened for a potential cytotoxic effect against Staphylococcus aureus in a high-throughput approach based on a resazurin assay.
Assay principle
Resazurin salt is a cell viability reagent that allows for both a fluorescent and colorimetric readout. Resazurin is an oxidation-reduction (REDOX) indicator that can be readily used in assays to assess cellular viability3. Active bacterial metabolism catalyzes resazurin irreversibly to the pink, fluorescent resorufin (Fig. 1).
The measurement of metabolically active bacteria via the resazurin assay can be used indirectly to screen for novel antibiotics in venom fractions.
Materials & methods
- 96-well plate clear, flat bottom (Nunc)
- S. aureus (DSM No.: 102262)
- Venom fractions in T-VDAmicrobe array (Venomtech Ltd.)
- 96-well plates, black, clear bottom, Sigma, for PI uptake
- FLUOstar® plate reader, BMG LABTECH
Experimental Procedure
Assay optimization and validation
To determine the optimal concentration of resazurin and bacterial cells, S. aureus was seeded with 1.25 x 106 – 1 x 107 cells/well. The resazurin stock was diluted with dH2O to obtain a final concentration in the wells of between 0.125 mM and 4 mM, respectively and the resazurin turnover was read over 120 min.
To validate the assay for high-throughput-screening, Z’ was calculated. The assay was based on 2.5 x 106 cells of S. aureus in LB broth in each well of a 96-well plate. Positive antibiotic controls contained 158 µg/mL streptomycin while negative control contained LB media only. After 1 h of incubation at 37°C, resazurin was added to a final concentration of 0.125 mM and the resazurin turnover was read over 120 min.
High-throughput-Screening of novel antibiotics in venoms Venom fractions were hydrated with 90 µL of dH2O. Each venom fraction was tested in triplicates. Therefore, 30 µL of each fraction (2 µg/treatment) was added to wells containing 2.5 x 106 bacterial cells. Plates were incubated for 2 h at 37°C. Afterwards, resazurin was added and fluorescence readings were taken every 30 min in the FLUOstar Omega to identify potential novel antibiotics. Therefore, the following settings were used:
Instrument settings
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Fluorescence intensity, plate mode kinetic
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||
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Optic settings
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Filters
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Ex 544-15
Em 595-10 |
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General settings
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Number of flashes
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50
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Gain
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800 |
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Kinetic settings
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Number of cycles
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4 |
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Cycle time
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1800 s |
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Incubation
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37°C
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Results & Discussion
Optimal concentration of bacteria and resazurin.
The optimal conditions to test for novel antibiotics were found in the combination of 0.125 mM resazurin and 2.5 x 106 bacterial cells/well as highlighted with the green frame in figure 2.
Validation of Assay for HTS
The conditions previously classified as ideal with 0.125 mM resazurin and 2.5 x 106 bacterial cells/well were used in the following to validate the assay for use in high-throughput screening of novel antibiotics from venom fractions. Z’ was calculated based on the positive control (158 µg/mL streptomycin for significant inhibition of bacterial metabolism) and the negative control (without cytotoxic reagent, Fig. 3). With a Z’ of 0.7 the assay was classified to be excellently suited for a HTS for novel antibiotics in venom fractions.
Identification of antimicrobial venoms with HTS Fluorescence readings of the resazurin turnover of S. aureus after the treatment with venom fractions from the T-VDA microbe array revealed 2 potential novel antibiotic candidates: #11 and #24 from Pandinus cavimanus (Fig. 4).
Conclusion
The optimized resazurin assay was validated as excellent option for the high-throughput screen of venoms for potential novel antibiotics to fight S. aureus resistance based on the evaluation of Z’. The high-throughput screen of antimicrobial activity using the FLUOstar Omega, identified two hits in Pandinus cavimanus. The promising candidates must subsequently be confirmed as novel antibiotics by investigating dose and response as well as determining the minimum inhibitory concentration.
References
- https://onlinelibrary.wiley.com/doi/full/10.1111/j.1469-0691.2006.01343.x
- https://pubmed.ncbi.nlm.nih.gov/12927960/
- https://link.springer.com/protocol/10.1007/978-1-4939-6960-9_1