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Description of Methodology for Testing the Synergistic and Additive Effects of Antibiotics in Vitro

Paweł Z. Kmiecikowski

Kmiecikowski, Paweł Z.

,

Gabriel, Aniela

,

y

29 ene 2025

Description of Methodology for Testing the Synergistic and Additive Effects of Antibiotics in Vitro's Cover Image

Advancements of Microbiology

Volumen 63 (2024): Edición 4 (Diciembre 2024)

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Publicado en línea: 29 ene 2025

Páginas: 199 - 222

Recibido: 01 ago 2024

Aceptado: 01 nov 2024

DOI: https://doi.org/10.2478/am-2024-0017

Palabras clave
antibiotic resistance, antibiotic synergism, checkerboard test, time-kill test

© 2024 Paweł Z. Kmiecikowski et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Broth microdilution method – preparation of a concentration pattern.

A– add Mueller-Hinton broth.
B– prepare a series of microdilutions of the first antibiotic.
C– prepare a series of microdilutions of the second antibiotic.
D– add antibiotics together.
E– re-add of Mueller-Hinton broth. — Broth microdilution method – preparation of a concentration pattern. A– add Mueller-Hinton broth. B– prepare a series of microdilutions of the first antibiotic. C– prepare a series of microdilutions of the second antibiotic. D– add antibiotics together. E– re-add of Mueller-Hinton broth.

The “time-kill” method – a series of dilutions.

A– prepare six 96-well plates (t0, t1, t2, t4, t6, t24) with 0.9% NaCl
B– add the bacterial suspension to test tubes 1st–4th.
C– transfer the suspension portion to the appropriate plate at predetermined times.
D– obtain a dilution of 1:10.
E– transfer a portion of the suspension to the plates using the “drop plate” method. — The “time-kill” method – a series of dilutions. A– prepare six 96-well plates (t0, t1, t2, t4, t6, t24) with 0.9% NaCl B– add the bacterial suspension to test tubes 1st–4th. C– transfer the suspension portion to the appropriate plate at predetermined times. D– obtain a dilution of 1:10. E– transfer a portion of the suspension to the plates using the “drop plate” method.

CombiANT method – the insert and experimental protocol.

A– the insert design: antibiotic reservoirs (a-c), interaction imaging area (d).
B– the assay protocol: „The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4–8°C. To activate an insert, add a second layer of 25 mL agar to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition are established within 16–24 hours.” (Fatsis-Kavalopoulos et al. 2020) — CombiANT method – the insert and experimental protocol. A– the insert design: antibiotic reservoirs (a-c), interaction imaging area (d). B– the assay protocol: „The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4–8°C. To activate an insert, add a second layer of 25 mL agar to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition are established within 16–24 hours.” (Fatsis-Kavalopoulos et al. 2020)

Assessment of the synergistic effect of antibiotics by the method of constant coefficients.

Assessment of the synergistic effect of antibiotics by cross-method.

Evaluation of the synergistic effect of antibiotics by the MIC:MIC ratio evaluation.

Idiomas:: Inglés, Polonais

Calendario de la edición:: 4 veces al año
Temas de la revista:: Ciencias de la vida, Microbiología y virología

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