96 well microtiter plate

Minimal inhibitory concentration (MIC) was determined as previously described in Fuchs et al. (2000 (link)), following the Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution MIC assay. The bacteria were incubated in a 96-well microtiter plate (Eppendorf, Germany) containing 90 μL of inoculum prepared in growth media at 10⁶ CFU/ml with 10 μL of twofold dilutions of the antibiotics. The results were evaluated after 24 h of cultivation at 37⁰C. The bacterial growth was assessed by scanning the absorbance data at 600 nm obtained by spectrophotometer (Multiscan GO, Thermo Fisher Scientific, US).

Membrane integrity was assessed using the fluorescent probes SYTO 9 and propidium iodide (PI) (LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes, USA). The test strains were grown to the mid-log phase. The obtained cultures were centrifuged at 10,000 rpm for 10 min, the supernatant was discarded and the pellet was dispersed in 10 mM HEPES buffer supplemented with 1.25 mM Ca ²⁺ to an optical density of 0.2 (OD ₆₀₀) that corresponded to 10⁸ CFU/ml. Bacterial cells in 50 μl were transferred to a 96-well microtiter plate (Eppendorf, US). SYTO 9 fluorescence kinetics was measured using Fluoroscan Ascent FL plate reader (Thermo Scientific, US) at 485 nm excitation and 535 nm of emission wavelengths as described earlier (Vasilchenko et al., 2020 (link)). The assay was performed twice in three technical replicates.
Microscopy was performed using a Zeiss Axio Imager A2 fluorescent microscope (Carl Zeiss, Germany) equipped with filter sets useful for simultaneous viewing of SYTO 9 and PI stains.

The minimum inhibitory concentration (MIC) of F105 was determined by the broth microdilution method in 96-well microtiter plates (Eppendorf) according to the EUCAST rules for antimicrobial susceptibility testing [35 (link)]. The concentrations of test compound ranged from 0.25 to 128 μg/mL. The minimal inhibitory concentration was determined as the lowest concentration of antimicrobial for which no visible bacterial growth could be observed after 24 h of incubation. Then, to determine a minimum bactericidal concentration, a culture liquid from wells without visible growth was diluted a thousand-fold using fresh medium and incubated for 24 h growth at 35 °C. MBC was assumed as a concentration where no viable cells were observed [36 (link)]. The development of bacterial resistance was tested using the serial passages approach as described in [37 (link)] with modifications as described in [38 (link)].

For miniaturization, samples and reactants were combined to a total volume of 100 µL in 96-well microtiter plates (Eppendorf, Hamburg, Germany) with 1–0.1 mg/mL antibody concentrations (Figure 1 and Figure 2) and incubated at RT. To avoid extensive evaporation within the Hamilton robotic devices, 2 mL deep-well plates were evaluated after sealing with lids or X-pierce seals, but process realization on a Tecan device in a laboratory with low ventilation resulted in a neglectable volume reduction. Matrix pipetting was carried out applying a customized protocol on a Tecan device as well as a customized solution for bulk database upload and tracking of the combinations.

The MIC of antimicrobials was determined by the broth microdilution method in 96-well microtiter plates (Eppendorf) according to the EUCAST recommendations [85 (link)]. Briefly, the bacterial suspension containing 10⁸ CFUs mL⁻¹ was subsequently diluted 1:300 with BM broth in microwell plates to obtain a 10⁶ cells mL⁻¹ suspension and incubated at 37 °C for 24 h. Antimicrobials were added in the concentration range of 0.25–512 mg L⁻¹. The MIC was determined as the lowest concentration of antimicrobial providing no visible bacterial growth after 24 h of incubation. To determine the MBC, 10 µL of culture liquid from wells without visible growth was seeded into 1 mL of fresh broth and incubated at 37 °C for 24 h. The antibiotic concentration corresponding to the absence of growth was considered as MBC.

The checkerboard assay was performed similarly to the MIC testing in 96-well microtiter plates (Eppendorf). Each plate contained serial dilutions of F105 in 0.1% pluoronic acid F-127 and different antibiotics in a checkerboard fashion as described previously (Eliopoulos and Moellering, 1996 ). Briefly, the final concentrations of both compounds ranged from 1/16× to 4× MIC for F105 and from 1/256× to 4× MIC for the antibiotics. In total, 11 dilution steps of antibiotics and 7 dilution steps of F105 were analyzed. The microwell plates were incubated at 35°C for 24 h. Each test was performed in triplicate and included a growth control with neither antibiotic nor F105 addition. The fractional inhibitory concentration index (FICI) for each double combination was calculated as
The FICIs were counted from the concentrations in the first non-turbid well found in each row and column along the turbidity/non-turbidity interface and the lowest FICI value was used to characterize the synergy. For the FICI interpretation we refer to den Hollander et al. (1998) (link) and Odds (2003) (link): FICI < 0.5 corresponds to synergy, 0.5 < FICI < 4 corresponds to either additive effects or indifference, while FICI > 4 corresponds to antagonism.