Mh broth

Clinical isolates of P. aeruginosa and S. aureus were obtained from the General University Hospital in Prague (Prague, Czech Republic). P. aeruginosa was resistant to ciprofloxacin, oxacillin, ticarcillin, colistin, gentamicin, and imipenem. S. aureus was resistant to ciprofloxacin, gentamicin, erythromycin, chloramphenicol, clindamycin, oxacillin, cefotaxime, and vancomycin. Bacteria were cultivated in Mueller–Hinton broth (MH broth, Merck). The susceptibility of the bacteria was evaluated by the minimum inhibitory concentrations (MIC). The MIC of derivatives in the presence of the antibiotic cut-off concentration was determined according to ISO 20776-1: 2020. The antibiotic cut-off concentration was chosen according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, Clinical breakpoints—bacteria (ver. 11.0), 1 January 2021). The 40 µM concentration of the derivatives was chosen as the highest concentration for the tests. Cell viability was determined by measuring absorbance (A590 nm) after 24 h incubation at 37 °C, 150 rpm.

The potential of cuminaldehyde to interact with ciprofloxacin was assessed on biofilm formation. Biofilm mass formation was quantified (as described above in the section Effects on biofilm formation). For this, 10 μl of each bacterial suspension were added per well into a 96-well cell culture plate containing cuminaldehyde (MIC/8) and ciprofloxacin (MIC/2) in 190 μl of MH broth (Merck Millipore). Vehicle (2% DMSO in saline)-treated bacteria and broth without bacteria were used as negative controls.

Cuminaldehyde (98% purity; Sigma-Aldrich) antimicrobial effects were investigated by the microdilution method [40 (link)]. For this, the bacteria previously cultured on MH agar were suspended in saline (~ 1.5 × 10⁸ colony forming units (CFU)/milliliter (ml)). For determining the MICs, 10 μl of the bacterial suspension were incubated in MH broth (190 μl/well; Merck Millipore) containing different concentrations of Cuminaldehyde (0.0234–24 mg/ml). Serial dilutions of ciprofloxacin (0.0009–200 μg/ml) were used as positive controls. Sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich; 2% in saline) was used to increase Cuminaldehyde solubility and as negative control. Samples were incubated for 24 h at 37°C, and the MICs (the lowest concentration at which no bacterial growth is observed) were evaluated. For this, the absorbances were read at 600 nm.
In parallel, the effects of the sub-inhibitory concentrations (MIC/2-MIC/8) of Cuminaldehyde and ciprofloxacin on bacterial viability were assessed and calculated by addition of the PrestoBlue® reagent (1:10; Life Technologies), according to the manufacturer’s instructions. The absorbance was read at 570 nm and 600 nm and cell viability expressed as Δ absorbance in nm.

Biofilm formation was quantified as previously described [38 (link)]. For this, 10 μl of each bacterial suspension (prepared as described above) were added per well into a 96-well cell culture plate containing sub-inhibitory concentrations of cuminaldehyde (MIC/2-MIC/8) or ciprofloxacin (MIC/2-MIC/8) in 190 μl of MH broth (Merck Millipore). Vehicle (2% DMSO in saline)-treated bacteria and broth without bacteria were used as negative controls. Samples were incubated at 37°C for 24 h, and then, the wells were washed three times with phosphate-buffered saline (PBS; Sigma-Aldrich). Samples were then, fixed with 200 μl of methanol (100%; Merck Millipore) for 15 min. Following, the methanol was removed and the plate wells allowed to air dry. Biofilm was stained with 5% crystal violet (Sigma-Aldrich) for 10 min at room temperature, and immediately solubilised with methanol (200 μl). The absorbance was read at 570 nm. Biofilm mass results are expressed as absorbance in nm. For analysis of biofilm viability, the PrestoBlue® reagent (1:10; Life Technologies) was used according to the manufacturer’s instructions. The absorbance was read at 570 nm and 600 nm and the results are expressed as Δ absorbance in nm.

Killing curve assays for G5-PDK were performed on representative isolates of P. aeruginosa, P. putida, and P. fluorescens as previously reported [62 (link)]. Experiments were performed over 24 h at G5-PDK concentrations of four times the MIC for all strains.
A mid logarithmic phase culture was diluted in Mueller–Hinton (MH) broth (Merck, Darmstadt, Germany) (10 mL) containing 4 × MIC of the dendrimer to give a final inoculum of 1.0 × 10⁵ CFU/mL. The same inoculum was added to cation-supplemented Mueller–Hinton broth (CSMHB) (Merck, Darmstadt, Germany), as a growth control. Tubes were incubated at 37 °C with constant shaking for 24 h. Samples of 0.20 mL from each tube were removed at 0, 2, 4, 6, and 24 h, diluted appropriately with a 0.9% sodium chloride solution to avoid carryover of G5-PDK being tested, plated onto MH plates, and incubated for 24 h at 37 °C. Growth controls were run in parallel. The percentage of surviving bacterial cells was determined for each sampling time by comparing colony counts with those of standard dilutions of the growth control. Results have been expressed as log10 of viable cell numbers (CFU/mL) of surviving bacterial cells over a 24 h period. Bactericidal effect was defined as a 3 log10 decrease of CFU/mL (99.9% killing) of the initial inoculum. All time-kill curve experiments were performed in triplicate.

An aliquot (200 μl) of each bacterial suspension was added to 2 ml of MH broth (Merck Millipore) containing ciprofloxacin (MIC/2) or cuminaldehyde (MIC/2-MIC/4) alone, or in combination. Vehicle (2% DMSO in saline)-treated bacteria were used as negative control. Cell growth was monitored by plating 10 μl of 10-fold-diluted suspensions from each sample at different time-points (0.15–8 h) in MH agar (Merck Millipore) plates. After 8 h of incubation at 37°C, the colonies were counted and then, the Log₁₀ CFU/ml was calculated. The bactericidal combinatory effects were assessed by variation on Log₁₀ CFU/ml (ΔLC). Synergy was defined as a decrease of ≥ 2 log₁₀ CFU/ml and antagonism as an increase of #x2265; 2 log₁₀ CFU/ml. If ΔLC was between 1 and 2 log₁₀ CFU/ml, the effects were recorded as additive, and as indifferent if ΔLC = ± 1 log₁₀ CFU/ml [40 (link)].