4.3. Antimicrobial Susceptibility Testing of Complexes on Pseudomonas aeruginosa Strains

All metal complexes and controls were tested for their activity on the clinical isolates (CF1–CF3) and laboratory strains (ATCC 27853 and PAO1) of P. aeruginosa, using the standard broth micro–dilution method to establish their minimum inhibitory concentration (MIC). The test complexes were two-fold serially diluted and mixed with equal volumes (100 µL) of diluted bacteria in 96-well plates (Cruinn), thus making a final concentration range of the complexes tested, and included the antibiotic control gentamicin, between 0.5 and 256 µg/mL. The MIC measurements were recorded on the basis of turbidity according to EUCAST guidelines [56], after 16–18 h of incubation at 37 °C with agitation.

The activity of all complexes and controls on biofilm formation of all P. aeruginosa strains were tested by measuring cellular viability and the total biomass in the cell culture wells. For both assays, 100 µL of the bacterial culture (made up in TSB medium) was distributed into each well of flat-bottomed 96–well polystyrene microtitre plates (Cruinn) and incubated for 48 h at 37 °C in the presence of all test compounds (100 µL), administered at a concentration range of 0.5–256 µg/mL. The supernatants from each well were removed, and the wells were then washed three times with phosphate buffered saline (PBS). Viable cells in the biofilm were assessed by a fluorometric assay that measures the metabolic capacity of cells [57]. TSB (250 µL) containing 5% (v/v) resazurin (Sigma-Aldrich) was added to the plates and incubated for 20 min at 37 °C. Fluorescence was then measured at λexcitation = 560 nm and λemission = 590 nm, with a Varioskan LUX (Thermo Scientific, Waltham, MA, USA) microplate reader. Then, the resazurin stain was removed and replaced by 250 µL of 0.1% (w/v) crystal violet solution on the same plate [58] to evaluate biofilm biomass. After 20 min, the crystal violet stain was removed, and the wells werewashed three times. The wells were dried and 250 µL of 30% acetic acid (Sigma–Aldrich) was added to release the bound crystal violet. Absorbance was measured on a Multiskan GO (Thermo Scientific, Waltham, MA, USA) microplate reader at 590 nm.

In addition to testing the effects of the metal-tdda-phen complexes on biofilm formation (pre-treatment) of the P. aeruginosa strains, their ability to weaken an established biofilm was also assessed with the post-treatment of mature biofilms. Moreover, 100 µL of the bacteria culture (in TSB) was added to each well of flat bottomed 96–well polystyrene microtitre plates (Cruinn) and incubated for 48 h at 37 °C, to allow for biofilm formation. After 48 h incubation, the test complexes and control were added over a range of concentrations (0.5–256 µg/mL) to mature biofilms and incubated for an additional 24 h. To the untreated control, 100 µL of fresh media was added to the wells. Cellular viability and biofilm biomass were measured using resazurin staining and crystal violet staining, as previously described in Section 4.3.2.

The anti-biofilm activity of individual metal-tdda-phen complexes alone and in combination with gentamicin were evaluated against the P. aeruginosa strains using the broth micro-dilution checkerboard technique [59]. Mature biofilms were prepared as previously described in Section 4.3.3. The preformed biofilms in the wells of separate 96-well microtitre plates were rinsed with PBS and 100 µL of the metal-tdda-phen complexes and gentamicin (two-fold serial dilutions); each was added to the wells containing the biofilms. Following incubation for 24 h at 37 °C, the biofilm biomass and cell viability were measured as previously described.

The fractional inhibitory concentration (FIC) index (FIC_I) for combinations of testing agents was calculated for selected P. aeruginosa strains PAO1 and CF3, according to the equation: FIC_I = FIC_{(metal-tdda-phen complex)} + FIC_{(gentamicin).} Where, FIC_{(metal-tdda-phen complex)} = (MBEC* of metal-tdda-phen complex in combination with gentamicin)/(MBEC* of metal-tdda-phen complex alone) and FIC_(gentamicin) = (MBEC* of gentamicin in combination with metal-tdda-phen complex)/(MBEC* of gentamicin alone). These strains were selected due to their contrasting activity profiles observed in the previous tests, and because they formed the strongest biofilms (evidenced by crystal violet staining). The following criteria were used to interpret the FIC_I: ≤0.5 synergy; 0.5–4.0 indifferent; >4.0 antagonism. *The minimum biofilm eradication concentration (MBEC) was defined as the minimal concentration of the compound required to eradicate the biofilm.