Time-kill assays.

To study the effect of this combination on the growth kinetics of Col-R Gram-negative bacteria, a time-kill assay was conducted in randomly selected Col-R E. coli (n = 2), K. pneumoniae (n = 2), E. cloacae (n = 2), and P. aeruginosa (n = 2) strains, as previously described (41). Briefly, bacteria were incubated with thymol and colistin alone or in combinations at 1 × 10⁶ CFU/mL. Tubes containing LB alone served as the negative control. The thymol monotherapy group with a final thymol concentration of 32 to 64 mg/L, the colistin monotherapy group with a final colistin concentration of 0.5 to 2 mg/L, and the monotherapy concentration of the corresponding strain was added to the combined group. The bacterial suspensions were incubated at 37°C with moderate shaking. CFU/were enumerated on MHA agar plates at 0, 2, 4, 6, 12, and 24 h.

Synergistic activity was defined as a ≥ 2 log₁₀ decrease in CFU/mL of the combination compared to the most active monotherapy.

The Col-R E. coli (n = 2), K. pneumoniae (n = 2), E. cloacae (n = 2), and P. aeruginosa (n = 2) strains we used for biofilm formation inhibition assays were based on the results of the checkerboard assay. The biofilm formation inhibition assays were performed as mentioned above with some modifications (42). Bacteria were inoculated on plates and cultured overnight. The bacterial suspension was adjusted to 0.5 McFarland and diluted 1:100 in fresh LB broth before being added to a 96-well plate. Thymol and colistin were then added to a 96-well plate at a final concentration of 32 to 64 mg/L and 0.5 to 1 mg/L, respectively, in combination and alone. The 96-well plates were then incubated at 37°C for 24 h. After 24 h of incubation, We washed the 96-well plate twice with 200 μL of 1× PBS (Sigma-Aldrich, Milan, Italy) to remove the planktonic bacteria. The stable biofilm quality was assessed using crystal violet staining (43). Read the absorbance at 595 nm on a microplate reader (MultiskanFC) (44). The experiment was repeated three times. P value < 0.05 was considered significant.