Galleria mellonella model of infection.

Galleria mellonella larvae were obtained from The Worm Lady. All larvae were used the same day. Bacteria were routinely cultured M9 medium with 0.4% glucose and 200 μM MgSO₄ with the appropriate antibiotics at 37°C for 16 to 18 h at 200 rpm and then subcultured in the same culture media. For infections, 1 ml of each culture was harvested by centrifugation and washed three times in 1× phosphate-buffered saline (PBS) (pH 7.4). The final, washed bacterial pellet was resuspended in 1 ml 1× PBS (pH 7.4) and adjusted to the designated number of CFU, which was confirmed by plate counting for each sample. Larvae were infected with one selected strain by injection into the hemolymph via the hindmost right proleg. For all experiments, larvae were incubated at 37°C in standard petri dishes for up to 72 h. All experiments were conducted at least in triplicate. Control larvae were injected with PBS (pH 7.4) to measure any lethal effects of the injection process. All organisms were monitored for survival for up to 72 h. Larvae were considered dead when they did not show any response to touch. Ten worms each were injected with the same sample, and each treatment result was based on at least three biological replicates.