HDP susceptibility.

The susceptibility of distinct graS mutants to individual HDPs was assessed using an established radial diffusion method (10). In brief, logarithmic-phase cells adjusted to 1 × 10⁶ CFU/ml were seeded into 10 ml of 1% agarose buffered to pH 7.5 using piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES, 10 mM). After 3 h of incubation, plates were overlaid with nutrient medium (Trypticase soy) containing 1% agarose and incubated for 24 h at 37°C. Zones of inhibition (ZOI) were measured to the nearest millimeter in diameter. A minimum of two independent experiments were conducted on separate days for statistical analysis.