Analysis of virulence factors production

S. aureus was cultured overnight at 37 °C in the presence and absence of UM-C162. Bacterial cells harvested from the overnight culture were prepared in 1X PBS. Rabbit blood plasma was separated from red blood cells by centrifugation at 900 × g for 5 minutes. Twenty µL of rabbit plasma was placed on the surface of a glass slide, followed by inoculation of 10 µL of S. aureus suspension. The mixture was mixed evenly for 10 seconds and macroscopic clumping/agglutination of bacterial cells was scored from 0 to 3+ (Fig. ^S5).

Lysis of red blood cells was assessed as previously described⁹ with minor modifications. Briefly, different concentrations of UM-C162 were added to a S. aureus culture with standardized inoculum size, followed by incubation at 37 °C with agitation for 16–18 hours. Rabbit blood was obtained from the Animal House Facility, Universiti Kebangsaan Malaysia. Red blood cells were separated from plasma by centrifugation at 900 × g for 5 minutes at 4 °C, washed 3 times with 1XPBS and diluted in PBS (330 µL of red blood cells in 10 mL of 1X PBS). To measure the haemolytic activity, 200 µL of S. aureus culture (OD₆₀₀ = 0.3) grown in the presence of compound was added into 10 mL of diluted red blood cells. The negative control (S. aureus supplemented with DMSO only) was run in parallel. The mixture was incubated shaking for 4 hours. Supernatants were collected by centrifugation at 16,600 × g for 10 minutes and the OD readings were measured at 543 nm.

The ability of UM-C162-treated and untreated S. aureus to produce protease was determined by spotting the bacterial culture on 3% skim milk agar (Oxoid, UK). S. aureus was grown overnight at 37 °C in the presence and absence of compound and adjusted to the standard inoculum size. Ten µL of standardized S. aureus inoculum was spotted onto skim milk agar, allowed to dry under sterile conditions and incubated at 37 °C for 24 hours. After 24 hours incubation, plates were observed for the formation of a clear zone/halo around the bacteria.