Omnilog instrument

The phenotypic analyses were performed using BioLog phenotypic microarray microplate PM1–2 (carbon sources), PM3 (nitrogen sources), and PM4 (phosphorus and sulphur sources), according to the manufacturer’s instruction (Biolog, Hayward, California, USA). In brief, B. pseudomallei WT and bipC mutant strains were cultured from glycerol stocks and passaged three times before the phenotypic microarray experiment. A single colony of these bacterial was picked and inoculated into the Biolog inoculating fluid (IF-0). Hundred microliters of the cells were transferred into each well and incubated in an OmniLog instrument (Biolog, Inc., Hayward, California, USA) at 37 °C for 48 hours. The substrate utilisation was measured via the reduction of a tetrazolium dye forming a purple formazan and is indicative of active cellular respiration, while the negative control wells remained colourless13 (link). The cellular respiration of wild type and mutant were recorded and the data were analysed using the OmniLog software. The option of A1 zero was selected during data processing to deduct the background signal with reference to the A1 negative control well in each plate. Three biological replicates were conducted for each strain.

Bacteria growth kinetics of the cells exposed to ARGIRIUM-SUNCs were assessed by the broth microdilution method. The experiment was done in 96-well polystyrene microplates. Also in this case, each strain was tested in 5 replicates and the experiment was repeated twice. The standardized inoculum (1 × 10⁷ CFU/mL) was exposed to a different ARGIRIUM-SUNCs concentration (0, 0.625, 1.25, 2.5, 3.0, 4.0, 5 and 10 ppm) and incubated at 37 °C. The growth increase was measured by using of OmniLog instrument (Biolog INC., Hayward, USA). The values were expressed as mean of five replicated growth curves.
Data obtained were also analyzed over time according to the Gompertz equation modified by Ref.^{25 (link)}: $$\text{Y}\left(\text{t}\right)=\text{b1}\times \text{exp}\left(-\text{exp}\left(\left(\text{b2}\times 2.7182/\text{b1}\right)\times \left(\text{b3}-\text{t}\right)+1\right)\right),$$ where Y is the number of CFU at each time of the experiment, b1 is the maximum CFU achieved during stationary phase, b2 is the maximum growth rate (1/h) and, b3 is the lag phase (hours) and t is the time. The data collected were the means of five independent repetitions.

Mitochondrial function assays were performed using MitoPlate S-1(Biolog Inc., Hayward, CA). The substrates on MitoPlate S-1 were first dissolved by incubating the plate with 30 µl of Assay Mix, consisting of 2x BMAS, 6x Redox Dye MC and saponin (30 µg/ml) necessary for cell permeabilization in a 5% CO2 incubator at 37°C for 1 h before inoculating 400,000 cells per well in a volume of 30 µl. Color changes are read kinetically during 24h and are performed with OmniLog instrument (Biolog Inc., Hayward, CA). Signal intensity was measured at 24h and corrected by background subtraction with the negative control condition without substrate. The relative intensity was computed by dividing each intensity by the global mean of a particular sample.