Omnilog parametric analysis software v1

The phenomic analysis of A. baumannii strains DR1, DR2, and AB067 were assayed using Biolog Phenotype MicroArray (PM) system as per manufacturer’s protocol to identify the sensitivities toward different stress (PM9 and PM10) and antimicrobial compounds (PM11–PM20). Briefly, the strains were streaked on LB agar plates overnight at 37°C, and the single colony cell suspension was made with 85% transmittance in Biolog IF-0 inoculation fluid (Biolog, Inc). Subsequently diluted in Biolog fluid containing dye A (Biolog, Inc.), further 100 μl of diluted cell suspension was added to each well of the Biolog microplates and incubated in Omnilog automatic plate reader (Biolog, Inc.) for 48 h at 37°C. Color formation from the redox-active tetrazolium-based dye (Biolog dye A) from colorless to formazan (violet) was monitored every 15 min. Data obtained from the respiration rate of A. baumannii strains were individually overlaid against the control (A. baumannii DR1) using the OmniLog File Management/Kinetic Analysis software v1.20.02 and analyzed using OmniLog Parametric Analysis software v1.20.02 (Biolog, Inc.). The growth curve height greater than 101 Omnilog units was considered as a positive phenotype, and higher values due to coloration of certain compounds were excluded from the analysis.

A. baumannii ΔadeRS cells were cultured on LB agar overnight at 37°C. A suspension of cells was made from a single colony in Biolog IF-0 inoculation fluid (Biolog, Inc.) to 85% transmittance and was subsequently diluted 1:200 in Biolog IF-0 containing dye A (Biolog, Inc.) and 0, 8, 16, 32 or 64 mg/L pentamidine. One hundred μL of each dilution was added to each well of the Biolog PM01 and PM02A MicroPlates and placed in an Omnilog automatic plate reader (Biolog, Inc.) for 72 h at 37°C. Color formation from the redox active dye (Biolog dye A) was monitored every 15 min. Data obtained from respiration of ΔadeRS under different pentamidine conditions were individually overlaid against the untreated control using the OmniLog File Management/Kinetic Analysis software v1.20.02, and analyzed using OmniLog Parametric Analysis software v1.20.02 (Biolog, Inc.).

The utilization of carbon sources was analyzed using Phenotype MicroArrays (Biolog, Hayward, CA, United States) as follows. The bacteria were cultured overnight on Luria-Bertani agar at 25°C, after which cells were harvested and suspended in inoculating fluid (IF-0). The transmittance of the suspension was adjusted to 42% using a Biolog turbidimeter. The cell suspension was mixed with IF−0 containing Dye Mix A (Biolog) to achieve a final transmittance of 85%. One hundred microliter aliquots of the adjusted cell suspension were inoculated into PM01 and PM02A plates, which were then incubated in an OmniLog Phenotype MicroArray System (Biolog) at 25°C for 48 h. The formation of formazan was recorded at 15 min intervals, and data were analyzed using OmniLog Parametric Analysis software v1.20.02 (Biolog). Relative growth of the studied strains was normalized to growth on D-glucose and visualized using Heatmapper (Babicki et al., 2016 (link)).