Fig 1.
Establishing a FCM assay by characterising RSG for assessing oxidative stress in antibiotic exposed A. baumannii.
The lab adapted strain, ATCC 19606, was first stained with RSG, before exposing to the ROS-generating reagents at room temperature. Bacteria were then assessed by FCM. Mode fluorescence intensities obtained from flow cytometric analyses were compared against untreated conditions as shown in panel (A). Flow cytometric histograms revealing the optimal fold change of RSG fluorescence intensities at 45 minutes were shown in panel (B).
Fig 2.
RSG fluorescence intensities in bacteria changes upon antibiotics exposure.
Panel (A) describes the labelling strategy to assess ROS in viable bacteria using a cocktail of dyes. SYTO-62 labels all bacteria by binding to nucleic acid, while propidium iodide (PI) only enters non-viable bacteria. Viable bacteria (takes up SYTO-62 but not PI) will be assessed for ROS. Basal ROS is generated as a by-product of respiration under homeostatic conditions, hence exhibiting RSG fluorescence. After bacteria were exposed to antibiotics, there will be an increase in ROS. This increase in intracellular ROS, increases the intensity of RSG fluorescence measurable by flow cytometry. (B) Representative flow cytometric histograms depicting the changes in RSG fluorescence intensities in clinical isolate (AB0047) upon exposure to various antibiotics. Flow cytometric histograms for clinical isolates (AB0356 and AB0603) are shown in S2 and S3 Figs in S1 File. Exposure to sufficient concentrations of antibiotics resulted in increased RSG fluorescence as indicated by rightward shifts of the histograms. The antibiotic concentration corresponding to the first histogram shift compared to untreated (no antibiotics) was determined to be the MIC of that antibiotic (circled in red). The ‘>‘ symbol indicates MIC is higher than the highest concentration tested in the customized microtiter panel.
Table 1.
Antibiotics susceptibility profiles of clinical AB isolates.
Table 2.
Summary of MICs comparisons between standard microtiter broth dilution method and MICs determined via FCM (using ROS as a marker).