Table 1.
Bacterial strains, cell lines and primers used in this study.
Fig 1.
Illustration of the FIC checkerboard scheme, 96-well plate set up.
The red wells are EDTA, blue wells are A-NO2-, and purple wells are the AB569 combination at various concentration as indicated.
Table 2.
Primers used in this study.
Table 3.
Antibiotic minimum inhibition concentration (MIC) and bacterial identification results.
Fig 2.
Acinetobacter spp. AB569 Average MIC/FIC.
Average FIC index results, average FIC, and MIC concentrations. Yellow wells indicate typical bacterial growth, blue indicates well that represents FIC with inhibited bacterial growth, and white wells indicated inhibited bacterial growth. EDTA concentrations are noted on the left, A-NO2- concentrations are annotated on the bottom, media only column 12 is listed on the bottom, and media plus bacteria column 11 is listed on the bottom. Each experiment was performed at least 3 times.
Table 4.
AB569 Fractional Inhibitory Concentration (FIC) testing.
Fig 3.
CFU counts transformed to Log10 and plotted in semi-log scale. Black line represents untreated control values, red lines are EDTA only samples, blue lines are A-NO2- only, and green lines are EDTA + A-NO2- (AB569) combination. (A). Log10 CFU/ml counts with 8-hr exposure time and treated with 1 mM EDTA only, 32 mM A-NO2- only, and 1 mM EDTA + 32 mM A-NO2- AB569 combination, n = 3. (B). Log10 CFU/ml counts with 48-hr exposure time and treated with 1.5 mM EDTA, 30 mM A-NO2-, or 1.5 mM EDTA + 30 mM A-NO2- AB569. Each experiment was performed at least 3 times.
Fig 4.
Quantitative PCR analysis of predicted genes affected by treatment of Ab with AB569.
Total RNA Isolation following treatment with EDTA, A-NO2-, or AB569 combination (n = 3). (A). Electrophoresis of purified RNA prior to RT-PCR, lane M is the ladder marker, lane 1 is genome control, lane 2 untreated control, lane 3 is sample treated with 1 mM EDTA, lane 4 is sample treated with 32 mM A-NO2-, and lane 5 is sample treated with 1 mM EDTA + 32 mM A-NO2- AB569 combination, note the absence of dsDNA in samples compared to the positive genome control in the first lane. (B). Ab relative gene expression (2-ΔΔCt Values). Black column represents untreated control, EDTA only is dark grey, A-NO2- only is light grey, and AB569 combination is the black outlined column. Genes tested are listed on the bottom and log scale 2-ΔΔCt values are listed on the left, n = 3. (C). Heat Map of qPCR 2-ΔΔCT values. The genes tested are listed on the left side and treatment conditions are noted on the bottom of the graph. Gene expression is indicated by green for down regulation, red for up regulation, and black for middle range of gene expression (adjusted for contrast, n = 3).
Fig 5.
Scanned 96-well plate following biofilm inhibition assay. EDTA row values are indicated on the left and A-NO2- column values are indicated on the bottom. Column 12 was the media only or background control and column 11 was media + bacteria only or the positive control. MIC values are marked with a green arrow. FIC concentrations and percent biofilm inhibition are annotated with a red arrow indicating the FIC well (n = 3).
Fig 6.
HDFa Sytox orange dead cell stain.
HDFa cells treated with various concentrations of EDTA, A-NO2- and AB569 combinations listed on the bottom axis. Each column represents a unique treatment condition. Percent cell death is listed on the left. Line indicates significant cell death at 37% compared to media only control (One way ANOVA F = 6.55, P Value = <0.0001).
Fig 7.
Comparison of Pa and Ab qPCR, gene expression following treatment with EDTA, A-NO2- and AB569 combination.
The black column represents the untreated control, EDTA is in dark grey, A-NO2- is in light grey, and the combination is the black outlined column 2-ΔΔCt values. Genes tested are listed on the x-axis and log scale values are listed on the y-axis. (A). Pa relative gene expression (2-ΔΔCt Values). (B). Ab relative gene expression (2-ΔΔCt Values).