TABLE 5.
Biological effects of PRMSE and catecholg
| Strain | Effect based on bioassays | Value for: |
Value for positive controle |
||||
|---|---|---|---|---|---|---|---|
| Untreated cells | Phenolic extract (PRMSE) | Pure compound (catechol) | Gentamicin (μg ml−1) | CCCP | CTAB | ||
| E. coli CFT073 | Minimum permeabilization concna (mg ml−1) (NPN assay) | 1.6 | 0.06 | 0.006 | NAf | NA | |
| % reduction in fluorescence intensity due to efflux of EtBrb | 36.9 | 22.7 | 34.3 | NA | 15.8 | NA | |
| % of cells with uncompromised membranec (BacLight assay) | 100 | 88.7 | 68 | NA | NA | 0 | |
| Change in bacterial colony-forming abilityd (log CFU ml−1) | 0 | 0.8 | 2.3 | NA | NA | 5.7 | |
| P. mirabilis HI4320 | Minimum permeabilization concn (mg ml−1) | 1.6 | 1 | 0.013 | NA | NA | |
| % reduction in fluorescence intensity due to efflux of EtBr | 35.9 | 23.4 | 33.9 | NA | 14.5 | NA | |
| % of cells with uncompromised membrane | 100 | 72.6 | 50.3 | NA | NA | 0 | |
| Change in bacterial colony-forming ability (log CFU ml−1) | 0 | 1.2 | 3.2 | NA | NA | 4.5 | |
| P. aeruginosa PAO1 | Minimum permeabilization concn (mg ml−1) | 0.8 | 0.5 | 1 | NA | NA | |
| % reduction in fluorescence intensity due to efflux of EtBr | 37.6 | 22.3 | 35.8 | NA | 19.3 | NA | |
| % cells with uncompromised membrane | 100 | 87.9 | 80.4 | NA | NA | 0 | |
| Change in bacterial colony-forming ability (log CFU ml−1) | 0 | 0.9 | 1.5 | NA | NA | 5.1 | |
| P. aeruginosa PA14 | Minimum permeabilization concn (mg ml−1) | 0.8 | 0.5 | 1 | NA | NA | |
| % reduction in fluorescence intensity due to efflux of EtBr | 30.9 | 19 | 29.9 | NA | 17 | NA | |
| % of cells with uncompromised membrane | 100 | 89.1 | 78.6 | NA | NA | 0 | |
| Change in bacterial colony-forming ability (log CFU ml−1) | 0 | 0.9 | 1.9 | NA | NA | 4.9 | |
Assay was performed at concentrations below the MICs of extract and pure compound compared to the MIC of the reference antibiotic (gentamicin at above and below the MICs). These concentrations led to a maximal increase in NPN (1-N-phenylnapthylamine) uptake based on fluorescence intensity recorded. A 2.5% membrane permeabilization was considered the baseline value for the determination of minimum permeabilization concentrations. Raw data are provided in Fig. S3 in the supplemental material.
The ratio of green to red fluorescence was normalized to that of the untreated control and expressed as a percentage of the control. Cells were treated at 4× MIC of pure compound and extract for 10 min.
Reduction in fluorescence intensity as a percentage of that at the first time point of recording. Cells were treated at 0.25 times the MIC of pure compound and extract overnight.
Bacterial colonies were counted from the LB agar plate, and the log decrease in CFU/ml compared to that of the untreated control was calculated.
CCCP, carbonyl cyanide m-chlorophenylhydrazone (100 μM); CTAB, cetyltrimethylammonium bromide (10 μM).
NA, not applicable.
Values are compared to those of reference antibacterial agents with known mechanisms of action for the ability to permeabilize the outer membrane, inhibit efflux pumps, and damage membrane integrity.