Table 1. The minimal inhibitory concentration and the growth efflux ratios for fusidic acid, oxacillin, dicloxacillin and piperacillin.
Both the growth efflux ratio and MIC (minimum inhibitory concentration) are obtained by fitting the OD600 of the MIC experiments applying the EC50 shift equation in GraphPad Prism. The efflux ratio is the EC50 of the individual AcrB substitution variants (I27A, N298A, L300A, Y327A, V340A, F332A, I335A, V340A, F380A, S630A and Q1000A) or wildtype AcrB, divided by the EC50 of inactive AcrB_D407N substitution variant. In bold are the MIC values differentiating by 2-dilution steps or more, and the EC50 values with statistically significant differences. The MIC experiments (for every substrate and clone) were repeated at least five times (biological replicates) with two technical replicates each. Indicated in parentheses are the number of biological replicates if done more than five times.
| Fusidic acid | Oxacillin | Dicloxacillin | Piperacillin | |||||
|---|---|---|---|---|---|---|---|---|
| AcrB Variants | MIC (μg ml−1) | Efflux ratio | MIC (μg ml−1) | Efflux ratio | MIC (μg ml−1) | Efflux ratio | MIC (μg ml−1) | Efflux ratio |
| D407N | 8–16 (9) | - | 2–4 (11) | - | 16 | - | 0.125–0.25 (8) | - |
| WT | 128–256 (8) | 8.64 ± 1.31 | 64–128 (9) | 20.49 ± 2.10 | 512 | 24.01 ± 4.04 | 0.50 | 2.44 ± 0.31 |
| I27A | 256 | 22.95 ± 3.48 | 256 | 51.11 ± 5.24 | 1024 | 52.19 ± 8.79 | 1.00 | 3.75 ± 0.48 |
| N298A | 64 | 4.35 ± 0.66 | 32–64 (8) | 10.31 ± 1.06 | 256 | 14.50 ± 2.44 | 0.25 | 1.30 ± 0.17 |
| L300A | 64–128 (7) | 6.56 ± 0.99 | 64 | 15.71 ± 1.61 | 512 | 21.01 ± 3.54 | 0.50 | 2.51 ± 0.32 |
| Y327A | 128–256 (7) | 13.18 ± 2.00 | 32–64 (8) | 13.06 ± 1.34 | 256 | 16.96 ± 2.86 | 0.25 | 1.75 ± 0.23 |
| F332A | 128 | 7.83 ± 1.19 | 64 (7) | 16.61 ± 1.79 | 256 | 16.93 ± 2.85 | 0.50 | 3.26 ± 0.42 |
| I335A | 128–256 (7) | 7.98 ± 1.21 | 64 (6) | 18.76 ± 1.93 | 256–512 (7) | 22.99 ± 3.87 | 0.25–0.50 (8) | 2.54 ± 0.33 |
| V340A | 64 | 4.25 ± 0.64 | 32 | 7.86 ± 0.81 | 256 | 10.86 ± 1.83 | 0.25 | 1.49 ± 0.19 |
| F380A | 64 | 4.53 ± 0.69 | 32–64 (7) | 8.60 ±0.88 | 256 | 12.03 ±2.03 | 0.25 | 1.42 ± 0.18 |
| S630A | 64–128 (8) | 5.60 ± 0.85 | 64 | 11.85 ± 1.22 | 256 | 15.56 ± 2.62 | 0.25 | 1.46 ± 0.19 |
| Q1000A | 128–256 (8) | 9.00 ± 1.36 | 64 (7) | 15.22 ± 1.56 | 256–512 (8) | 24.53 ± 4.13 | 0.50 | 3.33 ± 0.43 |