Figure 1. Changes in cell density have opposing effects on β-lactam efficacy in drug sensitive and drug resistant populations.

(A) Half-maximal inhibitory concentration (IC₅₀) of ampicillin as a function of inoculum density for resistant (red squares) and sensitive (blue circles) populations. IC₅₀ is estimated using a fit to Hill-like function $f\mathrm{}\mathrm{(}x\mathrm{)}\mathrm{=}{\mathrm{(}\mathrm{1}\mathrm{+}{\mathrm{(}x\mathrm{/}K\mathrm{)}}^h\mathrm{)}}^{\mathrm{-}\mathrm{1}}$, where $h$ is a Hill coefficient and $K$ is the IC₅₀. (B) Per capita growth rate of drug-sensitive populations held at a density of OD = 0.2 (open squares) and OD = 0.8 (filled circles) following addition of ampicillin at time 0. Growth rate is estimated, as in Karslake et al. (2016), from the average media flow rate required to maintain populations at the specified density in the presence of a constant drug concentration of 0.5 µg/mL. Flow rate is averaged over sliding 20 min windows after drug is added. Note that drug-resistant populations exhibit no growth inhibition over these density ranges, even for drug concentrations in excess of 10³ µg/mL. (C) Schematic of experimental setup. Cell density in planktonic populations is measured via light scattering from IR detector/emitter pairs calibrated to optical density (OD). Fresh media (containing appropriate drug concentrations) is introduced over time using computer-controlled peristaltic pumps, and waste is simultaneously removed to maintain constant volume (see Materials and methods). (D) Top panel: final cell density of drug sensitive populations exposed to constant drug influx over a 20 hr period. Experiments were started from either ‘high density’ (OD = 0.6, red) or ‘low density’ (OD = 0.1, blue) initial populations. Bottom panel: cell density time series for drug-resistant populations exposed to ampicillin influx of approximately 1200 µg/mL per hour. In all experiments media was refreshed and waste removed at a rate of µ₀ ≈ 0.1 hr⁻¹. E. In mixed populations containing both sensitive (green) and resistant (blue, ‘R’) cells, there are opposing density-dependent effects on drug efficacy. Increasing the density of resistant cells is expected to decrease drug efficacy as a result of increased β-lactamase production (left side). By contrast, increasing the density of the total cell population decreases the local pH and increases the efficacy of β-lactam antibiotics (right side).