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Population Density Modulates Drug Inhibition and Gives Rise to Potential Bistability of Treatment Outcomes for Bacterial Infections

Fig 1

Computer-controlled continuous culture devices can measure population growth at constant cell density.

A. Bacterial cultures (15 mL) are grown in glass vials with customized Teflon tops that allow inflow and outflow of fluid via silicone tubing. Cell density is monitored by light scattering using infrared LED/Detector pairs on the side of each vial holder. At the onset of the experiment, stationary phase bacterial cultures are diluted 500X and allowed to grow in the culture vials until a specific density is reached. At that point, drug is manually added at the desired concentration to both the culture vial and a connected chamber with fresh media. Flow between the media chamber, the culture vial, and a waste vial is managed by a series of computer-controlled peristaltic pumps that maintain constant cell density according to the pictured schematic. The entire system is controlled by custom Matlab software, and up to 18 cultures can be grown simultaneously using a multi-position magnetic stirrer. See also Figure A in S1 Text. B. Examples of a bacterial growth curve (red) and a constant cell-density experiment in which feedback from light scattering is used to maintain a constant cell density (blue). Lower inset: time series showing the status of the inflow/outflow pump, which provides fresh media and removed waste, during constant density (blue) experiment. Because total culture volume remains constant, the pump status time series can be used to calculate per capita growth rate, g, as a function of time when cell density is held constant: g = F/V, where F is the (time dependent) pump flow rate (mL/min) and V is the (constant) culture volume (mL). Upper inset: Calibration plot showing that voltage output from IR detectors is linearly related to optical density. C. Time dependent population growth rate is estimated from the relative pump flow rate F(t), which is the flow rate of the pumps required to maintain cell density (flow rates measured relative to maximum possible flow rate of approximately 1 ml/min). Drug is added at time 0, and following transient growth rate dynamics of approximately 200 minutes, growth rate reaches a steady state that is dependent on cell density. To reduce high-frequency noise, F(t) is estimated with a moving-average filter with window size of 15 minutes. D. To estimate growth rate relative to untreated cells, the steady state growth rate F(t) is averaged in the steady state and normalized by the same measurement in the absence of drug. Upper left inset, full growth curve for E. faecalis in the absence of drug. The densities measured here (0.2≤OD≤0.8) correspond to exponential phase growth, represented by a straight line (red) on a semi-log plot. Upper right inset, growth rate (not normalized) with and without drug. Without drug, growth varies by approximately 6% around the mean over these density ranges.

Fig 1