(586a) Trick and Treat: Pulse Dosing to Eradicate Persister Bacteria

Background: A small fraction of bacteria use persistence as a strategy to survive exposure to antibiotics. Persistence is conferred by dormancy (non-growth): while bacteria remain dormant, they cannot be killed by conventional antibiotics. When persistent bacteria (persisters) leave dormancy (recommence growth) they become again susceptible to antibiotics. Persisters are implicated in many chronic infections such as recurrent urinary tract infection or cystic fibrosis and are difficult to eradicate with existing antibiotics. Moreover, prolonged persistence favors the emergence of complete antimicrobial resistance. The state-of-the-art is prolonged treatment of persisters with antibiotics, hoping for eventual eradication with the help of a patient’s immune system. This strategy is not as successful as one would hope for. A few studies have hinted that pulse dosing is effective in treating persistent infections. Here we show both experimentally and computationally that pulse dosing outperforms constant dosing if done correctly.

Hypothesis/ Goals: Detailed computer simulations with calibrated models led us to the hypothesis that persisters can be eradicated by an alternating antibiotic dosing regimen, as follows: Expose bacteria to a repeated cycle of (a) high enough antibiotic concentration that quickly kills susceptible cells before they become dormant, and (b) a low (ideally zero) antibiotic concentration at an appropriate time within the cycle, enticing persisters to recommence growth and be susceptible to the antibiotic. We show that the critical parameter determining the success of pulse dosing is the ratio t_off/t_on where t_off (t_on) duration of zero (non-zero) antibiotic concentration.

Methods:

Experimental: For in vitro experiments, an E. Coli. (transformed with GFP) culture was treated with Ampicillin. All cultures were incubated at 37°C in LB liquid media. A typical cycle (Figure i) was (a) Expose bacteria to Ampicillin (100 µg/ml) for t_on hours, (b) wash treated cells and grow in fresh media for t_off hours. Bacteria population size was assessed by colony counting on LB-agar plates. A control population exposed at constant Ampicillin concentration was also used in all experiments.

Computational: MATLAB and Mathematica were used for simulations, modelling, parameter estimation, and optimization. Eigenvalue analysis of the dynamic model was used to assess the optimal rate of bacterial population decline as a function of t_on, t_off (Figure ii).

Results: The hypothesis was confirmed in vitro and was repeatable. Appropriately designed pulse dosing regimens outperformed conventional constant dosing regimens. Analysis using the fitted model resulted in the following predictions:

1. The ratio t_off/t_on is indicative of anticipated outcomes, i.e. bacterial population eradication or not (Figure ii);
2. A ratio of t_off/t_on < 5 is required for eventual eradication of the entire population whereas t_off/t_on > 5 fails (Figure ii and iii).;
3. At t_off/t_on ~ 0.5 the eradication is fastest (Figure ii and iii); and

These predictions were confirmed in vitro as shown in Figure (i, iv and v). Experiments also showed that pulse dosing is robust, in that random perturbations of the dosing period produce near-optimal results (Figure iv).

Implications:

Pulse dosing has a high potential for treating persistent infections with existing antibiotics, which otherwise would be ineffective. This would reduce drug load on patients and prevent development of antimicrobial resistance. This study successfully developed and tested a design procedure of such pulse dosing. Future work will concentrate on additional bacteria/antibiotic pairs, including combinations of antibiotics to treat stubborn infections.