Table 1.
Escherichia coli strains used in this study.
Figure 1.
Comparison between Ampicillin and Ofloxacin based persister cells isolation methods in 3 E. coli strains.
Using different antibiotics and methodologies for persister isolation generated markedly different survival fractions. Ampicillin and Ofloxacin were tested using exponentially growing cultures without dilution (1∶1) at an OD of 0.5. As a reference in this figure, we used the results of ampicillin treatment with an initial dilution of the culture (1∶100) reported by Balaban et al. in 2004 [5]. Error bars indicate the standard deviation (n = 3), except for the Ampicillin (1∶100) treatment in which the error bars indicate the range reported by Balaban for exponentially growing cultures.
Figure 2.
Persister cells isolation in E. coli strains using Ofloxacin.
The killing kinetics of Ofloxacin showed differences in stabilization time when comparing its activity in stationary and exponential growth phase, requiring almost double time when acting in stationary phase cells, even in the same E. coli strain (A). Survival fractions from stationary phase cultures of two different E. coli strains also exhibited markedly different stabilization times, even in the same physiological state (B). Error bars indicate the standard deviation (n = 3).
Figure 3.
Standardization of the lysis protocol for persister cells isolation.
For the characterization of our protocol for persister cells isolation we calculated persistence frequencies (survival fractions) in both stationary (A) and exponential (B) growth of three different E. coli strains, using 200 uL of each lytic solution for the exponentially growing cultures and 500 uL for the stationary cultures. We compared the results obtained using our protocol with those reported by Balaban et al. in 2004 (Ampicillin, 1∶100) for all the tested strains. Error bars indicate the standard deviation (n = 3), except for the Ampicillin (1∶100) treatment in which the error bars indicate the range reported by Balaban for cultures of the above mentioned strains.
Figure 4.
Persister cells isolation: Killing curves.
For all the tested strains our protocol reached stabilization shortly (15 minutes) after the addition of the enzymatic lytic solution and remained stable for more than 90 minutes in both exponentially (A) and stationary (B) growing cultures. For the exponentially growing cultures, 200 uL of each lytic solution was added to isolate persister cells as previously described, whereas for the stationary cultures we used 500 uL of each solution. Error bars indicate the standard deviation (n = 3).
Figure 5.
Differentiation between type I and type II persister cells from Stationary phase cultures.
Using different working concentration of our lysis solutions we obtain two marked plateaus in the killing curves. The first one corresponds to a mixture of type I and type II persister cells, while the second one corresponds to type I persister cells exclusively. The fraction of type I cells in the first plateau depends on the fraction of type I and type II persisters in the population, which in turn depends on strain and growth conditions.
Figure 6.
Time lapse microscopy validation of type I and type II persister cells isolation.
Using DIC microscopy we assessed the performance of our novel protocol in persister cells isolation and for differentiating between type I and type II persister cells. A) Type I and II persister cells were isolated from a stationary phase culture of E. coli DS1. Cell division time was found to be on average 159 minutes +/30 minutes (n = 36) for the cells that were growing. B) A stationary phase culture of hipA7 (TH1269) was treated with our protocol to differentially isolate type I persister cells. All cells were found to be non-growing whilst in the persistence state and after switching back to a normally growing state cell division occurred rapidly.