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Stationary-Phase Persisters to Ofloxacin Sustain DNA Damage and Require Repair Systems Only during Recovery.

Völzing KG, Brynildsen MP - MBio (2015)

Bottom Line: Persister survival is attributed to a transient state of dormancy in which a cell's growth and metabolism are significantly reduced and many essential processes are thought to be inactive.In this work, we show that in nongrowing populations, persisters to ofloxacin experience the same level of antibiotic-induced damage as cells that succumb to the treatment and that their survival critically depends on repair of this damage after the conclusion of treatment.We hypothesize that effective antipersister therapies may be developed on the basis of this knowledge.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, USA.

No MeSH data available.


Related in: MedlinePlus

Induction of the SOS response in stationary-phase persisters and nonpersisters is equivalent. (A) GFP induction from SOS transcriptional reporters with ofloxacin. Unshaded curves, ΔrecA mutant controls treated for 5 h with ofloxacin; shaded curves, samples treated for 5 h with ofloxacin. Events in gates C and D were considered to have responded to ofloxacin because ≤2% of the events from ofloxacin-treated ΔrecA mutant controls fell within those gates. Controls for each reporter are provided in Fig. S1 and S4 in the supplemental material. (B) Survival fractions of ofloxacin-treated cells from gates designated in panel A. T, total-population control passed through the sorter (all gates); α and β, samples diluted to postsorting cell density before and after sorting that did not themselves travel through the sorter. Sorting did not significantly reduce sample culturability. This was determined by a lack of significant difference in survival between the total sorted population (T) and the presorting (α) or postsorting (β) controls (by Student’s t test, two tails with unequal variance) for any of the SOS reporters tested. Survival fractions were calculated relative to the cell density of sorted samples, 3 × 105 cells/ml. Differences between the survival of PsulA (fractions or controls) and the other three SOS reporters were not significant for the majority of the comparisons, as determined by Student’s t test (two tails with unequal variance). Biphasic killing was observed for all strains (see Fig. S2 in the supplemental material), and reanalysis of FACS-segregated populations is provided in Fig. S5 in the supplemental material.
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fig2: Induction of the SOS response in stationary-phase persisters and nonpersisters is equivalent. (A) GFP induction from SOS transcriptional reporters with ofloxacin. Unshaded curves, ΔrecA mutant controls treated for 5 h with ofloxacin; shaded curves, samples treated for 5 h with ofloxacin. Events in gates C and D were considered to have responded to ofloxacin because ≤2% of the events from ofloxacin-treated ΔrecA mutant controls fell within those gates. Controls for each reporter are provided in Fig. S1 and S4 in the supplemental material. (B) Survival fractions of ofloxacin-treated cells from gates designated in panel A. T, total-population control passed through the sorter (all gates); α and β, samples diluted to postsorting cell density before and after sorting that did not themselves travel through the sorter. Sorting did not significantly reduce sample culturability. This was determined by a lack of significant difference in survival between the total sorted population (T) and the presorting (α) or postsorting (β) controls (by Student’s t test, two tails with unequal variance) for any of the SOS reporters tested. Survival fractions were calculated relative to the cell density of sorted samples, 3 × 105 cells/ml. Differences between the survival of PsulA (fractions or controls) and the other three SOS reporters were not significant for the majority of the comparisons, as determined by Student’s t test (two tails with unequal variance). Biphasic killing was observed for all strains (see Fig. S2 in the supplemental material), and reanalysis of FACS-segregated populations is provided in Fig. S5 in the supplemental material.

Mentions: To assess persister abundance in the subpopulations that did and did not elicit an ofloxacin-induced SOS response, we used FACS to segregate ofloxacin-treated cultures into four subpopulations on the basis of their induction of SOS promoters. Gates A and B were designed to capture the nR subpopulation, since ≥98% of the events from ofloxacin-treated ΔrecA mutant controls fell within these gates. The nR subpopulation was further separated such that quantile A contained a minimum number of events with the lowest GFP expression and quantile B contained the remainder. Gates C and D were designed to capture the R subpopulation, since ≤2% of the events from ofloxacin-treated ΔrecA mutant controls fell within these gates. The R subpopulation was further segregated such that quantile D included the top 10% of the cells with the highest GFP expression and quantile C contained the remainder of the R subpopulation. Surprisingly, and contrary to what was expected, we observed that persisters were equally distributed among all of the gates for each SOS reporter; in other words, survival was independent of SOS induction (Fig. 2). To confirm that these results were not due to imprecision in the sorting experiments, we reanalyzed the sorted fractions (A, B, C, and D) of each reporter (see Fig. S5 in the supplemental material). As anticipated, subpopulation A had the highest composition of nR cells upon reanalysis (~93% when averaged across all four reporters), whereas subpopulation D had the highest composition of R cells upon reanalysis (~78% when averaged across all four reporters). When this degree of imprecision was accounted for (see Text S1 in the supplemental material for details), the average frequencies of persisters in the nR and R subpopulations did not deviate by more than 13% from what was measured for the A and D subpopulations for each reporter (see Fig. S5 in the supplemental material), which confirmed that persisters arose from the nR and R subpopulations with approximately equal likelihood. These data suggested that persisters and nonpersisters experienced and responded to DNA damage equivalently. Further, these data implied that (i) the majority of stationary-phase E. coli cells, irrespective of whether they induced the SOS response, experienced a lethal level of DNA damage from ofloxacin and (ii) induction of the SOS response during ofloxacin treatment was inconsequential to persistence. The first of these implications is based on the fact that the majority of the cells in both the nR and R subpopulations died or lost their culturability during ofloxacin treatment, whereas the second is a direct corollary of equivalent persister frequencies in the nR and R subpopulations.


Stationary-Phase Persisters to Ofloxacin Sustain DNA Damage and Require Repair Systems Only during Recovery.

Völzing KG, Brynildsen MP - MBio (2015)

Induction of the SOS response in stationary-phase persisters and nonpersisters is equivalent. (A) GFP induction from SOS transcriptional reporters with ofloxacin. Unshaded curves, ΔrecA mutant controls treated for 5 h with ofloxacin; shaded curves, samples treated for 5 h with ofloxacin. Events in gates C and D were considered to have responded to ofloxacin because ≤2% of the events from ofloxacin-treated ΔrecA mutant controls fell within those gates. Controls for each reporter are provided in Fig. S1 and S4 in the supplemental material. (B) Survival fractions of ofloxacin-treated cells from gates designated in panel A. T, total-population control passed through the sorter (all gates); α and β, samples diluted to postsorting cell density before and after sorting that did not themselves travel through the sorter. Sorting did not significantly reduce sample culturability. This was determined by a lack of significant difference in survival between the total sorted population (T) and the presorting (α) or postsorting (β) controls (by Student’s t test, two tails with unequal variance) for any of the SOS reporters tested. Survival fractions were calculated relative to the cell density of sorted samples, 3 × 105 cells/ml. Differences between the survival of PsulA (fractions or controls) and the other three SOS reporters were not significant for the majority of the comparisons, as determined by Student’s t test (two tails with unequal variance). Biphasic killing was observed for all strains (see Fig. S2 in the supplemental material), and reanalysis of FACS-segregated populations is provided in Fig. S5 in the supplemental material.
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Related In: Results  -  Collection

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fig2: Induction of the SOS response in stationary-phase persisters and nonpersisters is equivalent. (A) GFP induction from SOS transcriptional reporters with ofloxacin. Unshaded curves, ΔrecA mutant controls treated for 5 h with ofloxacin; shaded curves, samples treated for 5 h with ofloxacin. Events in gates C and D were considered to have responded to ofloxacin because ≤2% of the events from ofloxacin-treated ΔrecA mutant controls fell within those gates. Controls for each reporter are provided in Fig. S1 and S4 in the supplemental material. (B) Survival fractions of ofloxacin-treated cells from gates designated in panel A. T, total-population control passed through the sorter (all gates); α and β, samples diluted to postsorting cell density before and after sorting that did not themselves travel through the sorter. Sorting did not significantly reduce sample culturability. This was determined by a lack of significant difference in survival between the total sorted population (T) and the presorting (α) or postsorting (β) controls (by Student’s t test, two tails with unequal variance) for any of the SOS reporters tested. Survival fractions were calculated relative to the cell density of sorted samples, 3 × 105 cells/ml. Differences between the survival of PsulA (fractions or controls) and the other three SOS reporters were not significant for the majority of the comparisons, as determined by Student’s t test (two tails with unequal variance). Biphasic killing was observed for all strains (see Fig. S2 in the supplemental material), and reanalysis of FACS-segregated populations is provided in Fig. S5 in the supplemental material.
Mentions: To assess persister abundance in the subpopulations that did and did not elicit an ofloxacin-induced SOS response, we used FACS to segregate ofloxacin-treated cultures into four subpopulations on the basis of their induction of SOS promoters. Gates A and B were designed to capture the nR subpopulation, since ≥98% of the events from ofloxacin-treated ΔrecA mutant controls fell within these gates. The nR subpopulation was further separated such that quantile A contained a minimum number of events with the lowest GFP expression and quantile B contained the remainder. Gates C and D were designed to capture the R subpopulation, since ≤2% of the events from ofloxacin-treated ΔrecA mutant controls fell within these gates. The R subpopulation was further segregated such that quantile D included the top 10% of the cells with the highest GFP expression and quantile C contained the remainder of the R subpopulation. Surprisingly, and contrary to what was expected, we observed that persisters were equally distributed among all of the gates for each SOS reporter; in other words, survival was independent of SOS induction (Fig. 2). To confirm that these results were not due to imprecision in the sorting experiments, we reanalyzed the sorted fractions (A, B, C, and D) of each reporter (see Fig. S5 in the supplemental material). As anticipated, subpopulation A had the highest composition of nR cells upon reanalysis (~93% when averaged across all four reporters), whereas subpopulation D had the highest composition of R cells upon reanalysis (~78% when averaged across all four reporters). When this degree of imprecision was accounted for (see Text S1 in the supplemental material for details), the average frequencies of persisters in the nR and R subpopulations did not deviate by more than 13% from what was measured for the A and D subpopulations for each reporter (see Fig. S5 in the supplemental material), which confirmed that persisters arose from the nR and R subpopulations with approximately equal likelihood. These data suggested that persisters and nonpersisters experienced and responded to DNA damage equivalently. Further, these data implied that (i) the majority of stationary-phase E. coli cells, irrespective of whether they induced the SOS response, experienced a lethal level of DNA damage from ofloxacin and (ii) induction of the SOS response during ofloxacin treatment was inconsequential to persistence. The first of these implications is based on the fact that the majority of the cells in both the nR and R subpopulations died or lost their culturability during ofloxacin treatment, whereas the second is a direct corollary of equivalent persister frequencies in the nR and R subpopulations.

Bottom Line: Persister survival is attributed to a transient state of dormancy in which a cell's growth and metabolism are significantly reduced and many essential processes are thought to be inactive.In this work, we show that in nongrowing populations, persisters to ofloxacin experience the same level of antibiotic-induced damage as cells that succumb to the treatment and that their survival critically depends on repair of this damage after the conclusion of treatment.We hypothesize that effective antipersister therapies may be developed on the basis of this knowledge.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, USA.

No MeSH data available.


Related in: MedlinePlus