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

Abstract

Chronic infections are a serious health care problem, and bacterial persisters have been implicated in infection reoccurrence. Progress toward finding antipersister therapies has been slow, in part because of knowledge gaps regarding the physiology of these rare phenotypic variants. Evidence shows that growth status is important for survival, as nongrowing cultures can have 100-fold more persisters than growing populations. However, additional factors are clearly important, as persisters remain rare even in nongrowing populations. What features, beyond growth inhibition, allow persisters to survive antibiotic stress while the majority of their kin succumb to it remains an open question. To investigate this, we used stationary phase as a model nongrowing environment to study Escherichia coli persistence to ofloxacin. Given that the prevailing model of persistence attributes survival to transient dormancy and antibiotic target inactivity, we anticipated that persisters would suffer less damage than their dying kin. However, using genetic mutants, flow cytometry, fluorescence-activated cell sorting, and persistence assays, we discovered that nongrowing ofloxacin persisters experience antibiotic-induced damage that is indistinguishable from that of nonpersisters. Consistent with this, we found that these persisters required DNA repair for survival and that repair machinery was unnecessary until the posttreatment recovery period (after ofloxacin removal). These findings suggest that persistence to ofloxacin is not engendered solely by reduced antibiotic target corruption, demonstrate that what happens following antibiotic stress can be critical to the persistence phenotype, and support the notion that inhibition of DNA damage repair systems could be an effective strategy to eliminate fluoroquinolone persisters.

Importance: In the absence of resistant mutants, infection reoccurrences can still occur because of persisters, rare bacterial cells that survive antibiotic treatments to repopulate infection sites. 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. Thus, dormancy is believed to protect persisters from antibiotic-induced damage and death. 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. These findings reveal that persistence to ofloxacin is not engendered solely by reduced antibiotic target corruption and highlight that processes following antibiotic stress are important to survival. We hypothesize that effective antipersister therapies may be developed on the basis of this knowledge.

FIG 1

Stationary-phase E. coli responds to ofloxacin by SOS response induction. (A) GFP induction from SOS transcriptional reporters upon ofloxacin treatment (shaded colored curves). R and nR subpopulations were delineated with respect to untreated controls (unshaded gray curves). nR cells are to the left of the dashed lines, and R cells are to the right. The average percentages of the total populations classified as R ± the standard deviation of at least three replicates are indicated for each promoter. Controls that confirm SOS-dependent induction in response to ofloxacin under these treatment conditions are provided in Fig. S1 in the supplemental material. Data that demonstrate biphasic killing of these strains in response to the ofloxacin treatment conditions used here are provided in Fig. S2 and S3 in the supplemental material. (B) Induction of mCherry from P_T5lacO occurs in both the R (shaded curves) and nR (unshaded curves) subpopulations. For each reporter, mCherry in R cells and that in nR cells largely overlap. Controls that confirm induction of mCherry during ofloxacin treatment as well as the gating strategies used are provided in Fig. S4 in the supplemental material.

FIG 2

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 P_sulA (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.

FIG 3

Persisters and nonpersisters induce an impressive SOS response within 2 h of recovery in liquid medium. (A) Shaded purple curve, WT containing P_recA-gfp treated in stationary phase for 5 h with ofloxacin, followed by 2 h of recovery in antibiotic-free LB medium. Unshaded curve, same as purple curve, except that the ΔrecA mutant containing P_recA-gfp was used. Vertical lines indicate the FACS gating strategy used. Additional controls are provided in Fig. S6 in the supplemental material. (B) The number of CFU per milliliter remained unchanged through 2 h of recovery in fresh LB medium. Numbers of CFU per milliliter are reported as average values ± the minimum and maximum values of two biological replicates. (C) Survival fractions from the A, B, C, and D gates designated in panel A. T, total population (all gates); α, pre-FACS control; β, post-FACS control. Survival fractions were calculated relative to the cell density of sorted samples, 3 × 10⁵ cells/ml. Data are average values ± standard errors from at least three biological replicates. (D) WT morphology and induction of P_recA in cells subjected to ofloxacin treatment (OFL tx) for 5 h in stationary phase after transfer to antibiotic-free LB medium for 0 and 2 h, along with untreated controls. Controls demonstrating that P_recA induction and filamentation are RecA dependent are provided in Fig. S6 in the supplemental material. All images are 87.36 by 66.56 µm.

FIG 4

DNA damage repair system mutants have reduced survival after ofloxacin treatment in stationary phase. Ofloxacin persisters decreased approximately 10⁴-fold in ΔrecA mutants, 10³-fold in ΔrecB mutants, and 50-fold in ΔruvA and ΔrecN mutants. ΔrecF, ΔtisAB, and ΔdksA resulted in persister levels comparable to those of the WT. Data are average values ± standard errors from at least three biological replicates.

FIG 5

Induction of transcription and translation during ofloxacin (OFL) treatment is not required for persister survival. (A) Treatment with CM (shaded blue curve) or RIF (shaded red curve) inhibits ofloxacin-induced translation or transcription, respectively, from P_recN-gfp. Unshaded gray curves, non-ofloxacin-treated control. (B) Treatment with CM or RIF does not alter ofloxacin persister levels or culturability; all ofloxacin-treated samples displayed biphasic killing. CM and RIF were dissolved in dimethyl sulfoxide (DMSO). (C) Ofloxacin persisters decreased 1,000-fold in the presence of LexA3 (gray curve) relative to the WT (black curve). Kill curve data are average values ± standard errors from at least three biological replicates.

FIG 6

RecA and the SOS response are critical to persistence only during recovery from ofloxacin treatment (OFL tx). (A) Ofloxacin persisters decreased >1,000-fold in the absence of RecA expression (dashed purple) relative to when RecA was expressed from 2 h pretreatment with ofloxacin through recovery (dashed blue). RecA induction on LB plates during recovery only (red) was sufficient to restore persisters to WT levels. (B) Ofloxacin persisters decreased 25-fold with LexA3 (blue) relative to the WT (black). LexA3 induction on LB plates only (red) maintained this reduced survival. We note that LexA3 induction only during recovery reduced the overall viability in both ofloxacin-treated and untreated samples, and this was accounted for by plotting the surviving fraction. Data are average values ± standard errors from at least three biological replicates. Numbers of CFU per milliliter for all samples and controls are provided in Fig. S7 in the supplemental material.