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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 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 PrecN-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.
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fig5: 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 PrecN-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.

Mentions: To begin to answer the above questions, we sought to provide evidence, in addition to the FACS analysis, that demonstrated that induction of the SOS response during ofloxacin treatment was inconsequential to persistence in stationary-phase cultures. To do this, we treated PrecN reporters with 100 µg/ml rifampin (RIF) or 50 µg/ml chloramphenicol (CM) to stop RNA or protein synthesis prior to and during ofloxacin treatment. Both RIF and CM, at these concentrations, prevented GFP induction by ofloxacin, confirming that the treatments used were sufficient to prevent RNA and protein synthesis (Fig. 5A). When RIF- or CM-treated cultures were assayed for persistence to ofloxacin, survival fractions were indistinguishable from the samples treated with ofloxacin only (Fig. 5B). This illustrated that transcription and translation during ofloxacin treatment, including but not limited to induction of the SOS response, are not required for persistence to ofloxacin in nongrowing populations.


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

Völzing KG, Brynildsen MP - MBio (2015)

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 PrecN-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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556807&req=5

fig5: 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 PrecN-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.
Mentions: To begin to answer the above questions, we sought to provide evidence, in addition to the FACS analysis, that demonstrated that induction of the SOS response during ofloxacin treatment was inconsequential to persistence in stationary-phase cultures. To do this, we treated PrecN reporters with 100 µg/ml rifampin (RIF) or 50 µg/ml chloramphenicol (CM) to stop RNA or protein synthesis prior to and during ofloxacin treatment. Both RIF and CM, at these concentrations, prevented GFP induction by ofloxacin, confirming that the treatments used were sufficient to prevent RNA and protein synthesis (Fig. 5A). When RIF- or CM-treated cultures were assayed for persistence to ofloxacin, survival fractions were indistinguishable from the samples treated with ofloxacin only (Fig. 5B). This illustrated that transcription and translation during ofloxacin treatment, including but not limited to induction of the SOS response, are not required for persistence to ofloxacin in nongrowing populations.

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