Non-Monotonic Survival of Staphylococcus aureus with Respect to Ciprofloxacin Concentration Arises from Prophage-Dependent Killing of Persisters

Abstract

Staphylococcus aureus is a notorious pathogen with a propensity to cause chronic, non-healing wounds. Bacterial persisters have been implicated in the recalcitrance of S. aureus infections, and this motivated us to examine the persistence of S. aureus to ciprofloxacin, a quinolone antibiotic. Upon treatment of exponential phase S. aureus with ciprofloxacin, we observed that survival was a non-monotonic function of ciprofloxacin concentration. Maximal killing occurred at 1 µg/mL ciprofloxacin, which corresponded to survival that was up to ~40-fold lower than that obtained with concentrations ≥ 5 µg/mL. Investigation of this phenomenon revealed that the non-monotonic response was associated with prophage induction, which facilitated killing of S. aureus persisters. Elimination of prophage induction with tetracycline was found to prevent cell lysis and persister killing. We anticipate that these findings may be useful for the design of quinolone treatments.

Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells

“…Beyond the effects of ϕ80, we showed that the use of intermediate concentrations of fluoroquinolones (such 0.5 to 1 µg/ml of ciprofloxacin) for E. coli persister assays is inappropriate, because—in full congruency with the results of Sandvik et al ( 26 ) for S. aureus —ca. 1 log of bacterial killing at these concentrations is caused not directly by the antibiotic but rather by the induction of resident prophages ( Fig.…”

“…Beyond these issues related to the growth conditions of bacteria studied in persistence assays, a recent publication by members of the Brynildsen laboratory on Staphylococcus aureus persister formation alerted us that the presence of resident prophages might affect measurements of persister frequencies under some conditions ( 26 ). In short, those authors showed that ciprofloxacin treatment at the commonly used concentrations of 0.5 to 1 µg/ml—more than an order of magnitude above the MIC and sufficient to kill all regular cells—eliminates a substantial fraction of persisters and that the effect is not due to the direct action of the antibiotic but is instead due to the secondary activation of prophages.…”

“…In short, those authors showed that ciprofloxacin treatment at the commonly used concentrations of 0.5 to 1 µg/ml—more than an order of magnitude above the MIC and sufficient to kill all regular cells—eliminates a substantial fraction of persisters and that the effect is not due to the direct action of the antibiotic but is instead due to the secondary activation of prophages. This effect could be overcome at very high concentrations of ciprofloxacin that cause a strong inhibition of cellular DNA processing and thus impair prophage development ( 26 ). Given that DNA damage is generally known to be a strong inducer of temperate prophages, including some of the cryptic prophages in the E. coli K-12 MG1655 chromosome ( 27 , 28 ), we suspected that a similar effect described by Sandvik et al ( 26 ) may also affect the persister results reported for E. coli .…”

“…This effect could be overcome at very high concentrations of ciprofloxacin that cause a strong inhibition of cellular DNA processing and thus impair prophage development ( 26 ). Given that DNA damage is generally known to be a strong inducer of temperate prophages, including some of the cryptic prophages in the E. coli K-12 MG1655 chromosome ( 27 , 28 ), we suspected that a similar effect described by Sandvik et al ( 26 ) may also affect the persister results reported for E. coli . We therefore compared the E. coli K-12 wild-type strain to a previously published mutant devoid of all nine cryptic prophages for tolerance to different concentrations of ciprofloxacin in a classical single-growth-time-point persister assay ( Fig.…”

Using an <i>Escherichia Coli</i> K-12/<i>recA-gfpmut2</i> Microbial Biosensor to Assess the Impact of Cyclophosphamide and L-ascorbic Acid Residues on Living Bacteria Cells

“…in chemical processes such as ROS generation. At higher concentrations, SOS signaling pathways are activated to protect against the effects of stressors [54][55].…”