Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin

ABSTRACT The ubiquitous opportunistic pathogen Pseudomonas aeruginosa is highly adaptive and refractory to several different classes of antibiotics. However, we found in this study that stationary-phase P. aeruginosa cultures exhibit greater sensitivity to the fluoroquinolone Levofloxacin (Levo) than other bactericidal antibiotics, including an aminoglycoside (Tobramycin) and β-lactam (Aztreonam). To understand the basis of this sensitivity, we conducted time-lapse fluorescence microscopy experiments of cells during Levo treatment. We discovered that stationary-phase P. aeruginosa cells die rapidly during treatment and undergo heterogeneous morphological changes, including explosive lysis, filamentation, and gradual loss of membrane integrity as evidenced by propidium iodide uptake. These morphologies are reminiscent of how the model organism Escherichia coli appears when recovering from fluoroquinolone treatment, a period when activation of the DNA damage-induced SOS response is crucial. Accordingly, we monitored the morphologies and survival of P. aeruginosa ΔrecA mutants and found that the SOS response is not involved in P. aeruginosa Levo sensitivity like it is for E. coli. We hypothesized that Levo sensitivity may be due to P. aeruginosa maintaining active metabolism in stationary phase. We determined that stationary-phase P. aeruginosa cells transcribe, maintain reductase activity, and accumulate reactive metabolic species which contribute to Levo-mediated death. By elucidating how P. aeruginosa cells sustain metabolic activity during the stationary phase, we can design strategies to sensitize these persistent subpopulations to Levo and maintain the efficacy of this clinically important fluoroquinolone antibiotic. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa is responsible for a variety of chronic human infections. Even in the absence of identifiable resistance mutations, this pathogen can tolerate lethal antibiotic doses through phenotypic strategies like biofilm formation and metabolic quiescence. In this study, we determined that P. aeruginosa maintains greater metabolic activity in the stationary phase compared to the model organism, Escherichia coli, which has traditionally been used to study fluoroquinolone antibiotic tolerance. We demonstrate that hallmarks of E. coli fluoroquinolone tolerance are not conserved in P. aeruginosa, including the timing of cell death and necessity of the SOS DNA damage response for survival. The heightened sensitivity of stationary-phase P. aeruginosa to fluoroquinolones is attributed to maintained transcriptional and reductase activity. Our data suggest that perturbations that suppress transcription and respiration in P. aeruginosa may actually protect the pathogen against this important class of antibiotics.

1st Editorial Decision Re: Spectrum03567-23 (Metabolic and Transcriptional Activities Underlie Stationary-Phase Pseudomonas aeruginosa Sensitivity to Levofloxacin) Dear Dr. Wendy W. K. Mok: Thank you for sending the manuscript to us.The manuscript was reviewed by two reviewers.Both reviewers found the work interesting, and the response was positive.Below you will find their comments as well as instructions from the Spectrum editorial office.The second reviewer directly commented on the manuscript (attached).
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Sincerely, Minsu Kim Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): The reviewer would like to thank the authors for their submission which is titled: "Metabolic and Transcriptional Activities Underlie Stationary-Phase Pseudomonas aeruginosa Sensitivity to Levofloxacin".In this work, the authors hypothesize that the apparent sensitivity of stationary-phase P. aeruginosa to Levofloxacin is mediated by the active metabolism of the cell and does not involve SOS response.They address this hypothesis by examining bactierial cell morphology and metabolism in the presence of Levo and making inferences from such data.The reviewer would like to provide a critique of the article on the basis of the stylistic aspects (major and minor) as well as the technical aspects (major and minor) Stylistic (minor) 1.If the authors could briefly clarify for the readers in the caption that the breakpoints are *not* conducted with stationary phase bacteria this might be best.The author understands what they intended to convey but at a cursory glance readers may mistake their data in red and blue for depicting resistance in a standard MIC assay.

Stylistic (major) No Major Stylistic Issues
Technical (minor) 1.In Figure 5A, the data representing PA14, there appear to be multiple peaks for RGS fluorescence in their flow data.Do the authors believe these are significant?Does RGS uptake and activity with respect to reductases generally exhibit an all-or-none response or are there variations in their experience?
2. The authors seem to suggest that there is no difference in survival among the wild-type and ΔrecA mutants.Examining Figure 4C, specifically PAO1 vs PAO1 ΔrecA, is this true for all concentrations?The reviewer would draw attention to the apparent 0.5 μg/mL data point.
3. As a follow-up to Major Technical Point 2, the specific line in the text reads, "stationary-phase P. aeruginosa recA knockouts and their wildtype counterparts across multiple Levo concentrations".Is the reviewer to interpret that the phrase "multiple" is used in place of "all" due to the point discussed above?

Technical (major) No Major Technical issues
Reviewer #2 (Comments for the Author): The authors have done extensive work on pseudomonas isolates with the aim of elucidating the mechanisms behind levofloxacin tolerance.The experiments are well-designed and executed.However, while the text reads well, the lack of tangible data made it difficult to read, particularly for someone who is coming from a clinical bacteriology background.An overall figure that explains the different experiments done (a conceptual framework if I may say so) with a hierarchical flow chart would make this work more easier to comprehend.Other comments are attached to the manuscript Editor's Comments: Thank you for sending the manuscript to us.The manuscript was reviewed by two reviewers.Both reviewers found the work interesting, and the response was positive.Below you will find their comments as well as instructions from the Spectrum editorial office.The second reviewer directly commented on the manuscript (attached).
Please address their comments and return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration by Spectrum.
Thank you for considering our manuscript and for the opportunity to revise our work according to the reviewers' feedback.

Reviewer 1:
The reviewer would like to thank the authors for their submission which is titled: "Metabolic and Transcriptional Activities Underlie Stationary-Phase Pseudomonas aeruginosa Sensitivity to Levofloxacin".In this work, the authors hypothesize that the apparent sensitivity of stationaryphase P. aeruginosa to Levofloxacin is mediated by the active metabolism of the cell and does not involve SOS response.They address this hypothesis by examining bactierial cell morphology and metabolism in the presence of Levo and making inferences from such data.The reviewer would like to provide a critique of the article on the basis of the stylistic aspects (major and minor) as well as the technical aspects (major and minor) We would like to thank Reviewer 1 for their suggestions on how to improve our manuscript.Please find our responses to each item below.

Stylistic (minor)
1.If the authors could briefly clarify for the readers in the caption that the breakpoints are *not* conducted with stationary phase bacteria this might be best.The author understands what they intended to convey but at a cursory glance readers may mistake their data in red and blue for depicting resistance in a standard MIC assay.
Thank you for this recommendation.We have now clarified that MIC assays are not conducted on stationary-phase bacteria and have adjusted the caption of Fig. 1 to read, "Minimum inhibitory concentrations of P. aeruginosa PAO1 and PA14, as measured by testing growth inhibition of low-density cultures with MIC test strips (n>3)."

Stylistic (major)
No Major Stylistic Issues Technical (minor) 1.In Figure 5A, the data representing PA14, there appear to be multiple peaks for RGS fluorescence in their flow data.Do the authors believe these are significant?Does RGS uptake and activity with respect to reductases generally exhibit an all-or-none response or are there variations in their experience?Our histograms of RSG fluorescence, like those from other publications (PMID: 29046671, 30332894), typically show a range of fluorescence intensities.Although the multiple peaks we observe are somewhat unusual, we do not know if they are biologically significant and will have to investigate further in future experiments.
2. The authors seem to suggest that there is no difference in survival among the wild-type and ΔrecA mutants.Examining Figure 4C, specifically PAO1 vs PAO1 ΔrecA, is this true for all concentrations?The reviewer would draw attention to the apparent 0.5 μg/mL data point.We appreciate the reviewer's careful review of the data in Fig. 3C.To determine if there were significant differences in survival between the PAO1 wildtype and ΔrecA at any of the Levo concentrations, we repeated our statistical analysis and confirmed that the differences in survival are not statistically significant (p > 0.05 by Mann-Whitney test with multiple test correction).The caption for Figure 3C has been updated to include this analysis.
3. As a follow-up to Major Technical Point 2, the specific line in the text reads, "stationary-phase P. aeruginosa recA knockouts and their wildtype counterparts across multiple Levo concentrations".Is the reviewer to interpret that the phrase "multiple" is used in place of "all" due to the point discussed above?The ΔrecA strain's Levo persistence is not different from the wildtype at any of the concentrations that we tested, but we did not want to use the word "all" because we did not exhaustively test all Levo concentrations from zero drug to complete killing.To make it clearer, we have adjusted the text from "…and their wildtype counterparts across multiple Levo concentrations," to "…and their wildtype counterparts across the range of Levo concentrations that we tested."

Reviewer 2:
The authors have done extensive work on pseudomonas isolates with the aim of elucidating the mechanisms behind levofloxacin tolerance.
The experiments are well-designed and executed.However, while the text reads well, the lack of tangible data made it difficult to read, particularly for someone who is coming from a clinical bacteriology background.An overall figure that explains the different experiments done (a conceptual framework if I may say so) with a hierarchical flow chart would make this work more easier to comprehend.Other comments are attached to the manuscript We would like to thank Reviewer 2 for their suggestions on how to improve our manuscript, especially in regards to improving accessibility for more clinical audiences.We have now included a supplemental figure that provides an overview of the key experiments we conducted in the manuscript to anchor readers (Fig. S1).
Main Text: "Antibiotic Recalcitrant": May be better to use a term that is more commonly used in describing bacterial caracteristics, as this term is specific to biofilms, to the best of my knowledge and it seems like the authors are implying general resistance than something specific to biofilms To clarify our text as the reviewer suggests, we have edited the wording to "antibiotic-refractory," which encompasses antibiotic treatment failure by resistance, tolerance, or persistence (PMID 20980069).
Is a biofilm considered as a reversible phenotypic change or a different growth pattern?Reconsider the wording please We consider a biofilm to be an example of a reversible phenotypic growth pattern because individual cells can reversibly express gene programs for attachment or motility/dispersal.We have tried to improve the sentence's phrasing to focus more on individual cells within biofilms than the biofilm itself: Changed from "…by undergoing reversible phenotypic changes, like forming biofilms" to "…by undergoing reversible phenotypic changes, as observed for bacteria in biofilms.""Stochastic": Consider using a term that is more commonly used in bacteriology We find the term "stochastic" is commonly used to describe the random/chance variations among single cells in isogenic populations in the field of persister biology.For example, one of the leaders in the field, Kim Lewis, published a paper in PLOS Biology in 2021 entitled, "Bacterial persisters are a stochastically formed subpopulation of low-energy cells."Other notable examples include PMIDs 27980159 and 19029898.
"Perturbations": May be better to use a term that is more commonly used in describing bacterial caracteristics We have re-written the sentence for clarity as the reviewer suggests: "In P. aeruginosa, metabolic heterogeneity due to differential nutrition and oxygenation has been shown to impact antibiotic tolerance."Lines 98-98: This para is a too lengthy.Better restrict to the key finding or two only, to avoid multiple duplucations We have removed sentences in this paragraph to shorten it and highlight the most important findings as recommended.
Line 102: While the title states stationary phase, the results does not state anything about the phase.There needs to be a better description indicating that this results are pertinent to the stationary phase experiments.We specified the phase of growth in the following places in the paragraph: "We began by asking whether the persistence of stationary-phase cultures of P. aeruginosa" (Lines 105-106) and "we performed survival assays at supra-MIC concentrations to determine how sensitive stationaryphase PAO1 and PA14 were to each antibiotic" (Lines 111-113).
"Highly sensitive": Too subjective Thank you for pointing this out.We have removed the word "highly" to more objectively state that stationary-phase P. aeruginosa is sensitive to Levo, but not the other antibiotic compounds.
Line 102: Was this experiment done for E.coli, if not why?This experiment was not done with E. coli because the focus of our investigation was on P. aeruginosa.E. coli was used in later experiments as a point of comparison against which we could highlight Levo-treated P. aeruginosa phenotypes.

Line 107: Differ in what? In response against antibiotics?
To enhance the clarity as the reviewer suggests, we have broken the sentence into three parts: "We began by asking whether the persistence of stationary-phase cultures of P. aeruginosa would differ against bactericidal antibiotics with different primary targets.For generalizability, we tested P. aeruginosa strains PAO1 and PA14-representing the two major genetic clades of this species-in each experiment.… Stationary-phase cultures were grown in chemically defined media with succinate as the sole carbon source before treatment."Line 111: Pls give the values of the MIC, it is difficult to make it out from the figure alone.As the reviewer suggested, we have added a new table to the supplement (Table S2) with the MIC values for Levo, Aztreonam, and Tobramycin so that the values can be elucidated more easily.
Line 114: Why were these multiplications used?The aztreonam MIC is approximately 4 ug/ml, according to the figure 1 they were tested in to 50 only.One has to then refer to the supplementary figures to find the data for this.This makes the paper difficult to read and comprehend.Remove figure S1 and include it within figure 1 itself where the complete concentration range tested are included in 1B,C and D. Also, give the exact reductions noted in Tobramycin in the figure As suggested, we now include the full datasets for Aztreonam and Tobramycin in the main figure panel 1A.
Line 115: Write in a quantitative manner.This is a subjective statement We have re-written the statement to be more objective and quantitative: "We found that P. aeruginosa was completely tolerant to Tobramycin at 15X MIC and Aztreonam up to 25-50X MIC (Fig. S1).Comparatively, P. aeruginosa was highly sensitive to Levo; we detected approximately six-log decreases in cell survival when Levo was administered at 15X MIC (5 μg/mL)."Line 116: State the actual descrease.We now state the decreased survival as "six-log" instead of "greater than five-log."Line 117: At stationary phase?We have changed the title to "Stationary-phase P. aeruginosa dies during Levo treatment" for clarity.
Lines 122-124: The results given this way without any data is not very convincing Was SytoxBlue completely removed before the cells were inoculated on the agarose pads?May need a statement here To clarify this section as suggested, the sentences in this paragraph have been rearranged to emphasize that the cells from SytoxBlue-containing cultures were diluted 30-fold in PBS before being seeded on the agarose pad for imaging, thus SytoxBlue is nearly absent from these samples.The data for SytoxBlue are shown in Fig. S4 in the Supplemental Materials and show that few cells are SytoxBlue-positive.
Line 126: What was the concentration of Levo used here?And what folds of the MIC?Why was SytoxBlue used in the earlier phase and PI here?As the reviewer suggests, the Levo concentration is now explicitly stated in the paragraph.To enhance our clarity, the paragraph has been re-written to emphasize the purpose of switching from SytoxBlue during growth to stationary phase and PI during treatment: Re: Spectrum03567-23R1 (Metabolic and Transcriptional Activities Underlie Stationary-Phase Pseudomonas aeruginosa Sensitivity to Levofloxacin) Dear Dr. Wendy W. K. Mok: Congratulation!Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
Thank you for submitting your paper to Spectrum.

Sincerely, Minsu Kim Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): The Authors have addressed this reviewers concerns adequately Reviewer #2 (Comments for the Author): Thank you for painstakingly going to each and every comments and modifying where needed.