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) tha...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2024-01-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03567-23 |
| _version_ | 1797357424836870144 |
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| author | Patricia J. Hare Juliet R. Gonzalez Ryan M. Quelle Yi I. Wu Wendy W. K. Mok |
| author_facet | Patricia J. Hare Juliet R. Gonzalez Ryan M. Quelle Yi I. Wu Wendy W. K. Mok |
| author_sort | Patricia J. Hare |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T14:43:50Z |
| format | Article |
| id | doaj.art-47bc5d0cf4b844cca3cedee8019b26d2 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-08T14:43:50Z |
| publishDate | 2024-01-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-47bc5d0cf4b844cca3cedee8019b26d22024-01-11T14:04:37ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972024-01-0112110.1128/spectrum.03567-23Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to LevofloxacinPatricia J. Hare0Juliet R. Gonzalez1Ryan M. Quelle2Yi I. Wu3Wendy W. K. Mok4Department of Molecular Biology & Biophysics, UConn Health , Farmington, Connecticut, USADepartment of Molecular Biology & Biophysics, UConn Health , Farmington, Connecticut, USADepartment of Molecular Biology & Biophysics, UConn Health , Farmington, Connecticut, USARichard D. Berlin Center for Cell Analysis and Modeling, UConn Health , Farmington, Connecticut, USADepartment of Molecular Biology & Biophysics, UConn Health , Farmington, Connecticut, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/spectrum.03567-23Pseudomonas aeruginosafluoroquinoloneantibiotic tolerancetranscriptionstationary phase |
| spellingShingle | Patricia J. Hare Juliet R. Gonzalez Ryan M. Quelle Yi I. Wu Wendy W. K. Mok Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin Microbiology Spectrum Pseudomonas aeruginosa fluoroquinolone antibiotic tolerance transcription stationary phase |
| title | Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin |
| title_full | Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin |
| title_fullStr | Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin |
| title_full_unstemmed | Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin |
| title_short | Metabolic and transcriptional activities underlie stationary-phase Pseudomonas aeruginosa sensitivity to Levofloxacin |
| title_sort | metabolic and transcriptional activities underlie stationary phase pseudomonas aeruginosa sensitivity to levofloxacin |
| topic | Pseudomonas aeruginosa fluoroquinolone antibiotic tolerance transcription stationary phase |
| url | https://journals.asm.org/doi/10.1128/spectrum.03567-23 |
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