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Research Article
Lindsay R. Triplett
The Connecticut Agricultural Experiment Station
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5560-9257
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This work is licensed under a CC BY 4.0 License. Read Full License
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Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of persistence to membrane-disrupting biological compounds is less well-understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin, and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population of redox and membrane permeability states. Sorting cells according to redox reporter intensity identified streptomycin persisters among the low-redox fraction, but tailocin persisters were only cultured from the fraction with intermediate redox activity. Cells from culturable fractions were able to infect host plants, while nonculturable redox-active cells were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct redox states associated with antibiotic persistence, tailocin persistence, and virulence, and demonstrates that tailocin is highly effective in eliminating dormant cells.
Keywords
Pseudomonas syringae, persistence, persister, VBNC, streptomycin, tailocin, bacteriocin
Figure 1
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Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Fig.S1.tif
Figure S1. Long-term growth curve of Pph 1448A. Overnight Pph cultures were inoculated 1/100 in fresh King’s B medium. CFUs were enumerated by plating on King’s B medium at indicated timepoints up to 120 hours.
- Fig.S2.tif
Figure S2. Proportional staining categories of log phase Pph cultures after treatment with streptomycin or tailocin. (A, B) Concentration and culturability effects. Cultures were treated with streptomycin (A) or tailocin (B) and enumerated by hemocytometer (grey bars) or by dilution plating (blue bars). Asterisks represent a significant difference from the T0 value (p<0.005). Tailocin-treated cultures averaged 54% fewer cells at T4 than T0. (C, D). Proportional composition of Pph individuals in five physiological categories before and after treatment with streptomycin (C) or tailocin (D). Categories were assigned after visual inspection of each cell under three filters. Each datapoint represents 1000 Pph cells counted across 10 images taken from at least three slides. The four datapoints for each treatment represent values from four independent experiments, and bars represent the means and standard deviation across experiments.
- Fig.S3.tif
Figure S3. Testing of triple physiological staining method. Log phase cultures of Pph were imaged with no treatment (top) and after ethanol treatment (bottom) after simultaneous staining with RSG, PI, and Hoechst 33342 stained exponential- and ethanol- treated Pph cells. Images were collected through FITC, Rhodamine, DAPI, and phase contrast channels, and combined into a multi-channel image. Scale bar = 20 µm.
- Fig.S4.tif
Figure S4. Intensity and roundness associated with physiological staining categories. (A) Average RSG signal intensities of Pph cells in Category 1 (redox-active) and Category 2 (redox-active, membrane permeable) staining categories. One hundred imaged cells were selected randomly from each of Categories 1 and 2. Cells were selected from across four images representing four independent streptomycin-treated stationary phase cultures. Intensity of the selected cells in the FITC channel was analyzed using the MicrobeJ plugin of Fiji. (B) Roundness of 100 randomly selected cells from each of the staining categories observed after streptomycin treatment with the exception of Category 5, for which only 70 cells could be observed. Roundness was analyzed in the phase contrast images using the MicrobeJ plugin.
- Fig.S5.tif
Figure S5. Stationary phase Pph cultures stained with RSG, PI, and Hoechst 33342 before (A), 3 minutes after (B), and 4 hours after (C) tailocin treatment. Inset highlights cells in which red stained material appeared externally from the cell. Scale bar = 20 µm.
- Fig.S6.tif
Figure S6. DRAQ7 staining is limited to redox-inactive cells. (A) A representative multichannel image of streptomycin-treated Pph cells after simultaneous staining with RSG, PI, and DRAQ7 for 10m. Scale bar= 20 µm. (B) Three single Pph cells imaged in three fluorescence channels. Scale bar = 2 µm.
- Fig.S7.tif
Figure S7. Persistence curve for CCCP. Pph cultures were treated with 5x MIC of CCCP (100 µg mL-1), and culturable populations were enumerated hourly for 5h. Error bars represent the standard deviation of the mean for three replicate cultures. The experiment was performed three independent times.
- Fig.S8.tif
Figure S8. (A, B) Stationary phase Pph culture treated with tailocin and streptomycin, washed and stained with RSG, PI, Hoechst 33342 and observed under fluorescence phase contrast microscope before treatment (A), and after treatment, followed by 50x concentration (B). Scale bar = 20 µm. (C,D) Bean leaves infiltrated with a water control (C) and Pph cultures sequentially treated with tailocin and streptomycin (D). Note that the leaf image in C is also shown as the negative control image in Fig. 5B, as these inoculations were performed on the same day under the same conditions.
- TableS1.docx
Table S1. Percentage of imaged log phase and stationary phase Pph cells in five fluorescent staining categories before and after antimicrobial treatment.
- TableS2.docx
Table S2. Percentage of imaged cells staining with RSG, DRAQ7, or Hoescht only, in microscopic analysis of stationary phase Pph before or after antimicrobial treatment.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Lindsay R. Triplett
The Connecticut Agricultural Experiment Station
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5560-9257
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
View the published article at mBio.
Version 1
Posted 20 Jan, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Applied & Industrial Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
View the published article at mBio.
Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of persistence to membrane-disrupting biological compounds is less well-understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin, and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population of redox and membrane permeability states. Sorting cells according to redox reporter intensity identified streptomycin persisters among the low-redox fraction, but tailocin persisters were only cultured from the fraction with intermediate redox activity. Cells from culturable fractions were able to infect host plants, while nonculturable redox-active cells were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct redox states associated with antibiotic persistence, tailocin persistence, and virulence, and demonstrates that tailocin is highly effective in eliminating dormant cells.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Fig.S1.tif
Figure S1. Long-term growth curve of Pph 1448A. Overnight Pph cultures were inoculated 1/100 in fresh King’s B medium. CFUs were enumerated by plating on King’s B medium at indicated timepoints up to 120 hours.
- Fig.S2.tif
Figure S2. Proportional staining categories of log phase Pph cultures after treatment with streptomycin or tailocin. (A, B) Concentration and culturability effects. Cultures were treated with streptomycin (A) or tailocin (B) and enumerated by hemocytometer (grey bars) or by dilution plating (blue bars). Asterisks represent a significant difference from the T0 value (p<0.005). Tailocin-treated cultures averaged 54% fewer cells at T4 than T0. (C, D). Proportional composition of Pph individuals in five physiological categories before and after treatment with streptomycin (C) or tailocin (D). Categories were assigned after visual inspection of each cell under three filters. Each datapoint represents 1000 Pph cells counted across 10 images taken from at least three slides. The four datapoints for each treatment represent values from four independent experiments, and bars represent the means and standard deviation across experiments.
- Fig.S3.tif
Figure S3. Testing of triple physiological staining method. Log phase cultures of Pph were imaged with no treatment (top) and after ethanol treatment (bottom) after simultaneous staining with RSG, PI, and Hoechst 33342 stained exponential- and ethanol- treated Pph cells. Images were collected through FITC, Rhodamine, DAPI, and phase contrast channels, and combined into a multi-channel image. Scale bar = 20 µm.
- Fig.S4.tif
Figure S4. Intensity and roundness associated with physiological staining categories. (A) Average RSG signal intensities of Pph cells in Category 1 (redox-active) and Category 2 (redox-active, membrane permeable) staining categories. One hundred imaged cells were selected randomly from each of Categories 1 and 2. Cells were selected from across four images representing four independent streptomycin-treated stationary phase cultures. Intensity of the selected cells in the FITC channel was analyzed using the MicrobeJ plugin of Fiji. (B) Roundness of 100 randomly selected cells from each of the staining categories observed after streptomycin treatment with the exception of Category 5, for which only 70 cells could be observed. Roundness was analyzed in the phase contrast images using the MicrobeJ plugin.
- Fig.S5.tif
Figure S5. Stationary phase Pph cultures stained with RSG, PI, and Hoechst 33342 before (A), 3 minutes after (B), and 4 hours after (C) tailocin treatment. Inset highlights cells in which red stained material appeared externally from the cell. Scale bar = 20 µm.
- Fig.S6.tif
Figure S6. DRAQ7 staining is limited to redox-inactive cells. (A) A representative multichannel image of streptomycin-treated Pph cells after simultaneous staining with RSG, PI, and DRAQ7 for 10m. Scale bar= 20 µm. (B) Three single Pph cells imaged in three fluorescence channels. Scale bar = 2 µm.
- Fig.S7.tif
Figure S7. Persistence curve for CCCP. Pph cultures were treated with 5x MIC of CCCP (100 µg mL-1), and culturable populations were enumerated hourly for 5h. Error bars represent the standard deviation of the mean for three replicate cultures. The experiment was performed three independent times.
- Fig.S8.tif
Figure S8. (A, B) Stationary phase Pph culture treated with tailocin and streptomycin, washed and stained with RSG, PI, Hoechst 33342 and observed under fluorescence phase contrast microscope before treatment (A), and after treatment, followed by 50x concentration (B). Scale bar = 20 µm. (C,D) Bean leaves infiltrated with a water control (C) and Pph cultures sequentially treated with tailocin and streptomycin (D). Note that the leaf image in C is also shown as the negative control image in Fig. 5B, as these inoculations were performed on the same day under the same conditions.
- TableS1.docx
Table S1. Percentage of imaged log phase and stationary phase Pph cells in five fluorescent staining categories before and after antimicrobial treatment.
- TableS2.docx
Table S2. Percentage of imaged cells staining with RSG, DRAQ7, or Hoescht only, in microscopic analysis of stationary phase Pph before or after antimicrobial treatment.
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