Persistence and Culturability of Escherichia coli under Induced Toxin Expression
<p>Growth, persistence, and culturability of <span class="html-italic">E. coli</span> BW25113/pRJW1. (<b>a</b>) Conventional plot showing OD<sub>600</sub>, viable cell count (total CFUs), and persister count during a 24-h time period. Means ± SE are shown (n = 3). (<b>b</b>) New plot showing the changes in culturable cell count and persister cell number during the same culturing period, with persistence lines (PL) of survival after ofloxacin challenge. Means ± SE are shown (n = 3).</p> "> Figure 2
<p>Persistence–Culturability (PC) plot of <span class="html-italic">E. coli</span> BW25113/pRJW1 during early culturing under <span class="html-italic">hipA</span> induction. Different moving directions of the plot indicate complex dynamics of growth and dormancy under different levels of induced toxin production by arabinose. Means ± SE are shown (n = 3).</p> "> Figure 3
<p>Culturability during the early stage of culturing under <span class="html-italic">hipA</span> induction (2 and 4 h after inoculation). (<b>a</b>) Total cell numbers based on counting chamber results. (<b>b</b>) Culturable counts based on CFU assay. (<b>c</b>) Total number of non-culturable cells. Means ± SE are shown (n = 3). Stars indicate the <span class="html-italic">p</span> values, **** <span class="html-italic">p</span> < 0.0001. “ns” indicates no significance. “ND” means not detected.</p> "> Figure 4
<p>Live/Dead staining of cells after 5 h incubation with or without arabinose induction. The percentage marked in each image indicates the amount of arabinose. Representative images from 4 biological repeats are shown.</p> "> Figure 5
<p>Growth of <span class="html-italic">E. coli</span> BW25113/pRJW1 under different levels of <span class="html-italic">hipA</span> induction. (<b>a</b>) Growth curves based on OD<sub>600</sub>. Means ± SE are shown (n = 4). (<b>b</b>) Growth rates (means of 4 biological repeats).</p> "> Figure 6
<p>Persistence and culturability under different levels of <span class="html-italic">hipA</span> induction. (<b>a</b>) Number of persister cells and total CFU with and without <span class="html-italic">hipA</span> induction. (<b>b</b>,<b>c</b>) Zoomed-in plots for 0.002% (<b>b</b>) and 0.02% (<b>c</b>) induction are included to illustrate various movements across the quadrants under these more dynamic conditions. (<b>d</b>) A 3D plot of the same data to better reveal the dynamic changes in culturability and persister cell count. The expression of <span class="html-italic">hipA</span> was induced by 0.0002%, 0.002%, 0.02%, 0.2%, or 2% arabinose. Persister cells were isolated by treating with 10 µg/mL ofloxacin for 5 h in PBS. Means ± SE are shown (n = 3). (<b>e</b>) Persister killing assay conducted with mitomycin C and colistin treatments for 1 h in PBS. Persisters were formed with 0.02% arabinose induction for 4 h. Means ± SE of culturable cell counts are shown (n = 3). Stars indicate <span class="html-italic">p</span> values, * <span class="html-italic">p</span> < 0.05, “ns” indicates no significance.</p> "> Figure 7
<p><span class="html-italic">hipA</span> expression with and without arabinose induction. The mRNA level of <span class="html-italic">hipA</span> relative to the empty vector control was quantified by qPCR after 2 and 5 h of culturing time. Housekeeping gene <span class="html-italic">rrsA</span> was used as a control for baseline expression. Means ± SE are shown (n = 3). Stars indicate the <span class="html-italic">p</span> values, **** <span class="html-italic">p</span> < 0.0001, ** <span class="html-italic">p</span> < 0.01, “ns” indicates no significance.</p> "> Figure 8
<p>Persister formation, growth rate, and cell count of arabinose-induced <span class="html-italic">E. coli</span> BW25113 <span class="html-italic">P<sub>rrnB</sub></span><sub>P1</sub>-GFP<sub>AGA</sub>/pRJW1. (<b>a</b>) Fluorescence images of dose-dependent 5 h arabinose induction along with fluorescence quantified with flow cytometry. (<b>b</b>) A plot of bright cell quantification from flow cytometry of <span class="html-italic">E. coli</span> BW25113 <span class="html-italic">P<sub>rrnB</sub></span><sub>P1</sub>-GFP<sub>AGA</sub>/pRJW1 fluorescence after 5 h of arabinose induction. Means ± SE are shown (n = 3). (<b>c</b>) The total, bright, and fluorescent (bright and dim) cell counts were determined based on the images in (<b>a</b>) using ImageJ version 1.53k. Means ± SE are shown (n = 3). (<b>d</b>) Membrane potential was determined after 5 h dose-dependent arabinose induction along with persister levels after ofloxacin challenge. Means ± SE are shown (n = 3). Stars indicate the <span class="html-italic">p</span> values, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001, “ns” indicates no significance.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Dynamic Changes in Persistence and Culturability under hipA Expression
2.2. Dynamic Changes in Culturability and Dormancy under hipA Induction
2.3. Dynamic Changes in Cell Counts Revealed More Information about Dormancy
2.4. Arabinose-Induced Expression of hipA
2.5. Corroborating the Results Using a Growth Reporter Strain
3. Discussion
4. Materials and Methods
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence |
---|---|
rrsA | FP: 5′-ctcttgccatcggatgtgccca-3′ RP: 5′-cagtgtggctggtcatcctctca-3′ |
hipA | FP: 5′-ggcggtacgggaatacacat-3′ RP: 5′-ccgggaatctcagcaccttt-3′ |
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Dhaouadi, Y.; Hashemi, M.J.; Ren, D. Persistence and Culturability of Escherichia coli under Induced Toxin Expression. Antibiotics 2024, 13, 863. https://doi.org/10.3390/antibiotics13090863
Dhaouadi Y, Hashemi MJ, Ren D. Persistence and Culturability of Escherichia coli under Induced Toxin Expression. Antibiotics. 2024; 13(9):863. https://doi.org/10.3390/antibiotics13090863
Chicago/Turabian StyleDhaouadi, Yousr, Mohamad Javad Hashemi, and Dacheng Ren. 2024. "Persistence and Culturability of Escherichia coli under Induced Toxin Expression" Antibiotics 13, no. 9: 863. https://doi.org/10.3390/antibiotics13090863