Fig 1.
Minocycline is more effective against E. coli HM22 persister cells than normal cells.
(A) Viability of E. coli HM22 persister and normal cells after tetracycline treatment in PBS. (B) Effects of minocycline (in PBS) on the viability of normal (black bars) and persister (white bars) cells of E. coli HM22. The untreated samples from each population were normalized as 100%. Means ± SE are shown (n = 5). (C) Different antibiotic treatments of E. coli HM22 persister cells including 100 μg/mL of ampicillin, 100 μg/mL of minocycline, and the combination of both treated in LB medium. (D) The effects were corroborated by a checkerboard assay. The exponential phase cells were treated with ampicillin for 3 h in LB, followed by treatment with minocycline for 1 h in PBS. (n = 2) (E) Intracellular concentration of minocycline based on the reporter bioassay. Minocycline concentration was calculated using a standard curve of reporter strain for each population (S1 Fig). Means ± SE are shown (n = 4). (F) Intracellular concentration of minocycline from treated and untreated samples in both normal (black bars) and persister (patterned bars) populations using LC-MS. Means ± SE are shown (n = 3). * p-value≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, ****p-value ≤ 0.0001.
Fig 2.
Persister formation led to reduced efflux pump activities.
(A) Induction of persister formation led to increased EtBr accumulation. Measurements were performed with a fluorescence microplate reader with excitation at 360 nm. (B-E) Flow cytometry analysis of EtBr staining. (B) EtBr stained uninduced E. coli Top10/pRJW1 (top) and EtBr stained arabinose induced E. coli Top10/pRJW1 (bottom). Induced E. coli Top10/pRJW1 had 13 ± 2.2% of the population shifted to stronger fluorescence, indicating more EtBr accumulation. (C) EtBr stained uninduced E. coli Top10 pBAD (top) and EtBr-stained arabinose induced E. coli Top10/pBAD (empty vector) (bottom). (D) E. coli ΔacrB without (top) and with (bottom) EtBr staining. (E) E. coli pUC19-acrB without (top) and with (bottom) EtBr staining. (F) Inactivation of efflux pumps sensitized normal cells to minocycline. Means ± SE are shown (n = 3). * p-value≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, ****p-value ≤ 0.0001.
Fig 3.
Persister formation led to lower membrane potential.
(A, B) Schematic of hipA mediated persister formation by the PBAD protmoter. Flow cytometry analysis of JC-1 stained samples was used to compare the membrane potential of induced (with arabinose and tetracycline) E. coli Top10/pRJW1 (A) vs. induced E. coli Top10/pBAD (empty vector) (B) cells. A shift to low red fluorescence was observed for 16 ± 2.5% of induced cells of E. coli Top10/pRJW1, while no change was observed in green fluorescence. (C) Persister count increased when induced with both arabinose and tetracycline. (D) E. coli HM22 normal population was pretreated with CCCP (100 μM) to reduce the membrane potential. The cells were then treated with 100 μg/mL of minocycline. Means ± SE are shown (n = 3). (E) Representative fluorescent images of control and minocycline treated E. coli HM22 normal cells with or without CCCP pretreatment. The cells were labeled with SYTO9 and propodium iodide (PI) (scale bar, 10 μM). (F) Cell viability based on mean fluorescence intensity quantified using Image J. Percentages are based on the ratios of green fluorescence vs. total fluorescence. * p-value≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, ****p-value ≤ 0.0001.
Fig 4.
Killing occurred upon wake-up of persister cells.
(A) Representative fluorescence images of control and minocycline treated E. coli HM22 normal and persister cells after LIVE/DEAD staining (scale bar = 10 μm). Fluorescence signals were used to compare the viability of normal (B) and persister (C) cells. Mean fluorescence intensity of SYTO9 and PI was quantified using ImageJ. (D) Representative fluorescence images of persister cells upon wake-up after minocycline treatment. The images show persister and normal cells at 0 and 30 min after spiking with LB medium. (E) Fluorescence signals of LB spiked E. coli HM22 persister cells. Three biological replicates were tested with 16 images randomly analyzed from each sample. (F) OD600 of E. coli HM22 persister cells during wake-up. Cells with and without minocycline treatment were compared. (G) Intracellular concentration of minocycline after minocycline was removed from the solution and replaced with LB medium. * p-value≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, ****p-value ≤ 0.0001.
Fig 5.
Viability of E. coli HM22 after treatment with rifamycin SV and eravacycline.
(A) Effects of 100 μg/mL rifamycin SV on the viability of normal (black bars) and persister (patterned bars) cells of E. coli HM22. Means ± SE are shown (n = 3). (B) Intracellular concentration of rifamycin SV based on the reporter bioassay. Rifamycin SV concentration was calculated using the standard curve of reporter strain for each population (S2B Fig). Means ± SE are shown (n = 3). (C) Effects of eravacycline on the viability of normal (black bars) and persister (patterned bars) cells of E. coli HM22. Means ± SE are shown (n = 3). (D) Intracellular concentration of eravacycline based on the reporter bioassay. Eravacycline concentration was calculated using a standard curve of reporter strain for each population (S2C Fig). Means ± SE are shown (n = 3). (E) Binding pocket of minocycline (top) and eravacycline (bottom) in the 30S ribosomal unit. Minocycline interacts with G966, C1195, U1196, G1197, and G1198 via hydrogen bonding and ionic interactions (black dashed lines) mediated by a pair of Mg ions. Eravacycline occupies the same binding pocket as minocycline but also binds to additional residues—C1054, C1195, and U1196. (c) Color scheme: uracil (teal), cytosine (light blue), guanine (gold), adenine (purple), C (green stick); O(red, stick); N (blue); F (cyan, stick); Mg ion (magenta, sphere). Solvent is omitted for clarity. (F) Different antibiotic treatments of E. coli HM22 persister cells including 100 μg/mL of ampicillin, 100 μg/mL of eravacycline, and the combination of both. Means ± SE are shown (n = 3). (G-H) Relative number of viable E. coli HM22 persister cells after eravacycline treatment (initial number normalized as 100%). The changes in OD600 (G) and CFU (H) were followed over time. Means ± SE are shown (n = 3 for OD600 and n = 4 for CFU). * p-value≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, ****p-value ≤ 0.0001.
Fig 6.
A conceptual model of persister control by leveraging reduced antibiotic efflux.
Persisters have reduced membrane potential and thus are difficult to penetrate by hydrophilic antibiotics and those require active transport. In comparison, antibiotics that can penetrate through lipid without active uptake can still target persister cells. Additionally, reduced drug efflux provides a favorable condition for accumulation of antibiotics in persister cells. This leads to killing if the internalized antibiotic molecules remain bound to the target during wake-up. The inactivated pathways in persister cells are indicated with lighter colors and/or marked with “X”. Minocycline, rifamycin SV, and eravacycline fit the criteria and are found effective in this study for persister control. The drugs targeting the 30S ribosomal subunit demonstrated in this study are shown as an example. Figures are drawn for Gram-negative species as tested in this study.