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. 2020 Apr 21;64(5):e02393-19.
doi: 10.1128/AAC.02393-19. Print 2020 Apr 21.

Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

Prashant Karki  1 , Sayed Golam Mohiuddin  1 , Pouria Kavousi  1 , Mehmet A Orman  2
Affiliations

Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

Prashant Karki et al. Antimicrob Agents Chemother. .

Abstract

Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

Keywords: Escherichia coli; antimicrobial activity; growth rate; osmolytes; pH; persisters; sodium nitrite; tolerance; urea.

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Figures

FIG 1
FIG 1
Eliminating preexisting persisters. (A) Overnight cultures were diluted 1:100-fold in modified LB and grown until mid-exponential phase for the dilution/growth cycle experiments. After the second cycle, the cells were diluted 1:100-fold in fresh modified LB and transferred to microarray plates. (B) The cells in the third dilution flask were treated with 5 μg/ml OFX. At designated time points, 1-ml samples were collected, washed to remove the antibiotic, serially diluted, and spotted in an LB agar plate to count the number of survived cells as measured by CFU.
FIG 2
FIG 2
Effects of sodium chloride, urea, sodium nitrite, and pH in PM plates. Prepropagated cells after the dilution/growth cycle experiments were transferred to PM plates and cultured for 24 h. Then, the cells from the plates were transferred to fresh media and treated with 5 μg/ml of OFX for 6 h. CFU measurements were performed before and after the OFX treatments. (A to D) The effects of sodium chloride, urea, sodium nitrite, and various pH conditions on E. coli cell survival and persistence are shown, respectively. *, the osmolyte or pH of interest significantly affects the E. coli cell viability compared to no-osmolyte or no-buffer controls before the antibiotic treatments (P < 0.05); #, the osmolyte or pH of interest significantly affects the OFX persister levels compared to no-osmolyte or no-buffer controls (P < 0.05); ^, CFU below the limit of detection.
FIG 3
FIG 3
Effects of various osmolytes in PM plates. (A to J) The effects of various osmolytes on E. coli survival and persistence are shown. *, the osmolyte of interest significantly affects the E. coli cell viability compared to no-osmolyte or no-buffer controls before the antibiotic treatments (P < 0.05); #, the osmolyte of interest significantly affects the OFX persister levels compared to no-osmolyte or no-buffer controls (P < 0.05); ^, CFU below the limit of detection.
FIG 4
FIG 4
Effects of urea (3.5%), sodium nitrite (60 mM), and pH 5 on E. coli persistence. Prepropagated cells were diluted 1:100-fold in fresh modified LB media with urea, sodium nitrite, and MES (pH 5) at indicated concentrations in baffled flasks. After growing the cultures for 24 h, the cells were diluted (1:100) to fresh, modified LB media with 5 μg/ml of OFX. At designated time points, samples were collected, washed to remove the antibiotics, and plated on agar media to quantify CFU. The kill-curve profile for each tested condition was generated and evaluated using the nonlinear model described by Windels et al. (12). **, statistical difference between the kill curve profiles of treatment and control groups (P < 0.0001).
FIG 5
FIG 5
Dependence of persister formation on cell growth for cultures prepropagated in modified LB. Prepropagated cells were transferred at a 1:100 ratio in modified LB containing osmolytes and pH buffers in baffled flasks and cultured for 24 h to monitor the persister levels. (A) At an hourly interval, samples were collected and diluted 1:2 in fresh media with osmolytes and buffers and treated with 5 μg/ml OFX for 6 h. The solid lines indicate log(CFU/ml) measured before antibiotic treatments, and the dashed lines indicate log(CFU/ml) measured after the antibiotic stress (persisters). The “log(CFU/ml) − normal cells” versus “log(CFU/ml) − persisters” plots were generated for all conditions: control (B), NaCl (1%) (C), urea (D), MES (pH 5) (E), and sodium nitrite (60 mM) (F). Each point in the graph represents a biological replicate for a time point, where the blue points represent exponential-phase time points and orange points represent stationary-phase time points. (G) Calculated Pearson correlations (R) and the corresponding P values. The threshold of significance is set to be P < 0.05.
FIG 6
FIG 6
Dependence of persister formation on cell growth for cultures prepropagated in modified LB in the presence of indicated osmolytes/buffers. Cells were prepropagated in the presence of osmolytes or buffers. After the dilution/growth cycle experiments, cells were transferred at a 1:100 ratio in modified LB containing osmolytes or pH buffers and cultured for 24 h to monitor the persister levels. At designated time points, samples were collected and diluted 1:2 in fresh media with osmolytes and buffers and treated with 5 μg/ml OFX for 6 h. (A) The solid lines represent log(CFU/ml) measured before antibiotic treatments, and the dashed lines represent log(CFU/ml) measured after the antibiotic stress (persisters). The “log(CFU/ml) − normal cells” versus “log(CFU/ml) − persisters” plots were generated for all conditions: control (B), urea (C), MES (pH 5) (D), and sodium nitrite (60 mM) (E) cultures. Each point in the graph represents a biological replicate for a time point, where the blue points represent exponential-phase time points and orange points represent stationary-phase time points. (F) Calculated Pearson correlations (R) and the corresponding P values. The threshold of significance was set to be P < 0.05.
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