The reference laboratory strains Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33591 were used in the study. Bacteria were grown in Luria-Bertani (LB) medium (Sigma-Aldrich, St. Louis, MO, United States) at 37°C with shaking for liquid cultures and on TSA (Oxoid, Basingstoke, United Kingdom) plates at 37°C for enumeration of colony forming units (CFU).

Carbonyl cyanide m-chlorophenylhydrazone was purchased from Sigma-Aldrich. CCCP was diluted in DMSO to obtain a stock solution of 40 mg/mL and stored in aliquots at -20°C.

Ciprofloxacin, colistin, levofloxacin and meropenem were provided as purified powder by the manufacturer (Sigma–Aldrich). Daptomycin (Cubicin) was purchased as purified powder from Novartis (Basel, Switzerland). Gentamicin was supplied as a 10-mg/mL sterile solution by Sigma–Aldrich. A stock solution of 4 mg/mL of each antibiotic was prepared in sterile milli-Q water and stored in aliquots at -20°C.

C5 and TB_L1FK were synthesized by Proteogenix (Schiltigheim, France). Den-SB056 was provided by PolyPeptide (Limhamn, Sweden). Analysis of synthetic peptides by high performance chromatography (HPLC) and mass spectrometry revealed purity ≥98%. Peptides were diluted in milli-Q water to obtain a stock solution of 1 mM and stored at -80°C. The main physicochemical features of the peptides are shown in Table 1.

The susceptibility of stationary-phase cultures of P. aeruginosa and S. aureus to CCCP was determined. Bacteria were grown in LB medium for approximately 18 h at 37°C to obtain stationary-phase cells, as assessed by monitoring bacterial growth by optical density. A volume of 1 mL of the overnight cultures was incubated with 10 μL of CCCP at different concentrations (from 25 to 400 μg/mL) for 3 h at 37°C with shaking. Bacterial cultures exposed to DMSO were used as cell viability control. Following the incubation, bacteria were washed twice in sodium-phosphate buffer (10 mM SPB, pH 7.4) by centrifugation (1700 × g for 10 min), subsequently diluted 10-fold in LB and plated on TSA to determine the number of CFU.

Concentration of CCCP and exposure time able to efficiently induce persistence in stationary-phase cultures of P. aeruginosa and S. aureus were investigated. Overnight cultures of P. aeruginosa and S. aureus were incubated with different concentrations of CCCP (from 25 to 400 μg/mL) for 1 or 3 h at 37°C with shaking. The number of CCCP-induced persisters was determined based on their survival to antibiotic treatments (Kwan et al., 2013). To this end, after the pretreatment with CCCP, bacteria were washed twice in SPB (1700 × g for 10 min) and re-suspended in SPB/LB (SPB supplemented with 1% LB) at a final density of 1 × 10⁶ CFU/mL. Hence, bacterial suspensions were exposed to different antibiotics for 3 h: ciprofloxacin (5 μg/mL) or meropenem (10 μg/mL) in the case of P. aeruginosa and levofloxacin (2.5 μg/mL) or gentamicin (10 μg/mL) in the case of S. aureus. CCCP-untreated and CCCP-pretreated bacteria re-suspended in SPB/LB alone were used as cell viability controls. The antibiotic concentrations used in the experiments were chosen based on preliminary assays in which stationary-phase cultures of P. aeruginosa and S. aureus were treated with increasing concentrations of each antibiotic. The threshold concentrations at which a killing plateau was reached and only spontaneous PCs survived were used for the experiments (Fauvart et al., 2011). Exposure of CCCP-pretreated cells to antibiotics was performed in SPB/LB in order to avoid their metabolic reactivation and replication after CCCP removal. Following the antibiotic treatment, bacteria were plated on TSA and the number of surviving PCs was determined by CFU counting after 48 h of incubation at 37°C.

The metabolic profile of CCCP-induced persisters of P. aeruginosa and S. aureus was evaluated by real-time monitoring of bacterial heat production through isothermal microcalorimetry. A 48-channel isothermal microcalorimeter (Thermal Activity Monitor, Model 3102 TAM III, TA Instruments, New Castle, DE, United States) was used for the study. Microcalorimetry glass ampoules were aseptically filled with 1 mL of the stationary-phase cultures and 10 μL of CCCP at a final concentration of 200 and 400 μg/mL for P. aeruginosa and S. aureus, respectively. Ampoules were inserted into the calorimeter, placed in the thermal equilibration position for 15 min and then lowered into the measuring position. After 45 min of signal stabilization, measurements of the heat flow were initiated and recorded for 24 h. Data analysis was performed with the manufacturer software (TAM Assistant) and GraphPad Prism (GraphPad Software, La Jolla, CA, United States), and results were plotted as heat flow (in microwatts) or total heat (in joules) versus time.

The metabolic activity of CCCP-induced PCs of P. aeruginosa and S. aureus was also evaluated by determining the oxidoreductase activity through flow cytometry. Stationary-phase cultures were incubated with CCCP at 200 and 400 μg/mL for P. aeruginosa and S. aureus, respectively. After a 3-h exposure, bacteria were washed twice in SPB (1700 × g for 10 min) and diluted to reach a cell density of 1 × 10⁶ CFU/mL. Then, bacterial suspensions were stained with the RedoxSensor^TM Green reagent (RSG) (BacLight^TM RedoxSensor^TM Green Vitality Kit, Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions. Briefly, 1 mL of the bacterial suspension was incubated with 1 μL (P. aeruginosa) or 0.1 μL (S. aureus) of 1 mM RSG for 10 min at 37°C in the dark. Stained samples were analyzed with a BD Accuri C6 flow cytometer (BD Biosciences, Mountain View, CA, United States) equipped with a 488 nm laser and a 533/30 nm optical filter. Data were collected and analyzed with BD Accuri C6 software (BD Biosciences), and results were plotted as cell counts versus fluorescence intensity.

The time required for the revival of CCCP-induced persisters was determined by evaluating bacterial heat production and bacterial growth after the removal of CCCP. Furthermore, the susceptibility of CCCP-treated cells to different antibiotics after the revival was assessed in terms of MIC and MBC values according to the standard microdilution method and compared to that of untreated cultures. Briefly, stationary-phase cultures were incubated for 3 h with CCCP at 200 and 400 μg/mL for P. aeruginosa and S. aureus, respectively. Following incubation, bacteria were washed twice in SPB (1700 × g for 10 min) in order to remove CCCP, re-suspended in fresh LB and diluted to a final density of 1 × 10⁶ CFU/mL. Microcalorimetry ampoules were aseptically filled with 1 mL of the bacterial cultures and heat production was continuously determined for 24 h as previously described. In parallel, bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) of the bacterial cultures at different incubation times. Once the early exponential phase of growth was reached (OD₆₀₀ of 0.5), bacteria were harvested, diluted in LB to a final density of 1 × 10⁶ CFU/mL and incubated into 96-well microplates with different concentrations of ciprofloxacin (0.125–2 μg/mL) or meropenem (2.5–40 μg/mL) in the case of P. aeruginosa and levofloxacin (0.125–2 μg/mL) or gentamicin (5–80 μg/mL) in the case of S. aureus. Bacterial cells suspended in LB alone were used as viability control. MIC values were defined as the lowest concentration of each antibiotic resulting in the complete inhibition of visible growth after 24 h of incubation at 37°C. For the determination of MBC values, bacteria from wells that did not exhibit growth were serially diluted and plated on TSA for CFU counting. The minimal concentration of antibiotic causing a CFU reduction of at least 3 Log₁₀ compared to the initial inoculum was taken as the MBC European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (2000). A number of 10 CFU/mL was taken as detection limit.

The activity of three different AMPs, colistin and daptomycin was tested against CCCP-induced persisters of P. aeruginosa and S. aureus. Persister-enriched cultures of P. aeruginosa and S. aureus were obtained by treatment with CCCP as described above. CCCP-treated and untreated bacteria were diluted in SPB/LB to a density of 1 × 10⁶ CFU/mL and incubated in the absence (viability control) or in the presence of different concentrations of C5, Den-SB056 and TB_L1FK (0.435–14 μM). Colistin (0.435–14 μM) and daptomycin (3.5–224 μM) were assayed against CCCP-treated cells of P. aeruginosa and S. aureus, respectively. For daptomycin testing, bacteria were diluted in deionized water supplemented with 1% LB and 75 μg/mL CaCl₂. After 3 h of incubation at 37°C, samples were serially diluted and plated on TSA to determine the number of CFU.

All experiments were performed at least in triplicate and differences between mean values of groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc test. A p-value < 0.05 was considered statistically significant. Data analysis was performed with GraphPad InStat (GraphPad Software, La Jolla, CA, United States).

We first assessed the susceptibility of stationary-phase cultures of P. aeruginosa and S. aureus to CCCP in order to identify the concentrations able to ensure maximum cell survival. As shown in Figure 1, exposure of P. aeruginosa cultures to CCCP at concentrations up to 200 μg/mL did not exert a significant killing effect. In the case of S. aureus, only a minor effect on cell viability was observed by treating cultures with CCCP at concentrations up to 400 μg/mL.

The optimal concentration of CCCP and exposure time required for the induction of persistence in stationary-phase cultures of P. aeruginosa and S. aureus were established based on the acquisition of tolerance to antibiotics following the treatment with CCCP. Indeed, the lack of specific markers for persistence makes antibiotic tolerance the only reliable evidence of the persister status (Brauner et al., 2016). Two fluoroquinolones, ciprofloxacin (5 μg/mL) and levofloxacin (2.5 μg/mL), were initially used to assess the acquisition of antibiotic tolerance in CCCP-pretreated cells of P. aeruginosa and S. aureus, respectively. When these antibiotics were used against bacteria in the absence of CCCP pretreatment, only a very low percentage of the initial population survived antibiotic exposure (Figures 2A,D). In particular, less than 0.01% of stationary-phase cultures of P. aeruginosa survived ciprofloxacin exposure (Figure 2A), while approximately 0.5% of the initial inoculum of S. aureus tolerated levofloxacin treatment (Figure 2D), in accordance with the presence of naturally occurring PCs within the cultures. Interestingly, the percentage of bacteria surviving antibiotic treatment markedly increased in a dose-dependent manner following CCCP pretreatment. In the case of P. aeruginosa, cultures pretreated with CCCP at 200 μg/mL for 3 h showed a 5000-fold increase in the number of persister-like cells as compared to the untreated control (Figure 2A). In the case of S. aureus, the highest percentage of persister-like cells was obtained by pretreating cultures with CCCP at 400 μg/mL. Indeed, after a 3-h pretreatment, approximately 60% of the initial population survived levofloxacin exposure (Figure 2D). Furthermore, a time-dependent increase in the number of persister-like cells emerged from the evaluation of the antibiotic susceptibility of both bacterial species following a pre-incubation of 1 or 3 h with the identified optimal concentrations of CCCP. Indeed, a 3-h exposure to CCCP produced a statistically significant increase in the survival rate of P. aeruginosa and S. aureus to fluoroquinolones as compared to a pretreatment of 1 h (Figures 2B,E).

The ability of CCCP to induce persistence was confirmed by exposing CCCP-pretreated cultures to antibiotics with a different mechanism of action than fluoroquinolones, namely the β-lactam meropenem and the aminoglycoside gentamicin against P. aeruginosa and S. aureus, respectively. As shown in Figures 2C,F, stationary-phase cultures pretreated with CCCP for 3 h exhibited a significantly increased tolerance to meropenem and gentamicin as compared to the untreated controls. In particular, treatment of P. aeruginosa cultures with CCCP at 200 μg/mL determined a 200-fold increase in the percentage of bacteria tolerant to meropenem (35% of the starting bacterial population) (Figure 2C). Analogously, survival of S. aureus cultures to gentamicin increased by approximately 500 times upon exposure to CCCP at 400 μg/mL, resulting in 20% of the cells becoming persisters (Figure 2F).

Since a 3-h exposure to CCCP at 200 and 400 μg/mL resulted to be efficient in the induction of a persister-like status in P. aeruginosa and S. aureus respectively, these optimal conditions were adopted for the generation of persister-enriched cultures in all the subsequent experiments.

Global metabolic activity of CCCP-treated cells of P. aeruginosa and S. aureus was quantitatively assessed in real time by isothermal microcalorimetry, a highly sensitive technique that allows to monitor microbial metabolic activity in the presence or absence of growth-inhibiting compounds through the measurement of heat production over time (Braissant et al., 2010; Di Luca et al., 2017). As shown in Figure 3, untreated stationary-phase cultures of both P. aeruginosa and S. aureus released heat over the time resulting in typical heat flow patterns. On the contrary, only a basal heat production was observed (<5 μWh) when both bacterial species were incubated with CCCP, suggesting an overall decrease in the metabolic activity of CCCP-treated cells.

The effect of CCCP treatment on bacterial oxidoreductase activity was assessed through cytofluorimetric analysis after staining of the cells with the fluorogenic dye RSG. RSG has been recently reported to be a convenient indicator of metabolic activity at the single cell level since it provides a stable green fluorescent signal when reduced by bacterial reductases in metabolically active cells (Orman and Brynildsen, 2013; Orman et al., 2016). In the case of both P. aeruginosa (Figure 4A) and S. aureus (Figure 4B), a 3-h exposure to CCCP determined a reduction of approximately three times in RSG fluorescence as compared to the untreated control, indicating a drop in bacterial oxidoreductase activity following CCCP treatment.

When incubated in a nutrient-rich environment, PCs have been shown to exhibit a characteristic lag phase before resumption of normal growth (Balaban et al., 2004; Gefen et al., 2008). Hence, in order to confirm further that CCCP pretreatment induced persistence, we evaluated the time required for the revival of the cultures upon removal of CCCP and exposure to fresh nutrients. The presence and duration of a time lag were initially determined by monitoring bacterial heat production through isothermal microcalorimetry. In the case of both P. aeruginosa (Figure 5A) and S. aureus (Figure 5C), identical heat flow patterns were obtained for CCCP-pretreated and untreated cultures. Nevertheless, CCCP-pretreated cells were characterized by a delay in heat production as compared to the untreated controls. Time of maximum heat production was extrapolated from the heat flow plots in order to determine the exact delay in the recovery of CCCP-pretreated bacteria. In this respect, a time lag of 1.5 h was recorded for CCCP-pretreated cultures of P. aeruginosa (Figure 5A), while a 1-h delay was observed in the case of S. aureus (Figure 5C). Similar results were also obtained when the revival was evaluated by measuring bacterial growth by optical density. Indeed, CCCP-induced persisters of both bacterial species displayed a lag phase of approximately 1 h before resuming replication at a normal rate (Figures 5B,D).

Susceptibility of CCCP-induced persisters to different classes of antibiotics was determined after the revival in order to verify the reversion of bacteria to a normal, antibiotic-sensitive phenotype. To this aim, the progeny of both CCCP-pretreated and untreated cells was harvested at early exponential growth phase and exposed to ciprofloxacin, gentamicin, levofloxacin or meropenem for the evaluation of the MIC and MBC values. As reported in Table 2, CCCP-pretreated and untreated cultures of both P. aeruginosa and S. aureus exhibited the same MIC and MBC values for each of the tested antibiotics, indicating that the loss of CCCP effect correlated with the sensitization to the action of antibiotics at the prior treatment levels.

The antimicrobial activity of three structurally different AMPs (Table 1) was determined by CFU counting against persister-enriched cultures of P. aeruginosa and S. aureus generated by CCCP treatment. Under the same experimental conditions, the anti-persister activity of the polypeptide colistin and the lipopeptide daptomycin was also assessed against CCCP-induced persisters of P. aeruginosa and S. aureus, respectively. In the case of both P. aeruginosa (Figure 6A) and S. aureus (Figure 6B), all three AMPs eradicated persister-enriched cultures to the limit of detection (10 CFU/mL) at concentrations ranging from 1.75 to 14 μM. Colistin displayed a bactericidal activity against CCCP-pretreated cells of P. aeruginosa at levels comparable to AMPs and determined the complete killing of the starting bacterial inoculum at concentrations as low as 3.5 μM (Figure 6A). On the contrary, a markedly reduced anti-persister activity against S. aureus was observed in the case of daptomycin, which exerted its bactericidal effect only at concentrations 32 to 64-fold higher than those of the tested AMPs (Figure 6B).

The susceptibility of CCCP-induced persisters to AMPs, colistin and daptomycin was compared to that of CCCP-untreated bacteria in terms of MBC values. As shown in Table 3, all tested AMPs and colistin exerted a bactericidal effect against CCCP-pretreated bacteria at levels comparable to those of CCCP-untreated bacteria. Indeed, the majority of the tested AMPs exhibited the same MBCs toward CCCP-pretreated and untreated cells of both P. aeruginosa and S. aureus. In the case of colistin, only a 2-fold increase in the MBC value against CCCP-pretreated bacteria of P. aeruginosa was recorded. Conversely, in the case of daptomycin, a 16-fold higher concentration was required to kill CCCP-induced persisters of S. aureus than that effective against untreated bacteria.