Experimental Therapeutics

Identification of 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol, a Novel Antibacterial Compound Active against Persisters of Pseudomonas aeruginosa

Veerle Liebens, Valerie Defraine, Wouter Knapen, Toon Swings, Serge Beullens, Romu Corbau, Arnaud Marchand, Patrick Chaltin, Maarten Fauvart, Jan Michiels
DOI: 10.1128/AAC.00836-17

ABSTRACT

Antibiotics typically fail to completely eradicate a bacterial population, leaving a small fraction of transiently antibiotic-tolerant persister cells intact. Persisters are therefore seen to be a major cause of treatment failure and greatly contribute to the recalcitrant nature of chronic infections. The current study focused on Pseudomonas aeruginosa, a Gram-negative pathogen belonging to the notorious ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and, due to increasing resistance against most conventional antibiotics, posing a serious threat to human health. Greatly contributing to the difficult treatment of P. aeruginosa infections is the presence of persister cells, and elimination of these cells would therefore significantly improve patient outcomes. In this study, a small-molecule library was screened for compounds that, in combination with the fluoroquinolone antibiotic ofloxacin, reduced the number of P. aeruginosa persisters compared to the number achieved with treatment with the antibiotic alone. Based on the early structure-activity relationship, 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009) was selected for further characterization. Combination of SPI009 with mechanistically distinct classes of antibiotics reduced the number of persisters up to 106-fold in both lab strains and clinical isolates of P. aeruginosa. Further characterization of the compound revealed a direct and efficient killing of persister cells. SPI009 caused no erythrocyte damage and demonstrated minor cytotoxicity. In conclusion, we identified a novel antipersister compound active against P. aeruginosa with promising applications for the design of novel, case-specific combination therapies in the fight against chronic infections.

INTRODUCTION

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen best known as the dominant cause of life-threatening chronic airway infections in cystic fibrosis (CF) patients (1). Infections are also commonly found in highly vulnerable patients, such as burn wound victims, immunocompromised individuals, and patients residing in intensive care units (2, 3). In addition to an intrinsic resistance toward a wide variety of antibiotics, P. aeruginosa has the remarkable ability to acquire additional resistance mechanisms. The rapidly increasing resistance to multiple antibiotic classes, including the so-called last-resort polymyxins, classifies the pathogen as multidrug-resistant or even pan-resistant and has caused P. aeruginosa infections to become increasingly difficult to treat (2, 4, 5). P. aeruginosa is the 4th most common cause of health care-associated infections in Europe (8.9%), with 14% of all isolates reported to be resistant to at least three antimicrobial classes (6, 7). The World Health Organization (WHO) has recently ranked P. aeruginosa as critical in the global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, emphasizing its clear clinical importance and encouraging the development of novel antibacterial therapies (8, 9).

Some infections, however, prove difficult to eradicate despite the absence of clinically detectable resistance against the antibiotic used (10). This can be explained by the presence of persister cells, a small fraction of phenotypic variants in a genetically homogeneous population that is tolerant to treatment with high doses of antibiotics (11, 12). It is generally assumed that, upon successful treatment with a bactericidal antibiotic, the host immune system is able to cope with this small fraction of surviving cells. However, in situations in which the immune system is compromised or bacteria are able to evade the immune system, persisters become a threat to human health and are considered a major culprit in chronic infections (13). Persister cells greatly contribute to the observed antibiotic tolerance in biofilms and are responsible for the recalcitrant nature of chronic infections (10, 14, 15), and recently, evidence is gathering that persister cells can facilitate the emergence of genetic resistance (16–19). The targeting of persisters is therefore likely to greatly improve outcomes for patients with chronic infectious diseases. Despite the clear clinical relevance of persisters, few antipersister compounds have been described in the literature (20). Contributing to this is the still limited knowledge of the mechanisms behind persister formation in P. aeruginosa, the fact that multiple, redundant pathways seem to be involved, and the observation that the processes implicated in persistence seem to be species specific (21, 22). Therefore, the rational design of target-based antipersister therapies remains difficult.

Here, we describe the screening of a diverse set of small-molecule compounds that resulted in the identification of the potent antipersister compound SPI009. This molecule strongly reduces or even completely eradicates persisters of P. aeruginosa when administered in combination with mechanistically distinct antibiotics. Importantly, SPI009 retains its activity against several clinical isolates, directly kills persister cells in a highly efficient manner, and causes no hemolytic effects.

RESULTS

Identification and structure-activity relationship of antipersister compound SPI009.A small-molecule library comprising 23,909 diverse molecules (23) was screened to identify compounds that reduce the persister fraction of P. aeruginosa when they are used in combination with the fluoroquinolone antibiotic ofloxacin (10 μg/ml). To confirm the antipersister activity of the identified compounds, a range of concentrations (0 to 200 μM) was tested and the efficacy was assessed through viable cell counting. From this analysis, SPI001 was selected for further characterization. To explore the effect of chemical modifications of the molecule on the observed activity, commercially available chemical analogues were purchased and evaluated for their antipersister effect (Table 1). On the basis of preliminary experiments (data not shown), the evaluation of the analogues was carried out using a single concentration of 200 μM, corresponding to 68 μg/ml, in combination with 10 μg/ml of ofloxacin. Among the analogues that significantly reduced the persister fraction, only SPI009, SPI015, and SPI016 showed an increase in antipersister activity compared to the original hit SPI001. SPI009, 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol, had the most pronounced effect, reducing the persister fraction ∼7,200-fold (P < 0.0001) compared to that achieved with treatment with ofloxacin alone. From a structure-activity relationship (SAR) point of view, it is worth noticing that the 7 most active compounds (those producing a fold decrease in the persister fraction of P. aeruginosa of >39 compared to that achieved with ofloxacin alone) are all secondary amines (R3 = hydrogen), whereas most of the other compounds are tertiary amines (the exceptions were SPI011, SPI014, and SPI020). Moreover, the hydroxyl residue (R2) does not seem to be essential for good biological activity (see the results for SPI015), although this should be confirmed by the evaluation of additional analogues. Finally, it is striking that the most active analogue (SPI009) was the only compound bearing a phenethyl residue on R4 instead of a benzyl residue, indicating that there is flexibility in the chain length. On the basis of all these results, SPI009 was selected for further characterization. The antibacterial activity of SPI009 was also evaluated by determination of the MIC value. The MIC value, defined as the minimal concentration required to completely inhibit bacterial growth, calculated for P. aeruginosa in 1:20 Trypticase soy broth (TSB) was 150 μM (corresponding to approximately 51 μg/ml).

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TABLE 1

SAR for SPI009 and chemical analogsa

SPI009 directly kills isolated persister cells.To investigate whether SPI009 needs to be administered simultaneously with ofloxacin for maximal activity, the compound was added at different time points during ofloxacin treatment. For this, P. aeruginosa stationary-phase cells were first treated with ofloxacin (10 μg/ml) and SPI009 was subsequently added after 0, 5, and 24 h (Fig. 1a to c). For each experiment, complete eradication of the bacterial population was achieved within 24 h after adding SPI009. These results indicate that SPI009 can be administered at any point during treatment without affecting its activity, thus broadening treatment options.

FIG 1
FIG 1

Timing of SPI009 addition to ofloxacin treatment does not influence activity. Stationary-phase cells of a PA14 wild-type culture were treated for 72 h with ofloxacin or the combination of ofloxacin and 68 μg/ml SPI009. SPI009 was added to the cultures at the beginning of treatment (a) or 5 h (b) or 24 h (c) after the onset of treatment, as indicated by the arrows. During treatment, the number of viable cells was determined at 24, 48, and 72 h by counting the number of CFU. Addition of the solvent DMSO (1%, vol/vol) did not result in any significant killing of the bacterial cells (data not shown). Data points represent the averages from three independent repeats, and error bars indicate standard errors of the means (SEM).

Based on these results, we concluded that SPI009 may act in two different ways. Either SPI009 wakes up persister cells, thereby rendering them sensitive to the bactericidal action of ofloxacin, or SPI009 kills the persister cells directly. Examples of both strategies have been reported in the literature (24–27). To discriminate between these two possibilities, persister cells were isolated and treated with either 10 μg/ml ofloxacin, 17 to 68 μg/ml SPI009, or a combination of ofloxacin and SPI009 (Fig. 2). As expected, treatment of the isolated persisters with ofloxacin alone caused only a minor decrease in the number of surviving cells, indicating the effective isolation of persister cells. In contrast, treatment of isolated persister cells with SPI009 caused a significant decrease in the number of surviving cells ranging between 0.71 ± 0.24 log unit and complete eradication compared to the number of surviving cells with ofloxacin treatment. Treatment with the combination of SPI009 at 34 μg/ml with ofloxacin was able to completely eliminate all bacterial cells (Fig. 2). These results show that SPI009 is capable of directly killing persister cells, even in the absence of antibiotics.

FIG 2
FIG 2

SPI009 directly kills isolated persister cells. Persister cells were isolated by means of ofloxacin treatment, after which they were treated for 5 h with either 1% DMSO, 10 μg/ml ofloxacin (OFX), 17 to 68 μg/ml SPI009, or the combination of ofloxacin with SPI009. After treatment, cells were washed, diluted, and plated out to determine the number of surviving persister cells. Data points correspond to the means from three independent repeats, and error bars represent SEM values. Significant differences from the results of ofloxacin treatment, unless indicated otherwise, were determined with log10-transformed data. *, P ≤ 0.05; ****, P ≤ 0.0001. ND, not detected.

SPI009 is capable of killing both normal and persister cells of P. aeruginosa.Previous experiments have clearly shown that SPI009 efficiently targets persister cells. To gain more information about the bactericidal effect on normal, nonpersister cells, stationary-phase cultures were treated with 10 μg/ml of ofloxacin or 17 to 68 μg/ml of SPI009. As expected, treatment with 10 μg/ml of ofloxacin caused a significant decrease in the number of surviving cells and allowed only persister cells to survive. Treatment of the culture with 34 or 68 μg/ml of SPI009 alone also significantly decreased the number of surviving cells compared to that of the untreated control, with reductions of 3.22 ± 0.38 and 6.36 ± 0.39 log units, respectively (Fig. 3). Since these decreases are larger than the expected persister fraction, the results obtained clearly show that SPI009 is capable of killing both persister and normal cells.

FIG 3
FIG 3

SPI009 targets both normal and persister cells. Stationary-phase cells of the P. aeruginosa PA14 wild type were treated for 5 h with 1% DMSO (control), 10 μg/ml ofloxacin (OFX), or 17 to 68 μg/ml of SPI009. After treatment, the cells where washed, diluted, and plated onto solid medium to determine the number of surviving cells. Data points represent the means from three independent repeats. Error bars represent SEM. Statistical analysis was performed on log-transformed CFU data comparing the effects of the different treatment conditions with the untreated control. ****, P ≤ 0.0001. ND, not detected.

Killing kinetics of SPI009 as mono- and cotherapy.To assess the killing kinetics of SPI009, stationary-phase cultures were exposed to different treatments from 15 min to 24 h. In order to evaluate both the antibacterial and the antipersister effects of the compound, cells were treated with SPI009 alone (17 to 34 μg/ml) or with the combination of SPI009 and ofloxacin (10 μg/ml), respectively. The time-kill curves obtained showed a slight biphasic pattern, where most killing was obtained in the first 3 to 4 h (Fig. 4). When the treatments were compared, treatment with the combination of ofloxacin and SPI009 always outcompeted the monotherapies. The combination of ofloxacin with 17 or 34 μg/ml SPI009 completely eradicated the culture after 24 h or 5 h of treatment, respectively. In comparison, 5 h of treatment with 10 μg/ml ofloxacin or 100 μM SPI009 alone caused 2.87 ± 0.35-log-unit and 4.14 ± 0.35-log-unit decreases in the number of surviving cells, respectively. As suspected, use of SPI009 alone also had an effect on the bacterial culture, causing maximal 2.36 ± 0.38- and 4.38 ± 0.42-log-unit decreases after 3 h of treatment with 17 or 34 μg/ml, respectively. The effect of SPI009 was comparable to that of ofloxacin, with a minor increase in the number of bacterial CFU being seen after 3 h for SPI009.

FIG 4
FIG 4

Killing kinetics of SPI009 as a monotherapy or combination therapy. Stationary-phase cells were treated with 1% DSMO (control), 10 μg/ml ofloxacin (OFX 10), 17 or 34 μg/ml SPI009, or the combination of ofloxacin with 17 or 34 μg/ml SPI009. Samples were taken at 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 24 h after the onset of treatment, after which they were washed and appropriate dilutions were plated out to assess the number of surviving cells. Data points represent the averages from at least three independent repeats, with error bars indicating SEM values.

The activity of SPI009 is antibiotic independent.In addition to ofloxacin, other antibiotics used to treat P. aeruginosa infections in the clinic include the aminoglycoside amikacin and the cephalosporin ceftazidime (28). As shown in Fig. 5a, 5 h of treatment with SPI009 in combination with 75 μg/ml amikacin completely eradicated the stationary-phase culture, while treatment with the combination of SPI009 at 17 or 34 μg/ml and ofloxacin resulted in significant 3.36 ± 0.45- and 5.58 ± 0.45-log-unit decreases, respectively. The treatment with the combination of ceftazidime and 34 μg/ml SPI009 also significantly reduced the persister fraction (>3,500-fold) in an exponentially growing culture (Fig. 5b). These results clearly show that SPI009 can be combined with different classes of antibiotics and thus possesses antibiotic-independent activity.

FIG 5
FIG 5

The combination of SPI009 with antibiotics of distinct classes reveals an antibiotic-independent effect. Stationary-phase cells (a) and exponential-phase cells (b) of the P. aeruginosa PA14 wild type were treated for 5 h with ofloxacin (OFX; 10 μg/ml), amikacin (AMK; 75 μg/ml), or ceftazidime (CAZ; 30 μg/ml) in combination with 1% DMSO (black bars) or 17 to 34 μg/ml SPI009 (white bars). Data points correspond to the means from three independent repeats. Error bars represent SEM. Statistical analysis compared the effect of antibiotic treatment alone with that of the different combination treatments. ****, P ≤ 0.0001. ND, not detected.

SPI009 shows potent activity against several clinical isolates.To test whether SPI009 is also active against other clinically relevant strains, we selected five P. aeruginosa strains to obtain a panel of isolates that originated from different human sources, such as burn wounds, urine, throat, and bronchus, and that had different resistance patterns. Being one of the most dominant bacterial species in the lungs of CF patients, several P. aeruginosa isolates from the sputum or bronchus of CF patients were included in the panel. Ofloxacin concentrations were optimized for each strain (data not shown). Stationary-phase cells of the different cultures were treated with 10 or 100 μg/ml ofloxacin and the combination of ofloxacin with 17 or 34 μg/ml of SPI009. Six out of the eight isolates proved extremely susceptible to killing by the combination treatment, resulting in a (nearly) complete eradication of the bacterial population (Fig. 6). For the least sensitive strain, strain AA249, which was resistant to aztreonam, ciprofloxacin, ceftazidime, imipenem, and meropenem, the combination of ofloxacin with 17 or 34 μg/ml SPI009 still resulted in a significant 2.06 ± 0.55- and 4.69 ± 0.55-log-unit reduction in the number of surviving cells, respectively. Evaluation of isolates derived from CF patients showed modest sensitivity to ofloxacin and demonstrated the potent activity of SPI009, resulting in 4.1 ± 0.3-, 4.8 ± 0.3-, and 3.7 ± 0.5-log-unit decreases in the number of surviving cells when cells were treated with the combination of ofloxacin and 34 μg/ml SPI009 for DAF87-203, PA1256, and PA1255, respectively. In summary, SPI009 showed potent antipersister activity against all clinical isolates tested, independently of the source of isolation, and was even capable of strongly reducing the survival of multidrug-resistant strains and isolates from CF patients.

FIG 6
FIG 6

Effect of SPI009 against several clinical P. aeruginosa isolates. (a) Stationary-phase cells of several clinical isolates were treated for 5 h with ofloxacin (OFX) alone or combined with SPI009. After treatment, the cells were washed, diluted, and plated onto solid medium to determine the number of surviving cells. Data points represent the means from at least three independent repeats, and error bars represent SEM. Statistical analysis was done on log10-transformed CFU data. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. ND, not detected. (b) Profiles of resistance of the different strains to imipenem (IPM), meropenem (MEM), ceftazidime (CAZ), aztreonam (ATM), ciprofloxacin (CIP), piperacillin (PIP), ticarcillin (TIC), ofloxacin (OFX), colistin (CST), and piperacillin-tazobactam (TZP).

SPI009 causes no significant cytotoxicity or hemolysis.The in vitro activity of SPI009 was clearly demonstrated in different experimental setups. An important factor determining its in vivo potential is the cytotoxicity of SPI009 in eukaryotic cell lines. For this, embryonic kidney HEK293T cells were treated with increasing concentrations of SPI009 ranging from 4.25 to 136 μg/ml for 24 h. The cytotoxicity was determined by spectroscopic detection of lactate dehydrogenase (LDH), with three independent repeats resulting in a 50% inhibitory concentration (IC50) of 32.3 ± 0.81 μg/ml. Another important factor to be considered for the clinical use of SPI009 is possible hemolytic activity. This was evaluated by exposing horse erythrocytes to increasing concentrations of SPI009 (8.5 to 34 μg/ml) for 1 h. The absorbance at 540 nm was measured, and the percent hemolysis relative to that for the positive control (treated with 0.1% Triton X-100) was determined. The results indicated no significant difference between the results obtained with the negative control or dimethyl sulfoxide (DMSO; carrier control; 0.5%) and concentrations of up to 34 μg/ml SPI009 or between the results obtained with the different testing agents and 0% hemolysis (see Fig. S1 in the supplemental material). While the lack of any hemolytic activity is promising, additional adaptation of the chemical structure of SPI009 is desirable to further decrease the moderate cytotoxic effects without affecting its antibacterial properties when applications other than the topical treatment of infections are envisioned.

DISCUSSION

The rate of resistance of P. aeruginosa to the most commonly used antibiotics is rapidly increasing worldwide, and even resistance to colistin and polymyxin B, the antibiotics currently used as a last resort in the treatment of P. aeruginosa infections, has been reported (4, 29, 30). Besides this rapidly increasing multidrug resistance, treatment of P. aeruginosa infections is further compromised by the ability of the organism to form biofilms and the presence of an antibiotic-tolerant persister fraction. Persister cells are able to withstand antibiotic treatment, thereby greatly hampering efficient eradication of the bacterial infection, contributing to the recalcitrant nature of chronic infections and increasing the chance of resistance development (13, 16, 31). Multiple international organizations and research groups acknowledge the increasing threat of bacterial infections, predicted to cause over 10 million deaths annually by 2050 (9), and research into new antibacterial and antipersister therapies is increasing. In this study, a screen to search for novel antipersister compounds capable of significantly decreasing the persister fraction in combination with the fluoroquinolone antibiotic ofloxacin led to the identification of 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009).

Small persister cell numbers, the different parallel mechanisms behind their formation and conservation, and the still limited knowledge greatly impede the rational design of target-based antipersister therapies. Nevertheless, several antipersister molecules have been described in the literature. Theoretically, three approaches may be used to combat bacterial persister cells: (i) waking up persisters and thereby rendering them sensitive to an antibiotic, (ii) directly killing persister cells, and (iii) preventing the formation of persister cells. Experiments both on isolated persister cells (Fig. 2) and at the population level (Fig. 3) revealed the ability of SPI009 to significantly decrease the number of surviving cells when it was added alone. These results clearly demonstrate that SPI009 does not depend on ofloxacin to exert its activity and is capable of directly killing persister cells, categorizing SPI009 in the second class of antipersister molecules. Other examples of this class include membrane-acting molecules, such as peptides shown to target Escherichia coli cells, including persisters (32), and NH125, a compound identified through large-scale screening capable of eradicating methicillin-resistant Staphylococcus aureus persisters through membrane permeabilization (33). ADEP4, an acyldepsipeptide, promotes self-digestion in S. aureus through constitutive activation of the ClpP protease, and combination of ADEP4 with rifampin completely eradicated biofilm infections in vitro and in vivo (25). More recently, the anticancer drug mitomycin C was described to actively kill persister cells of E. coli, S. aureus, and P. aeruginosa (34). A more rational approach was used in the development of the artilysin Art-175, consisting of a bacteriophage genome-encoded endolysin coupled to a peptide for guidance through the bacterial outer membrane. Art-175 is capable of puncturing the peptidoglycan, resulting in cell lysis, and was shown to be active against persister cells of both P. aeruginosa (24) and Acinetobacter baumannii (35). Another target-based approach led to the identification of a group of quorum-sensing (QS) inhibitors that specifically target the P. aeruginosa MvfR system. Besides disrupting cell-to-cell communication and decreasing infection, these compounds were the first molecules identified to limit the formation of persister cells in P. aeruginosa (36).

When a normal bacterial population consisting of both persister and nonpersister cells was treated with SPI0009, it was observed that the activity of the compound was not restricted to nondividing persister cells but also encompassed normal, nonpersister cells (Fig. 3). MIC assays, however, revealed a relatively high concentration of 150 μM for SPI009, corresponding to 51 μg/ml, well above the MIC values for most conventional antipseudomonal antibiotics (37). Taken together, these results suggest a primary activity of SPI009 against nondividing or persister cells, with SPI009 having an advantageous secondary effect against normal, actively dividing cells. Coates and coworkers even suggested the use of alternative, more relevant parameters, such as the minimal stationary cidal concentration (MSC) or the minimal dormicidal concentration (MDC), for evaluation of the activities of compounds against nondividing cells (38–40). Experiments were further focused on stationary-phase cultures, since the absence of active growth, a higher persister fraction, and nutritional starvation result in a more pronounced antibiotic tolerance and, partly due to the similarities to biofilms, increased clinical relevance (13, 41). Other antipersister molecules primarily aimed at targeting nonmultiplying cells have been described for Mycobacterium tuberculosis (42) and S. aureus (43).

While the initial screening was done in combination with ofloxacin, further experiments were undertaken to explore the combination spectrum of SPI009. The limitation of described antipersister therapies often lies in the fact that they increase the susceptibility of persister cells to one or a limited number of antibiotics. It has, however, been suggested that the multidrug-tolerant persister population is actually composed of several subpopulations of persister cells, each of which is characterized by its own tolerance profile. This hypothesis is supported by the observed differences in the persister fraction upon treatment of a population with distinct classes of antibiotics (44–47). Previous results already indicated an antibiotic-independent effect, and additional experiments showed that SPI009 is capable of reducing the persister fraction in combination with at least two additional mechanistically different classes of antibiotics (Fig. 5). Combination of SPI009 with the commonly used aminoglycoside amikacin, which acts by inhibiting translation, or ceftazidime, a cephalosporin antibiotic acting on cell wall biogenesis, resulted in complete eradication or significant decreases in the number of surviving cells, proving that SPI009 is capable of targeting multiple subpopulations of persisters. For all antibiotics tested, the addition of SPI009 clearly enhanced bacterial killing, making it a good candidate for the case-specific design of antibacterial cotherapies. The increasing rates of resistance to most antibiotics, together with the current lack of novel antipseudomonal compounds, have recently resulted in the increased use of antibiotic combination therapies. Although the possible negative side effects need to be studied in more detail, addition of a different antibiotic or nonantibiotic adjuvant to conventional therapy has the potential to lower the concentrations of both agents, reduce treatment times, combine different modes of action, and facilitate the treatment of resistant strains (48–50).

The currently described antipersister molecules can be classified into three broad categories on the basis of their mechanisms of action: (i) compounds that directly kill persister cells, (ii) compounds that sensitize persister cells to antibiotic killing, and (iii) compounds that prevent or decrease persister formation. The ability of SPI009 to directly kill persister cells, combined with its excellent activity in combination with distinct classes of antibiotics, suggests that SPI009 belongs to the first class. Several compounds capable of directly killing persister cells have been described and use different strategies to kill persister cells, such as depolarization and destruction of the cell membrane, DNA cross-linking, inhibition of essential enzymes, and generation of reactive oxygen species (20, 51, 52). Additional research will be necessary to further unravel the mode of action, but preliminary data suggest that SPI009 acts primarily by causing membrane damage (V. Defraine et al., unpublished data).

In conclusion, we identified 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol to be a promising compound for use in the development of future antipersister therapies. SPI009 is capable of significantly reducing or even eliminating the persister fraction of the Gram-negative bacterial pathogen P. aeruginosa by directly killing antibiotic-tolerant persister cells. Due to its activity against both nondividing and dividing cells and the possibility to combine SPI009 with mechanistically different classes of antibiotics, this molecule has the potential to serve as an adjuvant in antibacterial combination therapies to treat infections containing a mix of bacteria in different growth phases. The absence of any hemolytic effects and the limited cytotoxicity, together with the possibility to further adapt the chemical structure to increase the selectivity index, encourage further research into SPI009 and the development of novel antibacterial strategies in the fight against chronic infections.

MATERIALS AND METHODS

Bacterial strains, human cell lines, and culture conditions.Bacterial strains were cultured in 1:20-diluted Trypticase soy broth (TSB; 1:20 TSB) at 37°C with shaking at 200 rpm. For solid medium, TSB was supplemented with 1.5% agar. Human HEK293T cells were cultivated in Dulbecco modified Eagle medium containing 5% heat-inactivated fetal calf serum and kept at 37°C with 5% CO2. The following antibiotics were used: ofloxacin and ceftazidime (Sigma-Aldrich) and amikacin (Acros). Concentrations are indicated throughout the text. The small-molecule library was provided by the Centre for Drug Design and Discovery (CD3) of KU Leuven (23, 53). The bacterial strains used in this study are listed in Table 2.

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TABLE 2

Strains used in this study

Antipersister screening assay.The small-molecule library was screened for antipersister activity as previously described (54). Briefly, stationary-phase cells of P. aeruginosa cultured in 1:20 TSB were treated for 5 h with a combination of ofloxacin (10 μg/ml) and the test compound (10 μg/ml, corresponding to 20 to 50 μM compound). Treated cells were diluted 100-fold into fresh TSB growth medium and incubated in an automated optical density (OD) plate reader (Bioscreen C; Oy Growth Curves Ab Ltd.) at 37°C with aeration. Previous research revealed a linear relationship between the number of cells incubated in the automated plate reader and the time that it takes to reach an OD at 600 nm (OD600) of 0.6, which was t(OD600 = 0.6) = −2.1574x + 28.792 (where t is the time needed to reach an OD600 of 0.6, and x is the logarithmic value of the number of cells incubated in the automated plate reader); the R2 value was 0.987 (55). On the basis of this relationship, 105 compounds that produced delayed growth and thus decreased the number of surviving persister cells were selected. Of the 15 most interesting compounds producing a significant reduction in the persister fraction, 5 compounds belonging to three structurally distinct families were further confirmed via plate counting. Family 1 proved to be the most active, and 19 chemical analogues of compound SPI001 were subjected to a structure-activity relationship study (Table 1).

MIC assay.MICs were determined using the EUCAST standards for broth microdilution (56). The starting inoculum was adapted to approximately 5 × 105 cells/ml and incubated in the presence of a 2-fold dilution series of SPI009 for 24 h with shaking at 37°C. The MIC was determined to be the lowest concentration completely inhibiting bacterial growth.

Treatment of isolated persister cells.Persister cells were isolated as described previously, with minor modifications (57). Stationary-phase cells were treated with ofloxacin (10 μg/ml) for 5 h. Higher ofloxacin concentrations or increased incubation times did not lead to a further reduction of the number of surviving cells (data not shown). Persisters were washed twice with 0.85% NaCl (centrifugation at 5,200 × g, 15 min, 4°C) and subsequently used for the killing assays, as described below.

Killing assays.Killing assays were performed as previously described (58). Briefly, stationary-phase cultures were treated with a combination of antibiotic and compound (to determine the antipersister effect) or compound alone (to determine the bactericidal effect). Antibiotic concentrations were chosen to allow only persister cells to survive, indicated by a drop in the killing rate. Volumes of 200 μl of the treated culture were dispensed in a 96-well plate, and the plate was incubated for 5 h at 37°C with shaking at 200 rpm. To explore the effects of different treatment regimens, SPI009 was added 0, 5, or 24 h after the onset of ofloxacin treatment, and the cells were treated for a total of 72 h. For time-kill curves, different treatment durations between 15 min and 24 h were chosen, and the treatments comprised DMSO (0.5%; solvent control), ofloxacin (10 μg/ml), SPI009 alone (17 or 34 μg/ml), and the combination of ofloxacin with SPI009. To assess the effects of different classes of antibiotics, stationary-phase or exponential-phase cultures were treated with 75 μg/ml amikacin or 30 μg/ml ceftazidime for 5 h. After treatment, the cells were washed twice in 10 mM MgSO4 (centrifugation at 3,300 × g, 10 min, 4°C), after which appropriate dilutions were plated on solid growth medium to determine the number of viable cells. The plates were monitored for 72 h to ensure detection of slow-growing colonies.

Cytotoxicity assay.Cytotoxicity was evaluated through colorimetric determination of the lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells (Cytotoxicity Detection kit Plus). HEK293T cells were seeded at concentrations of 1.25 × 104 cells/well in 50 μl of appropriate medium, and adhesion was allowed overnight. The eukaryotic cells were exposed to increasing concentrations of SPI009 for 24 h, after which LDH activity was measured according to the manufacturer's guidelines. The relative cytotoxicity (in percent) was determined as follows: [(value for the sample − value for the low control)/(value for the high control − value for the low control)] 100, where low control and high control refer to the controls treated with medium and lysis buffer, respectively.

Hemolytic assay.Hemolytic activity was assessed as described previously (59), with minor modifications. Defibrinated whole blood from horses (Oxoid) was washed three times with 10 mM Tris-HCl, 0.9% NaCl, pH 7.4 (centrifugation at 1,000 × g, 10 min, 4°C) with sequential removal of the buffy coat. Washed erythrocytes were diluted to a final concentration of 2% in 10 mM Tris-HCl, 0.9% HCl, pH 7.4, and preincubated in 1-ml volumes for 10 min (37°C). Erythrocytes (190 μl) were mixed with 10 μl of the different testing agents, and the mixture was incubated at 37°C for 1 h. Tris-HCl (10 mM), 0.9% HCl, pH 7.4, and 0.1% Triton X-100 were used as negative and positive controls, respectively. The test concentrations of SPI009 ranged from 8.5 to 34 μg/ml. After incubation, the erythrocyte solutions were centrifuged for 5 min (3,000 × g), and the absorbance of the supernatant was measured at 540 nm (BioTek multimode reader) to assess hemolytic damage. The values for the background controls were subtracted from the OD540 values, and percent hemolysis relative to that for the positive control (0.1% Triton X-100) was determined. Statistical analysis was performed on control-corrected OD540 values using unpaired, one-way analysis of variance (ANOVA) testing with appropriate correction for multiple comparisons (Dunnett's test) (significance level, α = 0.05).

Statistical analysis.Unless mentioned otherwise, all statistical calculations were performed on log10-transformed data using GraphPad Prism software (version 6.01). The effect of the different treatments on the number of CFU was analyzed using unpaired, one-way ANOVA testing with appropriate correction for multiple comparison (Dunnett's test) (significance level, α = 0.05). Averages are the results from at least three independent repeats.

ACKNOWLEDGMENTS

We thank Pierre Cornelis, Jean-Paul Pirnay, and Françoise van Bambeke for providing us with the P. aeruginosa PA14 wild-type strain and P. aeruginosa clinical isolates and Alex O'Neill and Liam Sharkey (School of Molecular and Cellular Biology, University of Leeds) for helpful discussions and comments on a previous version of the manuscript. We thank Annelies Van der Leyden for technical assistance.

V.L., V.D., W.K., and T.S. are the recipients of a Ph.D. grant from the Agency for Innovation through Science and Technology (IWT). This work was supported by grants from the KU Leuven Excellence Center (PF/2010/07), the KU Leuven Research Council (PF/10/010, NATAR), the Belgian Science Policy Office (BELSPO; IAP P7/28), and the Fund for Scientific Research, Flanders (FWO; G047112N, G0B2515N, G055517N).

FOOTNOTES

    • Received 20 April 2017.
    • Returned for modification 5 June 2017.
    • Accepted 9 June 2017.
    • Accepted manuscript posted online 19 June 2017.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00836-17 .

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KEYWORDS

Anti-Bacterial Agents
Pseudomonas aeruginosa
Pseudomonas aeruginosa
antibiotic tolerance
combination therapy
antipersister therapies

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