Fighting bacterial persistence: Current and emerging anti-persister strategies and therapeutics.

In addition to the well-known strategies of antibiotic resistance and biofilm formation, bacterial populations possess an additional survival strategy to endure hostile environments or antibiotic exposure. A small fraction of transiently antibiotic-tolerant phenotypical variants, called persister cells, is capable of surviving treatment with high doses of antibiotics. When antibiotic pressure drops, persisters that switch back to a normal phenotype can resume growth, ensuring survival of the bacterial population. Persister cells have been identified in every major pathogen, contribute to the antibiotic tolerance observed in biofilms, and are responsible for the recalcitrant nature of chronic infections. Also, evidence is accumulating that persister cells can contribute to the emergence of antibiotic resistance. Consequently, effective treatment of persister cells could greatly improve patient outcome. The small number of persisters and the redundancy in mechanisms of persister formation impede target-based development of anti-persister therapies. Nonetheless, the armory of anti-persister molecules is increasing. This review presents a comprehensive overview of anti-persister molecules and strategies described in literature to date and offers perspectives on potential anti-persistence targets and methods for the development of future therapies. Furthermore, we highlight in vivo model systems for pre-clinical testing and summarize ongoing clinical trials of candidate anti-persister therapeutics.

1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol Kills Pseudomonas aeruginosa through Extensive Membrane Damage.

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.

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

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.

Antibacterial Activity of 1-[(2,4-Dichlorophenethyl)amino]-3-Phenoxypropan-2-ol against Antibiotic-Resistant Strains of Diverse Bacterial Pathogens, Biofilms and in Pre-clinical Infection Models.

We recently described the novel anti-persister compound 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol (SPI009), capable of directly killing persister cells of the Gram-negative pathogen Pseudomonas aeruginosa. This compound also shows antibacterial effects against non-persister cells, suggesting that SPI009 could be used as an adjuvant for antibacterial combination therapy. Here, we demonstrate the broad-spectrum activity of SPI009, combined with different classes of antibiotics, against the clinically relevant ESKAPE pathogens Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterococcus faecium and Burkholderia cenocepacia and Escherichia coli. Importantly, SPI009 re-enabled killing of antibiotic-resistant strains and effectively lowered the required antibiotic concentrations. The clinical potential was further confirmed in biofilm models of P. aeruginosa and S. aureus where SPI009 exhibited effective biofilm inhibition and eradication. Caenorhabditis elegans infected with P. aeruginosa also showed a significant improvement in survival when SPI009 was added to conventional antibiotic treatment. Overall, we demonstrate that SPI009, initially discovered as an anti-persister molecule in P. aeruginosa, possesses broad-spectrum activity and is highly suitable for the development of antibacterial combination therapies in the fight against chronic infections.

Repurposing Toremifene for Treatment of Oral Bacterial Infections.

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 µM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 µM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

Efficacy of Artilysin Art-175 against Resistant and Persistent Acinetobacter baumannii.

Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility of Pseudomonas aeruginosa to Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistant Acinetobacter baumannii, even resulting in a complete elimination of large inocula (≥10(8) CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing of A. baumannii upon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against both A. baumannii and P. aeruginosa, two of the most life-threatening pathogens of the order Pseudomonadales.

A Whole-Cell-Based High-Throughput Screening Method to Identify Molecules Targeting Pseudomonas aeruginosa Persister Cells.

Despite its clinical relevance and the fact that the phenomenon of persistence was discovered in the 1940s, little is known about the mechanisms behind persister cell formation. Research in this field has mainly focused on the model organism Escherichia coli and few genetic determinants of persistence have been described in other bacterial species, impairing the development of target-based strategies to combat these antibiotic-tolerant cells. In this chapter we describe a top-down large-scale screening method capable of specifically identifying small molecule compounds that, in combination with conventional antibiotics, significantly reduce the persister fraction in Pseudomonas aeruginosa. The method is readily adaptable for other species. Further characterization and analysis of the mode of action of the identified compounds can provide additional insight into the mechanisms behind persister formation and can guide the development of future anti-persister therapies.

Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa.

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (∼ 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa.

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.