A new class of synthetic retinoid antibiotics effective against bacterial persisters.

A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant 'persister' subpopulations that exhibit high levels of tolerance to antibiotics and have a role in chronic or recurrent infections. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans-MRSA infection screen to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.

Activity of a novel protonophore against methicillin-resistant Staphylococcus aureus.

AIM: Compound 1-(4-chlorophenyl)-4,4,4-trifluoro-3-hydroxy-2-buten-1-one (compound 1) was identified as a hit against methicillin-resistant Staphylococcus aureus (MRSA) strain MW2. METHODS & RESULTS: The MIC of compound 1 against MRSA was 4 µg/ml. The compound showed enhanced activity at acidic pH by lowering bacterial intracellular pH and exhibited no lysis of human red blood cells at up to 64 µg/ml and its IC50 against HepG2 cells was 32 µg/ml. The compound reduced 1-log10 colony forming units of intracellular MRSA in macrophages and prolonged the survival of MRSA-infected Caenorhabditis elegans (p = 0.0015) and Galleria mellonella (p = 0.0002). CONCLUSION: Compound 1 is a protonophore with potent in vitro and in vivo activity against MRSA and no toxicity in mammalian cells up to 8 µg/ml that warrants further investigation as a novel antibacterial.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

NH125 kills methicillin-resistant Staphylococcus aureus persisters by lipid bilayer disruption.

BACKGROUND: NH125, a known WalK inhibitor kills MRSA persisters. However, its precise mode of action is still unknown. METHODS & RESULTS: The mode of action of NH125 was investigated by comparing its spectrum of antimicrobial activity and its effects on membrane permeability and giant unilamellar vesicles (GUVs) with walrycin B, a WalR inhibitor and benzyldimethylhexadecylammonium chloride (16-BAC), a cationic surfactant. NH125 killed persister cells of a variety of Staphylococcus aureus strains. Similar to 16-BAC, NH125 killed MRSA persisters by inducing rapid membrane permeabilization and caused the rupture of GUVs, whereas walrycin B did not kill MRSA persisters or induce membrane permeabilization and did not affect GUVs. CONCLUSION: NH125 kills MRSA persisters by interacting with and disrupting membranes in a detergent-like manner.

Inhibition of bacterial and fungal pathogens by the orphaned drug auranofin.

BACKGROUND: We identified auranofin as an antimicrobial compound utilizing a high-throughput screen using a Caenorhabditis elegans-Staphylococcus aureus infection model. Results/methodology: Treatment of infected nematodes with auranofin resulted in a prolonged survival rate of 95%, reached with 0.78 µg/ml. Further investigation of the antimicrobial activity of auranofin found inhibition against S. aureus, Enterococcus faecium and Enterococcus faecalis. Importantly, the fungal pathogens Cryptococcus neoformans was also effectively inhibited with an MIC at 0.5 µg/ml. Auranofin appears to target the thioredoxin system. CONCLUSION: This work provides extensive additional data on the antibacterial effects of auranofin that includes both reference and clinical isolates and reports a novel inhibition of fungal pathogens by this compound.

Micafungin Elicits an Immunomodulatory Effect in Galleria mellonella and Mice.

The echinocandin family of drugs is well characterized for antifungal function that inhibits ß-D-glucan synthesis. The aim of this work was to study whether micafungin, a member of the echinocandin family, elicits additional activities that prime the host's immune response. We found that in a Galleria mellonella model, prophylactic treatment with micafungin extended the life of Staphylococcus aureus-infected larvae (a pathogen to which the drug demonstrates no direct antimicrobial activity) compared to insects that did not receive micafungin (P < 0.05). The inhibition of pathogens in the G. mellonella infection model was characterized by a 2.43-fold increase in hemocyte density, compared to larvae inoculated with PBS. In a murine model where animals were provided micafungin prophylaxis 3 days prior to macrophage collection, macrophages were found associated with an average 0.9 more fungal cells per macrophage as compared to saline-treated animals. Interestingly, micafungin-stimulated macrophages killed 11.6 ± 6.2 % of fungal cells compared to 3.8 ± 2.4 % of macrophages from saline-treated animals. The prophylactic provision of micafungin prior to Candida albicans infection was characterized by an increase in the proinflammatory cytokines CXCL13 and SPP1 by 11- and 6.9-fold, respectively. In conclusion, micafungin demonstrated the ability to stimulate phagocytic cells and promote an immune response that can inhibit microbial infections.

Antibacterial properties of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile.

The emergence of multidrug-resistant bacterial strains has heightened the need for new antimicrobial agents based on novel chemical scaffolds that are able to circumvent current modes of resistance. We recently developed a whole-animal drug-screening methodology in pursuit of this goal and now report the discovery of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC) as a novel antibacterial effective against resistant nosocomial pathogens. The minimum inhibitory concentrations (MIC) of PSPC against Staphylococcus aureus and Enterococcus faecium were 4 µg/mL and 8 µg/mL, respectively, whereas the MICs were higher against the Gram-negative bacteria Klebsiella pneumoniae (64 µg/mL), Acinetobacter baumannii (32 µg/mL), Pseudomonas aeruginosa (>64 µg/mL), and Enterobacter spp. (>64 µg/mL). However, co-treatment of PSPC with the efflux pump inhibitor phenylalanine arginyl ß-naphthylamide (PAßN) or with sub-inhibitory concentrations of the lipopeptide antibiotic polymyxin B reduced the MICs of PSPC against the Gram-negative strains by >4-fold. A sulfide analog of PSPC (PSPC-1S) showed no antibacterial activity, whereas the sulfoxide analog (PSPC-6S) showed identical activity as PSPC across all strains, confirming structure-dependent activity for PSPC and suggesting a target-based mechanism of action. PSPC displayed dose dependent toxicity to both Caenorhabditis elegans and HEK-293 mammalian cells, culminating with a survival rate of 16% (100 µg/mL) and 8.5% (64 µg/mL), respectively, at the maximum tested concentration. However, PSPC did not result in hemolysis of erythrocytes, even at a concentration of 64 µg/mL. Together these results support PSPC as a new chemotype suitable for further development of new antibiotics against Gram-positive and Gram-negative bacteria.

Identification of an Antimicrobial Agent Effective against Methicillin-Resistant Staphylococcus aureus Persisters Using a Fluorescence-Based Screening Strategy.

Persisters are a subpopulation of normal bacterial cells that show tolerance to conventional antibiotics. Persister cells are responsible for recalcitrant chronic infections and new antibiotics effective against persisters would be a major development in the treatment of these infections. Using the reporter dye SYTOX Green that only stains cells with permeabilized membranes, we developed a fluorescence-based screening assay in a 384-well format for identifying compounds that can kill methicillin-resistant Staphylococcus aureus (MRSA) persisters. The assay proved robust and suitable for high throughput screening (Z`-factor: >0.7). In screening a library of hits from a previous screen, which identified compounds that had the ability to block killing of the nematode Caenorhabditis by MRSA, we discovered that the low molecular weight compound NH125, a bacterial histidine kinase inhibitor, kills MRSA persisters by causing cell membrane permeabilization, and that 5 µg/mL of the compound can kill all cells to the limit of detection in a 108 CFU/mL culture of MRSA persisters within 3h. Furthermore, NH125 disrupts 50% of established MRSA biofilms at 20 µg/mL and completely eradicates biofilms at 160 µg/mL. Our results suggest that the SYTOX Green screening assay is suitable for large-scale projects to identify small molecules effective against MRSA persisters and should be easily adaptable to a broad range of pathogens that form persisters. Since NH125 has strong bactericidal properties against MRSA persisters and high selectivity to bacteria, we believe NH125 is a good anti-MRSA candidate drug that should be further evaluated.

A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus.

The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC) of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16-64 µg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl2 respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT) at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 µg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 µg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 µg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.

Repurposing salicylanilide anthelmintic drugs to combat drug resistant Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to de novo development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong in vivo and in vitro activity against methicillin resistant S. aureus (minimum inhibitory concentration (MIC): 0.125 and 0.5 µg/ml respectively; minimum effective concentration: ≤ 0.78 µg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria Enterococcus faecium (MIC 0.25 and 2 µg/ml respectively), but not against the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes. The in vitro antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant S. aureus clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 µg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its in vitro MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.

Insect-derived cecropins display activity against Acinetobacter baumannii in a whole-animal high-throughput Caenorhabditis elegans model.

The rise of multidrug-resistant Acinetobacter baumannii and a concomitant decrease in antibiotic treatment options warrants a search for new classes of antibacterial agents. We have found that A. baumannii is pathogenic and lethal to the model host organism Caenorhabditis elegans and have exploited this phenomenon to develop an automated, high-throughput, high-content screening assay in liquid culture that can be used to identify novel antibiotics effective against A. baumannii. The screening assay involves coincubating C. elegans with A. baumannii in 384-well plates containing potential antibacterial compounds. At the end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z' factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhance C. elegans survival in the presence of A. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquito Aedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low MIC (5 µg/ml) against A. baumannii. BR003-cecropin A causes membrane permeability in A. baumannii, which could be the underlying mechanism of its lethality.

On the Mechanism of Berberine-INF55 (5-Nitro-2-phenylindole) Hybrid Antibacterials.

Berberine-INF55 hybrids are a promising class of antibacterials that combine berberine and the NorA multidrug resistance pump inhibitor INF55 (5-nitro-2-phenylindole) together in one molecule via a chemically stable linkage. Previous studies demonstrated the potential of these compounds for countering efflux-mediated antibacterial drug resistance but they didn't establish whether the compounds function as originally intended, i.e. with the berberine moiety providing antibacterial activity and the attached INF55 component independently blocking multidrug resistance pumps, thereby enhancing the activity of berberine by reducing its efflux. We hypothesised that if the proposed mechanism is correct, then hybrids carrying more potent INF55 pump inhibitor structures should show enhanced antibacterial effects relative to those bearing weaker inhibitors. Two INF55 analogues showing graded reductions in NorA inhibitory activity compared with INF55 were identified and their corresponding berberine-INF55 hybrids carrying equivalent INF55 inhibitor structures synthesised. Multiple assays comparing the antibacterial effects of the hybrids and their corresponding berberine-INF55 analogue combinations showed that the three hybrids all show very similar activities, leading us to conclude that the antibacterial mechanism(s) of berberine-INF55 hybrids is different from berberine-INF55 combinations.

A multi-host approach for the systematic analysis of virulence factors in Cryptococcus neoformans.

A multi-host approach was followed to screen a library of 1201 signature-tagged deletion strains of Cryptococcus neoformans mutants to identify previously unknown virulence factors. The primary screen was performed using a Caenorhabditis elegans-C. neoformans infection assay. The hits among these strains were reconfirmed as less virulent than the wild type in the insect Galleria mellonella-C. neoformans infection assay. After this 2-stage screen, and to prioritize hits, we performed serial evaluations of the selected strains, using the C. elegans model. All hit strains identified through these studies were validated in a murine model of systemic cryptococcosis. Twelve strains were identified through a stepwise screening assay. Among them, 4 (CSN1201, SRE1, RDI1, and YLR243W) were previously discovered, providing proof of principle for this approach, while the role of the remaining 8 genes (CKS101, CNC5600, YOL003C, CND1850, MLH3, HAP502, MSL5, and CNA2580) were not previously described in cryptococcal virulence. The multi-host approach is an efficient method of studying the pathogenesis of C. neoformans. We used diverse model hosts, C. elegans, G. mellonella, and mice, with physiological differences and identified 12 genes associated with mammalian infection. Our approach may be suitable for large pathogenesis screens.

Effector triggered immunity.

Pathogenic bacteria produce virulence factors called effectors, which are important components of the infection process. Effectors aid in pathogenesis by facilitating bacterial attachment, pathogen entry into or exit from the host cell, immunoevasion, and immunosuppression. Effectors also have the ability to subvert host cellular processes, such as hijacking cytoskeletal machinery or blocking protein translation. However, host cells possess an evolutionarily conserved innate immune response that can sense the pathogen through the activity of its effectors and mount a robust immune response. This "effector triggered immunity" (ETI) was first discovered in plants but recent evidence suggest that the process is also well conserved in metazoans. We will discuss salient points of the mechanism of ETI in metazoans from recent studies done in mammalian cells and invertebrate model hosts.

Whole animal automated platform for drug discovery against multi-drug resistant Staphylococcus aureus.

Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States, is also pathogenic to the model nematode Caenorhabditis elegans. The C. elegans-S. aureus infection model was previously carried out on solid agar plates where the bacteriovorous C. elegans feeds on a lawn of S. aureus. However, agar-based assays are not amenable to large scale screens for antibacterial compounds. We have developed a high throughput liquid screening assay that uses robotic instrumentation to dispense a precise amount of methicillin resistant S. aureus (MRSA) and worms in 384-well assay plates, followed by automated microscopy and image analysis. In validation of the liquid assay, an MRSA cell wall defective mutant, MW2ΔtarO, which is attenuated for killing in the agar-based assay, was found to be less virulent in the liquid assay. This robust assay with a Z'-factor consistently greater than 0.5 was utilized to screen the Biomol 4 compound library consisting of 640 small molecules with well characterized bioactivities. As proof of principle, 27 of the 30 clinically used antibiotics present in the library conferred increased C. elegans survival and were identified as hits in the screen. Surprisingly, the antihelminthic drug closantel was also identified as a hit in the screen. In further studies, we confirmed the anti-staphylococcal activity of closantel against vancomycin-resistant S. aureus isolates and other Gram-positive bacteria. The liquid C. elegans-S. aureus assay described here allows screening for anti-staphylococcal compounds that are not toxic to the host.