Anti-MRSA agent discovery using Caenorhabditis elegans-based high-throughput screening.

Staphylococcus aureus is a leading cause of hospital- and community-acquired infections. Despite current advances in antimicrobial chemotherapy, the infections caused by S. aureus remain challenging due to their ability to readily develop resistance. Indeed, antibiotic resistance, exemplified by methicillin-resistant S. aureus (MRSA) is a top threat to global health security. Furthermore, the current rate of antibiotic discovery is much slower than the rate of antibiotic-resistance development. It seems evident that the conventional in vitro bacterial growth-based screening strategies can no longer effectively supply new antibiotics at the rate needed to combat bacterial antibiotic-resistance. To overcome this antibiotic resistance crisis, screening assays based on host-pathogen interactions have been developed. In particular, the free-living nematode Caenorhabditis elegans has been used for drug screening against MRSA. In this review, we will discuss the general principles of the C. elegans-based screening platform and will highlight its unique strengths by comparing it with conventional antibiotic screening platforms. We will outline major hits from high-throughput screens of more than 100,000 small molecules using the C. elegans-MRSA infection assay and will review the mode-of-action of the identified hit compounds. Lastly, we will discuss the potential of a C. elegans-based screening strategy as a paradigm shift screening platform.

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.

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.

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.