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.

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.

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.

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.

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.

No abstract

BackgroundAbundant populations of bacteria have been observed on Mir and the International Space Station. While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how different microorganisms will respond to the microgravity environment. In order to build our understanding of how spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed differences are due to varied methods, experimental conditions, or organism specific responses.ResultsHere, we have explored how phosphate concentration, carbon source, oxygen availability, and motility affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls when low concentrations of phosphate in the media were combined with decreased oxygen availability. In contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in swimming motility. However, the final cell densities observed with the motility mutant were consistent with those observed with wild type for all conditions tested.ConclusionsThese results indicate that differences in bacterial final cell densities observed between spaceflight and normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology.

No abstract