No abstract

Candida albicans is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50s ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato (Solanum lycopersicum) and exhibited high levels of fungistatic activity against Candida species (MIC50s ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated C. albicans cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole-genome sequencing identified 2 nonsynonymous mutations in ERG6 (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

Background. Thymol is an important component of essential oils found in the oil of thyme, is extracted mainly from Thymus vulgaris, and was shown to act synergistically with streptomycin against Klebsiella pneumoniae biofilms. Additionally, thymol could be encapsulated into poly (lactic-co-glycolic acid) (PLGA) nanoparticles to overcome issues related to its low water solubility and high volatility. The present study aimed to investigate the antibiofilm activity of thymol-loaded PLGA nanoparticles (Thy-NPs) alone and in combination with streptomycin against biofilms of K. pneumoniae isolates. Methods. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The antibiofilm activities were determined by the safranin dye assay. The synergistic effect of Thy-NPs with streptomycin was assessed by the checkerboard method. The kinetic study of the biofilm biomass and time-kill assay were further performed. Results. Thy-NPs exhibited the highest antibacterial activity against K. pneumoniae isolates, with MIC values ranging from 1 to 8 µg/mL. Additionally, Thy-NPs showed the highest antibiofilm activity against K. pneumoniae isolates with minimal biofilm inhibitory concentration (MBIC) and minimal biofilm eradication concentration (MBEC) values ranging from 16 to 64 µg/mL and from 32 to 128 µg/Ml, respectively. The combination treatment combining Thy-NPs with streptomycin showed a synergistic effect against the inhibition of biofilm formation and eradication of biofilms of K. pneumoniae isolates with fractional inhibitory concentration index values ranging from 0.13 to 0.28. In addition, the MBIC and MBEC values of streptomycin against K. pneumoniae isolates were dramatically reduced (up to 128-fold) in combination with Thy-NPs, suggesting that Thy-NPs would enhance the antibiofilm activity of streptomycin. The biomass and time-kill kinetics analysis confirmed the observed synergistic interactions and showed the bactericidal activity of streptomycin in combination with Thy-NPs. Conclusions. Our results indicate that the synergistic bactericidal effect between streptomycin and Thy-NPs could be a promising approach in the control of biofilm-associated infections caused by K. pneumoniae.