High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of approximate to 20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Ion channel behavior of amphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes investigated by electrochemistry and spectroscopy

Amphotericin B (AmB) is a popular drug frequently applied in the treatment of systemic fungal infections. In the presence of ruthenium (II) as the maker ion, the behavior of AmB to form ion channels in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes were studied by cyclic votammetry, AC impedance spectroscopy, and UV/visible absorbance spectroscopy. Different concentrations of AmB ranging from a molecularly dispersed to a highly aggregated state of the drug were investigated. In a fixed cholesterol or ergosterol content (5 mol %) in glassy carbon electrode-supported model membranes, our results showed that no matter what form of AmB, monomeric or aggregated, AmB could form ion channels in supported ergosterol-containing phosphatidylcholine bilayer model membranes. However, AmB could not form ion channels in its monomeric form in sterol-free and cholesterol-containing supported model membranes. On the one hand, when AmB is present as an aggregated state, it can form ion channels in cholesterol-containing supported model membranes; on the other hand, only when AmB is present as a relatively highly aggregated state can it form ion channels in sterol-free supported phosphatidylcholine bilayer model membranes. The results showed that the state of AmB played an important role in forming ion channels in sterol-free and cholesterol-containing supported phosphatidylcholine bilayer model membranes.

Effects of the “all-fish” GH (growth hormone) transgene expression on resistance to Ichthyophthirius multifiliis infections in common carp, Cyprinus carpio L.

A study was undertaken on the susceptibility of the F-4 generation of "all-fish" growth hormone transgenic carp, Cyprinus carpio L, against Ichthyophthirius multifiliis infections. When 1-year old, transgenic carp, with non-transgenic carp and non-manipulated carp (controls) were split into three batches, and experimental infections were performed throughout the 3-month period. All 72 fish were successfully infected. It was shown that there was a significant difference (P<0.01) on infection level between transgenics and non-transgenics, and transgenics and controls. It possibly resulted from transgenics that had stronger non-specific immune functions. In addition, fish surface area affected significantly infection level (P<0.001). Carp with larger surface area harboured more parasites for each type of fish, but transgenic with larger surface area than non-transgenics and controls (P<0.01), loaded fewer parasites than others. Besides, the time of infection also greatly influenced (P<0.001) infection level. Results showed that there was a significant decline in parasite infectivity through October to November (P<0.001). It was likely to suggest that there existed senescence resulted in failure of any I. multifiliis isolate maintenance. Significant difference in infectivity between isolate G from grass carp and isolate H from gold fish suggested that different parasite strains may exist. (C) 2009 Elsevier B.V. All rights reserved.

Host sex and ectoparasite infections of plateau pika (Ochotona curzoniae, Hodgson) on the Qinghai-Tibetan Plateau

A total of 449 plateau pika (Ochotona curzoniae Hudgson) individuals were sampled with rattraps from 21 plots (size 1 ha) randomly scattered over the area of the species distribution at the altitude 3275-4807 in a.s.l. in the Qinghai-Tibetan Plateau (West China). Two main ectoparasite species Hypoderma satyrus Brauer and Ixodes crenulatus Neumann of plateau pika were surveyed, and the relations between host sex and parasitism were analyzed. The results were: (i) although not significantly, the infection rate of female young was close to zero and lower than that of male young (6%), while the infection rate of female sub-adults (19%) was contrarily - higher than that of male sub-adults (11%); adult females had significantly higher (41%) infection rate than that of males (18%) (P<0.001); (ii) the parasite infection rates for both males and females increased with increasing age, but female age-groups had obviously steeper slope. We suggested that the differences of body mass, growth rate and home range between males and females had mainly caused the sex-biased parasitism (SBP) of plateau pika at each age stage. Also, due to the higher increases of body mass and maybe as well as of the home range differences between consecutive age-groups, the parasite infections of females became more sensitive to the influences of age than that of males.