No abstract

bWith voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain of Aspergillus flavus, the second leading cause of IA after Aspergillus fumigatus, has emerged. The VRC-resistant strain of A. flavus was isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain of A. flavus were cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in the cyp51C gene. To further determine the role of this mutated gene for VRC resistance in A. flavus, an Agrobacterium tumefaciens-mediated gene replacement approach was applied. Consequently, the mutated cyp51C gene from this A. flavus strain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in the cyp51C gene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in the cyp51C gene was identified as responsible for VRC resistance in A. flavus. These findings indicated that the detection of this mutation in A. flavus could serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance in A. flavus causing IA, would be required to fully understand this mechanism.

No abstract

We investigated the antifungal effect of non-thermal plasma, as well as its combination with common antifungal drugs, against Candida biofilms. A direct current atmospheric pressure He/O2 (2%) plasma microjet (PMJ) was used to treat Candida biofilms in a 96-well plate. Inactivation efficacies of the biofilms were evaluated by XTT assay and counting colony forming units (CFUs). Morphological properties of the biofilms were evaluated by Scanning Electron Microscope (SEM). The sessile minimal inhibitory concentrations (SMICs) of fluconazole, amphotericin B, and caspofungin for the biofilms were also tested. Electron Spin Resonance (ESR) spectroscopy was used to detect the reactive oxygen species (ROS) generated directly and indirectly by PMJ. The Candida biofilms were completely inactivated after 1 min PMJ treatment, where severely deformed fungal elements were observed in SEM images. The SMICs of the tested antifungal drugs for the plasma-treated biofilms were decreased by 2–6 folds of dilution, compared to those of the untreated controls. ROS such as hydroxyl radical (•OH), superoxide anion radical (•O2 -) and singlet molecular oxygen (1O2) were detected by ESR. We hence conclude that He/O2 (2%) plasma alone, as well as in combination with common antifungal drugs, is able to inactivate Candida biofilms rapidly. The generation of ROS is believed to be one of the underlying mechanisms for the fungicidal activity of plasma.

The cholesterol-lowering agents known as statins have in vitro activities against human pathogenic fungi, such as Candida species, Cryptococcus neoformans, and Zygomycetes. Synergy between statins and azoles against these fungi has also been reported. We evaluated the in vitro activities of two statins, lovastatin and simvastatin, alone and in combination with azoles and amphotericin B, against clinical isolates of Aspergillus spp. A disk diffusion assay showed that both statins were active against Aspergillus spp. The minimal inhibitory concentration (MIC) ranges for lovastatin and simvastatin against Aspergillus spp. were 16 to >256 microg/ml and 4 to >256 microg/ml, respectively. Although both statins were fungicidal for A. fumigatus, the MICs were vastly higher than clinically achievable concentrations. The results of a combined agar dilution-Epsilometer test as well as a disk diffusion assay showed that neither statin had any effect on the in vitro activities of itraconazole, voriconazole, or amphotericin B against Aspergillus spp.