716. Characterization of Azole Susceptibility Profiles of Aspergillus spp. Isolates Obtained From Immunocompromised Patients in Cali, Colombia

Background Aspergillus spp. are opportunistic filamentous fungi causing a spectrum of diseases described aspergillosis. Aspergillosis is treated with triazole antifungals of second-generation, mostly voriconazole or itraconazole, which inhibit the ergosterol synthesis, an important component of the fungal membrane. However, the efficacy of these drugs has been affected by the presence of resistance found in different Aspergillus spp. species, as a mutations in CYP51 gene We describe the azole susceptibility profiles of Aspergillus spp., isolated from clinical samples in a hospital in Cali, Colombia. Methods A total of 63 Aspergillus spp. clinical isolates were identified at a phenotypic and protein level through matrix-assisted laser ionization (MALDI-TOF-MS), susceptibility profiles against voriconazole and itraconazole were subsequently characterized. Following the guidelines for susceptibility determination issued by EUCAST, 96-well plates were prepared with the azole antifungals itraconazole (ITZ) and voriconazole(VCZ), using concentrations ranging from 0.06 to 32 mg/L. Each well was then inoculated with 100μL of the fungal inoculum previously diluted in distilled water and 20% tween adjusted to a concentration of 0.5 on the Mcfarland scale. The plates were incubated at 37 °C and readings were taken after 48 hours of incubation. Results A total of 63 clinical isolates of Aspergillus spp were collected, of these 44% corresponded to isolates of A. fumigatus, 25% A. flavus/oryzae, 18% A. niger, 5% A. parasiticus, 3% A. terreus and 3% A. versicolor. In vitro characterization of the susceptibility profiles revealed variable phenotypes, with a predominance of susceptible strains for the two antifungals tested, however, of the total isolates, 8% (5/63) were resistant to itraconazole (ITZ), and 6% (4/63) to voriconazole (VRZ). Figure 1. Distribution or Frequency of Aspergillus spp. Species and Their Resistance Profiles Conclusion Azole resistance was low 6-8% . Susceptibility profiles of the strains isolated from clinical samples is important to carry out an accurate identification of each one of the agents involved in infections caused by Aspergillus spp in order to differentiate common species from cryptic ones, since these have increasingly acquired importance at the clinical level,. Disclosures All Authors: No reported disclosures

Antifungal activity of fibrate-based compounds and substituted pyrroles inhibiting the enzyme 3-hydroxy-methyl-glutaryl-CoA reductase of Candida glabrata (CgHMGR), and decreasing yeast viability and ergosterol synthesis

ABSTRACTDue to the emergence of multi-drug resistant strains of yeasts belonging to the Candida genus, there is an urgent need to discover antifungal agents directed at alternative molecular targets. The aim of the current study was to evaluate the capacity of synthetic compounds to inhibit the Candida glabrata enzyme denominated 3-hydroxy-methyl-glutaryl-CoA reductase (CgHMGR), and thus affect ergosterol synthesis and yeast viability. One series of synthetic antifungal compounds were analogues to fibrates, a second series had substituted 1,2-dihydroquinolines and the third series included substituted pyrroles. α-asarone-related compounds 1c and 5b with a pyrrolic core were selected as the best antifungal candidates. Both inhibited the growth of fluconazole-resistant C. glabrata 43 and fluconazole-susceptible C. glabrata CBS 138. A yeast growth rescue experiment based on the addition of exogenous ergosterol showed that the compounds act by inhibiting the mevalonate synthesis pathway. A greater recovery of yeast growth occurred for the C. glabrata 43 strain and after the 1c (versus 5b) treatment. Given that the compounds decreased the ergosterol concentration in the yeast strains, they probably target the ergosterol synthesis. According to the docking analysis, the inhibitory effect of the 1c and 5b could possibly be mediated by their interaction with the amino acid residues of the catalytic site of CgHMGR. Since 1c displayed higher binding energy than α-asarone and 5b, it is a good candidate for further research, which should include structural modifications to increase its specificity and potency as well as in vivo studies on its effectiveness at a therapeutic dose.HIGHLIGHTSFibrate-based and pyrrole-containing compounds were tested as C. glabrata inhibitors.The best inhibitor from fibrate was 1c and from pyrroles was 5b.These agents inhibited C. glabrata growth better than the reference antifungals.They also inhibited ergosterol synthesis by the two C. glabrata strains tested. Experimental

Long Terminal Repeat Retrotransposon Afut4 promotes azole resistance of Aspergillus fumigatus by enhancing the expression of sac1 gene

Aspergillus fumigatus causes a series of invasive diseases, including the high-mortality invasive aspergillosis, and has been a serious global health threat because of its increased resistance to the first-line clinical triazoles. We analyzed the whole-genome sequence of 15 A. fumigatus strains from China and found that long terminal repeat retrotransposons (LTR-RTs), including Afut1 , Afut2, Afut3, and Afut4 , are most common and have the largest total nucleotide length among all transposable elements in A. fumigatus . Deleting one of the most enriched Afut4 977-sac1 in azole-resistant strains decreased azole resistance and downregulated its nearby gene, sac1 , but it did not significantly affect the expression of genes of the ergosterol synthesis pathway. We then discovered that 5'LTR of Afut4 977-sac1 had promoter activity and enhanced the adjacent sac1 gene expression. We found that sac1 is important to A. fumigatus , and the upregulated sac1 caused the elevated resistance of A. fumigatus to azoles. Finally, we showed that Afut4 977-sac1 has an evolution pattern similar to that of the whole genome of azole-resistant strains due to azoles; phylogenetic analysis on both the whole genome and Afut4 977-sac1 suggests that the insertion of Afut4 977-sac1 might have preceded the emergence of azole-resistant strains. Taking these data together, we found that LTR-RT Afut4 977-sac1 might be involved in the regulation of azole resistance of A. fumigatus by upregulating its nearby sac1 gene.

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives

A newly synthetized series of N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone (5d), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone (5i), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone (5k) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MICT = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MICP = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol (8h–i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c–d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.

Thidiazuron, a phenyl-urea cytokinin, inhibits ergosterol synthesis and attenuates biofilm formation of Candida albicans

Candida albicans is a commensal human fungal pathogen that colonizes and develops dental biofilm which cause Oral candidiosis. This study investigates the effects of a new molecule Thidiazuron against the growth and biofilm formation properties of C. albicans. This study applied computational and in vitro approaches such as broth microdilution, SEM, time-kill dynamics, crystal violet assay, XTT reduction assay, ergosterol quantification and quantitative RT PCR analysis of gene expression to validate the growth and biofilm inhibitory potential of thidiazuron against C. albicans. Preliminary molecular docking study revealed potential interaction between thidiazuron and amino acids residues of CYP51. Further in vitro anti-fungal susceptibility test, SEM and time kill analysis revealed anti-fungal potency of thidiazuron in dose and time dependent passion. Crystal violet staining, XTT reduction assay and Acridine Orange staining visually confirmed biofilm inhibitory potential of thidiazuron. Gene expression study shows that thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis, cell adhesion and hyphae development in C. albicans. This study identified thidiazuron as CYP51 inhibitor and a new antibiofilm agent against C. albicans.

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole–resistant strains of A. fumigatus. The TR34/L98H and TR46/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR34/L98H resistance allele was the most common in all regions except South America where the TR46/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.

Phytomedicine from Middle Eastern Countries: An Alternative Remedy to Modern Medicine against Candida spp Infection

Candida spp are capable of infecting both normal and immunocompromised individuals. More recently, Candida infections have spread considerably in healthcare settings, especially in intensive care units, where it is the most frequently encountered pathogen. Candida albicans is the commonest species encountered, although infections by non-albicans species have also risen in the past few years. The pathogenicity of Candida is credited to its aptitude to change between yeast and hyphal modes of growth. Candida spp produce biofilms on synthetic materials that protect them and facilitate drug resistance and act as a source for chronic and recurrent infections. Primarily, azoles antifungal agents are utilized to treat Candida infection that targets the ergosterol synthesis pathway in the cell wall. The development of antifungal resistance in Candida species is a major reason for treatment failure, and hence, there is a need to develop newer antifungal molecules and/or modifications of existing antifungals to make them more effective and less toxic. This has led researchers to oversee the plants to discover newer antimicrobials. Middle Eastern countries are well known for their landscape ranging from dry and sandy deserts to snow-capped mountains. However, they comprise enormous plant diversity with over 20,000 different species showing various types of bioactivities, such as anticancer, antidiabetic, and antimicrobial activities. Especially, the antifungal potential of these phytoproducts could be exploited in the clinical setting for therapy. The present review examines some of the promising alternative natural compounds that have been tested and found effective in treating Candida infections in vitro in some Middle Eastern countries.

The transcriptome analysis on urea response mechanism in the process of ergosterol synthesis by Cordyceps cicadae

Nitrogen source is required for the growth of Cordyceps cicadae and involved in the regulation of metabolite synthesis. In order to further investigate the regulatory effects of nitrogen sources on the ergosterol synthesis by C. cicadae. We first confirmed that urea could significantly increase the ergosterol synthesis. The transcriptome analysis showed that compared with biomass cultured in the control fermentation medium (CFM), 1340 differentially expressed genes (DEGs) were obtained by Gene Ontology (GO) annotation, and 312 DEGs were obtained by Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation from the biomass cultured in CFM + CO(NH2)2. Urea up-regulated D-3-phosphoglycerate dehydrogenase gene transcription level and down-regulated enolase and L-serine/L-threonine ammonialyase gene transcription level, increased serine synthesis, allosterically activate pyruvate kinase, to promote the synthesis of pyruvate and CH3CO ~ SCOA, the primer of ergosterol; Urea increase the genes transcription related with ergosterol synthesis by up-regulating the steroid regulatory element binding protein gene transcription levels. The transcriptome results were provided by those of qRT-PCR. Collectively, our finding provided valuable insights into the regulatory effect of nitrogen source on the ergosterol synthesis by C. cicadae.

Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae

Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species of the Ceratocystidaceae were identified. Ergosterol and some of the intermediates in the pathway were also detected in seven species (Ceratocystis manginecans, C. adiposa, Huntiella moniliformis, Thielaviopsis punctulata, Bretziella fagacearum, Endoconidiophora polonica and Davidsoniella virescens), using gas chromatography-mass spectrometry analysis. The average ergosterol content differed among different genera of Ceratocystidaceae. We also identified all possible terpene related genes and possible biosynthetic clusters in the genomes used in this study. We found a highly conserved terpene biosynthesis gene cluster containing some genes encoding ergosterol biosynthesis enzymes in the analysed genomes. An additional possible terpene gene cluster was also identified in all of the Ceratocystidaceae. We also evaluated the sensitivity of the Ceratocystidaceae to a triazole fungicide that inhibits ergosterol synthesis. The results showed that different members of this family behave differently when exposed to different concentrations of triazole tebuconazole.