Synthesis of Antibacterial Hybrid Hydroxyapatite/Collagen/Polysaccharide Bioactive Membranes and Their Effect on Osteoblast Culture.

Inspired by the composition and confined environment provided by collagen fibrils during bone formation, this study aimed to compare two different strategies to synthesize bioactive hybrid membranes and to assess the role the organic matrix plays as physical confinement during mineral phase deposition. The hybrid membranes were prepared by (1) incorporating calcium phosphate in a biopolymeric membrane for in situ hydroxyapatite (HAp) precipitation in the interstices of the biopolymeric membrane as a confined environment (Methodology 1) or (2) adding synthetic HAp nanoparticles (SHAp) to the freshly prepared biopolymeric membrane (Methodology 2). The biopolymeric membranes were based on hydrolyzed collagen (HC) and chitosan (Cht) or κ-carrageenan (κ-carr). The hybrid membranes presented homogeneous and continuous dispersion of the mineral particles embedded in the biopolymeric membrane interstices and enhanced mechanical properties. The importance of the confined spaces in biomineralization was confirmed by controlled biomimetic HAp precipitation via Methodology 1. HAp precipitation after immersion in simulated body fluid attested that the hybrid membranes were bioactive. Hybrid membranes containing Cht were not toxic to the osteoblasts. Hybrid membranes added with silver nanoparticles (AgNPs) displayed antibacterial action against different clinically important pathogenic microorganisms. Overall, these results open simple and promising pathways to develop a new generation of bioactive hybrid membranes with controllable degradation rates and antimicrobial properties.

Integrating morphological, biochemical, behavioural, and molecular approaches to investigate developmental toxicity triggered by tebuthiuron in zebrafish (Danio rerio).

Tebuthiuron (TBU), a phenylurea herbicide, is widely applied in agricultural and non-agricultural soils. Because TBU resists degradation, it can contaminate water and reach the biota once it is released into the environment. However, the potential toxic effects of TBU on aquatic developing organisms have been poorly studied. By taking advantage of the early-life stages of zebrafish (Danio rerio), we have combined morphological, biochemical, behavioural, and molecular approaches to investigate the developmental toxicity triggered by environmentally relevant concentrations (from 0.1 to 1000 µg/L) of TBU. Exposure to TBU did not elicit morphological abnormalities but it significantly delayed hatching. In addition, TBU altered the frequency of tail coils in one-day post-fertilization (dpf) old embryos. Moreover, TBU exposure during four days significantly inhibited the whole body AChE activity of larvae. At the molecular level, TBU did not significantly affect the mRNA levels of four genes (elavl3, gfap, gap43, and shha) that play key roles during the neurodevelopment of zebrafish. By assessing the motor responses to repeated light-dark stimuli, 6 dpf larvae exposed to TBU displayed hyperactivity, showing greater travelling distance during the dark periods. Our categorization of swimming speed revealed an interesting finding - after the light was turned off, the exposed larvae abandoned the freezing mode (<2 mm/s) and travelled mainly at cruising speed (2-20 mm/s), showing that the larval hyperactivity did not translate into higher swimming velocity. Overall, our results offer new insights into the TBU toxicity to developing organisms, namely effects in AChE activity and hyperactivity, providing support data for future studies considering environmental risk assessment of this herbicide.

Genetic diversity assessed using PFGE, MLP and MLST in Candida spp. candidemia isolates obtained from a Brazilian hospital.

Candida spp. are the main causative agents of invasive fungal infections in immunocompromised patients. Candidemia has attributable mortality rates of 15 to 35% and increases hospitalisation time and costs, thus making this disease a public health concern. This study aimed to use pulsed-field gel electrophoresis (PFGE), microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST) to analyse the genetic relationships among 65 Candida spp. bloodstream isolates, including 35 Candida albicans, 15 Candida glabrata and 15 Candida tropicalis isolates, all of which were obtained from patients in a Brazilian hospital. Moreover, patient clinical data were assessed. All techniques resulted in high discriminatory indexes. C. albicans and C. tropicalis isolates showed high genetic variability, while C. glabrata isolates had relatively low genetic variability. Moreover, a cluster of C. glabrata isolates was identified in a hospital unit. New MLST sequence types, diploid sequence types and alleles are described. Relationships were not observed between the molecular typing results and clinical characteristics. The molecular typing of clinical strains increases our understanding of candidemia epidemiology and promotes the development of strategies that can reduce the incidence of this disease. Moreover, this study is the first to combine these techniques to genotype these three species in Brazil.

Nopal cladode as a novel reinforcing and antioxidant agent for starch-based films: A comparison with lignin and propolis extract.

The potential use of nopal cladode flour (NC) as reinforcing/bioactive agent in cassava starch-based films was evaluated and compared with the use of propolis extract or lignin, which are commonly used for these purposes. Cassava starch-based films containing untreated NC (S-NC), NC treated at pH 12 (S-NC12), aqueous propolis extract at two different concentrations (SP1 or SP2), or lignin (S-L) were produced by the casting technique; glycerol was used as plasticizer. NC12 and NC affected the mechanical properties of the cassava starch-based film similarly as compared to propolis extract and lignin. Moreover, NC and NC12 had different performance as reinforcing and antioxidant agent in cassava starch-based film. Thus, S-NC12 film was more elongable (28.5 ± 6.5%), more hydrophobic (contact angle: 70.8° ± 0.1), less permeable to water vapor (0.8 ± 0.0 × 10-10 g·m-1·s-1·Pa-1) and had better antioxidant activity by ABTS•+ (44.70 ± 0.3 µM Trolox·g-1 of film) than the S-NC film. SEM and TGA analysis of films showed that NC12 was better incorporated into the cassava starch matrix than NC, lignin and propolis extract. Overall, nopal cladode flour has potential use in the production of active biodegradable packaging for the food preservation with high oxidation rate.

Genetic Diversity of Multidrug-Resistant CMY-Producing Escherichia coli from Feces and Soil in a Small-Scale Pig Farm.

Diarrheagenic Escherichia coli cause diarrheal diseases, which are a public health concern and affect mainly developing countries. Multidrug-resistant (MDR) pathogens have been spreading in different sources, including animals and the environment. E. coli strains were obtained from a small-scale pig farm and 33 antimicrobials were tested. All strains were classified as MDR and harbored several antimicrobial resistance genes (ARGs) [blaCMY, blaOXA-1-like, blaSHV, tet(A), tet(B), aadA, aac(6')-Ib, aph(3')-Ia, sul1, sul2, sul3, floR, and cmlA] and plasmids. Besides, mutations in quinolone resistance-determining region of GyrA (Ser83Leu and Asp87Asn) and ParC (Glu84Asp) were detected. Among the MDR E. coli, nine strains (52%) presented diarrheagenic virulence genes, including genes related to Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC). The pulsed-field gel electrophoresis results showed a high genetic diversity among the MDR E. coli strains. Multilocus sequence typing (MLST) analyses revealed different sequence types phylogenetically related to each other, including ST10 and ST56. Subtyping of MLST by fimH gene showed different fimH type. This study shows a high genetic diversity among MDR ARG-producing E. coli belonging to STEC, EIEC, and EAEC pathotypes obtained from a small-scale pig farm and contributes to the monitoring of antimicrobial-resistant pathogens worldwide, mainly in environmental samples, which are associated with One Health framework.

Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA.

The protein phosphatase calcineurin is an important mediator connecting calcium-dependent signalling to various cellular responses in multiple organisms. In fungi calcineurin acts largely through regulating Crz1p-like transcription factors. Here we characterize an Aspergillus fumigatus CRZ1 homologue, CrzA and demonstrate its mediation of cellular tolerance to increased concentrations of calcium and manganese. In addition to acute sensitivity to these ions, and decreased conidiation, the crzA null mutant suffers altered expression of calcium transporter mRNAs under high concentrations of calcium, and loss of virulence when compared with the corresponding complemented and wild-type strains. We use multiple expression analyses to probe the transcriptional basis of A. fumigatus calcium tolerance identifying several genes having calA and/or crzA dependent mRNA accumulation patterns. We also demonstrate that contrary to previous findings, the gene encoding the Aspergillus nidulans calcineurin subunit homologue, cnaA, is not essential and that the cnaA deletion mutant shares the morphological phenotypes observed in the corresponding A. fumigatus mutant, DeltacalA. Exploiting the A. nidulans model system, we have linked calcineurin activity with asexual developmental induction, finding that CrzA supports appropriate developmental induction in a calcineurin and brlA-dependent manner in both species.

Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase.

Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The DeltaaifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 microM FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 microM FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.

Phenotypic analysis of genes whose mRNA accumulation is dependent on calcineurin in Aspergillus fumigatus.

Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory subunit B. A mutant of Aspergillus fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and DeltacalA mutant strains. Our results showed that the mitochondrial copy number is reduced in the DeltacalA mutant strain, and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified four genes that encode transcription factors that have increased mRNA expression in the DeltacalA mutant. Deletion mutants for these transcription factors had reduced susceptibility to itraconazole, caspofungin, and sodium dodecyl sulfate (SDS).

Functional characterization of the Aspergillus fumigatusPHO80 homologue.

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P(i)) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P(i) at normal or high external P(i) concentrations) and a high-affinity (activated in response to P(i) starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB(PHO80). We show that the DeltaphoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and DeltaphoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the DeltaphoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1mM P(i). Epifluorescence microscopy revealed accumulation of poly-phosphate in DeltaphoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, DeltaphoB(PHO80) and DeltaphoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis.

cipC is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is the main causative agent of invasive aspergillosis, a disease that affects immunocompromised patients and has a high mortality rate. We previously observed that the transcription of a cipC-like gene was increased when A. fumigatus encountered an increased CO2 concentration, as occurs during the infection process. CipC is a protein of unknown function that might be associated with fungal pathogenicity. In this study, the cipC gene was disrupted in A. fumigatus to evaluate its importance for fungal pathogenicity. The gene was replaced, and the germination, growth phenotype, stress responses, and virulence of the resultant mutant were assessed. Although cipC was not essential, its deletion attenuated A. fumigatus virulence in a low-dose murine infection model, suggesting the involvement of the cipC gene in the virulence of this fungus. This study is the first to disrupt the cipC gene in A. fumigatus.

Evaluation of fluconazole resistance mechanisms in candida albicans clinical isolates from HIV-infected patients in Brazil.

In this study, we describe resistance mechanisms in fluconazole-resistant isolates of C. albicans isolated from AIDS patients from nine Brazilian hospitals. These mechanisms include the presence of point mutations in the ERG11 gene and overexpression of ERG11, and several genes encoding efflux pumps, as measured by quantitative real-time reverse transcriptase polymerase chain reaction. Several fluconazole-resistant strains had multiple mechanisms of resistance. Four mutations previously described, Y132F, K143R, E266D, and V437I, were identified among the strains, whereas some isolates contained more than one mutation. Fourteen novel mutations were identified. Interestingly, all Brazilian fluconazole-resistant isolates showed homozygosity at mating-type loci (MTL) associated with fluconazole resistance. This is the first comprehensive assessment at molecular level of mechanisms of fluconazole resistance in C. albicans isolates from South America.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA.

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the DeltaddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The DeltaddbA mutation can genetically interact with uvsB (ATR), atmA(ATM), nkuA (KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne's syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP::DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

Molecular characterization of the Aspergillus fumigatus NCS-1 homologue, NcsA.

Here, we characterize the Aspergillus fumigatus homologue ncsA Neuronal Calcium Sensor. We showed that ncsA is not an essential gene and ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm and its cellular localization is not affected by the cellular response to either calcium chloride or EGTA. The ncsA mutant strain is more sensitive to voriconazole, itraconazole, and amphotericin. Polar growth in the DeltancsA mutant was also considerably more affected by lovastatin than in the wild type strain. The Spitzenkörper can be visualized in both strains and although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the DeltancsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.

Transcriptome analysis of the Aspergillus nidulans AtmA (ATM, Ataxia-Telangiectasia mutated) null mutant.

ATM is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of DNA damage response in eukaryotes. In humans, mutations in ATM cause the devastating neurodegenerative disease Ataxia-Telangiectasia. Previously, we characterized the homologue of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Our results suggested that AtmA probably regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. Here, we extended these studies by investigating which pathways are influenced by AtmA during proliferation and polar growth by comparatively determining the transcriptional profile of A. nidulans wild-type and DeltaatmA mutant strains in different growth conditions. Our results indicate an important role of the pentose phosphate pathway in the fungal proliferation during endogenous DNA damage and polar growth monitored by the AtmA kinase. Furthermore, we identified several genes that have decreased mRNA expression in the DeltaatmA mutant that are involved in the formation of a polarized hyphae and control of polar growth; in the synthesis of phosphatidic acid (e.g. phospholipase D); in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts); and in intracellular trafficking.

Functional characterization of the Aspergillus fumigatus calcineurin.

Aspergillus fumigatus is an aggressive opportunistic pathogen of humans as well as a major allergen. Environmental sensing and retrieving essential nutrients from the environment are general metabolic traits associated with the growth of this saprophytic fungus. Two important mediators of calcium signals in eukaryotic cells are the Ca(2+)-binding protein calmodulin and the Ca(2+)/calmodulin-dependent phosphatase calcineurin. Calcineurin is a heterodimer that consists of a catalytic subunit A and a Ca(2+)/calmodulin binding unit. We deleted the A. fumigatus calA gene, which encodes the calcineurin A catalytic subunit, and demonstrated that this gene is not essential in this fungus. The DeltacalA mutant strain has severe defects in growth extension, branching and conidial architecture. Furthermore, the A. fumigatus DeltacalA mutant strain has decreased fitness in a low dose murine infection and cannot grow in fetal bovine serum (FBS). After potassium phosphate was added to liquid FBS, the DeltacalA mutant strain could grow with the characteristic phenotype of the DeltacalA mutation. When A. fumigatus calcineurin is inhibited by tacrolimus in a phosphate depleted medium, there is a reduction in the inorganic phosphate transport and six putative phosphate transporter genes have altered mRNA levels. However, there is no effect on the acid phosphatase activity. These results suggest that calcineurin is involved in the regulation of the PHO pathway in A. fumigatus. Our work on calcineurin opens new venues for the research on sensing and nutrient acquisition in A. fumigatus.

Fungal metabolic model for tyrosinemia type 3: molecular characterization of a gene encoding a 4-hydroxy-phenyl pyruvate dioxygenase from Aspergillus nidulans.

Mutations in the human HPD gene (encoding 4-hydroxyphenylpyruvic acid dioxygenase) cause hereditary tyrosinemia type 3 (HT3). We deleted the Aspergillus nidulans homologue (hpdA). We showed that the mutant strain is not able to grow in the presence of phenylalanine and that it accumulates increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid, mimicking the human HT3 phenotype.

The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus.

To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB(KU80)). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuB(KU80) mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB(KU80) had no influence on pathogenicity in a low-dose murine infection model.

Transcriptome analysis of Aspergillus fumigatus exposed to voriconazole.

For a comprehensive evaluation of genes that have their expression modulated during exposure of the mycelia to voriconazole, we performed a large-scale analysis of gene expression in Aspergillus fumigatus using a microarray hybridization approach. By comparing the expression of genes between the reference time and after addition of voriconazole (30, 60, 120, and 240 min), we identified 2,271 genes differentially expressed in the wild-type strain. To validate the expression of some of these genes during exposure to voriconazole, we analyzed 13 genes showing higher expression in the presence of voriconazole by real-time RT-PCR. Although the magnitudes of induction differed between the two experimental systems, in about 85% of the cases they were in good agreement with the microarray data. To our knowledge this is the first study of microarray hybridization analysis for a filamentous fungus exposed to an antifungal agent. In our study, we have observed: (i) a decreased mRNA expression of various ergosterol biosynthesis genes; (ii) increased mRNA levels of genes involved in a variety of cell functions, such as transporters, transcription factors, proteins involved in cell metabolism, and hypothetical proteins; and (iii) the involvement of the cyclic AMP-protein kinase signaling pathway in the increased mRNA expression of several of these genes.

Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

In vitro evolution of itraconazole resistance in Aspergillus fumigatus involves multiple mechanisms of resistance.

We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.