Adriana Celis

Physiological and Molecular Characterization of Atypical Isolates of Malassezia furfur

ABSTRACT The species constituting the genus Malassezia are considered to be emergent opportunistic yeasts of great importance. Characterized as lipophilic yeasts, they are found in normal human skin flora and sometimes are associated with different dermatological pathologies. We have isolated seven Malassezia species strains that have a different Tween assimilation pattern from the one typically used to differentiate M. furfur, M. sympodialis, and M. slooffiae from other Malassezia species. In order to characterize these isolates of Malassezia spp., we studied their physiological features and conducted morphological and molecular characterization by PCR-restriction fragment length polymorphism and sequencing of the 26S and 5.8S ribosomal DNA-internal transcribed spacer 2 regions in three strains from healthy individuals, four clinical strains, and eight reference strains. The sequence analysis of the ribosomal region was based on the Blastn algorithm and revealed that the sequences of our isolates were homologous to M. furfur sequences. To support these findings, we carried out phylogenetic analyses to establish the relationship of the isolates to M. furfur and other reported species. All of our results confirm that all seven strains are M. furfur; the atypical assimilation of Tween 80 was found to be a new physiological pattern characteristic of some strains isolated in Colombia.

Virulence Gene Expression in Malassezia spp from Individuals with Seborrheic Dermatitis

TO THE EDITOR Seborrheic dermatitis (SD) is a chronic inflammatory disease that compromises skin areas rich in sebaceous glands, such as the face, scalp, and upper trunk. The condition is frequently observed in acquired immune deficiency syndrome patients (30–85% compared with 3–5% of immunocompetent adults). Its appearance is considered to be an early marker of the evolutionary trend of HIV infection (Gupta et al., 2001; Gupta and Bluhm, 2004; Ashbee, 2007; Naldi and Rebora, 2009). The cause or causes of SD and the pathogenic role of Malassezia spp are, at present, not completely understood (Gupta et al., 2000; DeAngelis et al., 2005; Tajima et al., 2008). At this time, we can only rely on hypotheses of the pathogenicity mechanisms and the pathogenic determinants (Ran et al., 1993; Brunke and Hube, 2006; Xu et al., 2007). Analyses of the complete genome of Malassezia globosa and the partial genome of M. restricta (Xu et al., 2007) have presented gene-encoding enzymes of the lipase and phospholipase families that could explain the lipid dependency of the genus. The secretion of enzymes by human pathogenic fungi has been considered an important factor in the invasion and dissemination in the host (Staib et al., 1999; Descamps et al., 2002) and, thus, it is suggested that lipases and phospholipases are involved in the mechanisms of pathogenicity of Malassezia spp. However, except for an early approximation of M. globosa lipase gene expression on human scalp, the expression of these genes has never been tested during disease development (Xu et al., 2007). In order to assess the induction of lipases and phospholipases in SD, we analyzed the expression profiles of candidate virulence genes of Malassezia spp during host infection. A total of 40 Malassezia isolates were obtained from four groups of individuals (non-Malassezia lesion (NML), SD, NMLþHIV, and SDþHIV) (Table 1). The selection of SD and SDþHIV patients was based on dermatologist evaluation of the presence of erythema and peeling in areas such as the face and scalp. We assessed isolates for morphological and physiological features, and we confirmed their identification by amplification and sequencing of 5.8S rDNA-ITS2 regions (Supplementary Materials and Methods online). Six Malassezia spp strains were used as a reference (M. furfur CBS 1878, M. sympodialis CBS 7222, M. globosa CBS 7966, M. restricta CBS 7877, M. slooffiae CBS 7956, and M. pachydematis CBS 1879). All of the Malassezia species isolated from NML, SD, NMLþHIV, and SDþHIV individuals exhibited typical morphological, physiological, and molecular features (Supplementary Table S1 online), with the exception of one: M. Furfur, which presented atypical Tween assimilation pattern (Gonzalez et al., 2009). We found M. restricta to be the predominant species in all groups of individuals, and M. furfur was found in all groups except the SD group. M. furfur with an atypical Tween assimilation pattern was present only in SDþHIV individuals, and M. globosa was present in SD and SDþHIV individuals. These results coincide with previous reports (Tajima et al., 2008; Oh et al., 2010). For the expression assays, four genes, Mgl0797/M. globosa LIP1, Mgl0798/M. globosa hypothetical secretory lipase, Mgl3326/M. globosa hypothetical phospholipase, and Mflip 1/M. furfur MfLIP1 were used for a quantitative analysis using real-time PCR (Supplementary Table S2 online). All the genes were detected in all samples.

Metal Complex Derivatives of Azole: a Study on Their Synthesis, Characterization, and Antibacterial and Antifungal Activities

Four new zinc complex derivatives of azoles and ligands were synthesized and isolated as white air-stable solids and characterized by elemental analyses, thermogravimetric analysis (TGA), infrared spectroscopy, nuclear magnetic resonance (NMR) and mass spectra. The elemental analysis, theoretical calculations and NMR show that the complexes likely have a 1:1 (M:L) stoichiometry and tetrahedral geometry. To evaluate the biological activity of the complexes and to discuss the role of metal ions and structural properties, the ligands and their metal complexes have been studied. Their antimicrobial activity was determined in vitro by agar-well diffusion and broth microdilution against nine bacterial strains and seven fungal strains with clinical relevance. In vitro assays showed that the complexes exhibited moderate antibacterial and/or antifungal activities. The antimicrobial activity was found to be more active for the metal complexes than the ligands. The metal complexes that contained copper and cobalt, respectively, displayed notable antibacterial and antifungal effects against all the tested bacterial strains. The minimum inhibitory concentration 50 (MIC50) values were in the range 2454-0.7 µg mL-1. Metal complexes were more effective at inhibiting bacteria than fungi. The results could provide a high-potential solution for antimicrobial growth resistance, for both bacteria and fungi.

Antibacterial Activities of Azole Complexes Combined with Silver Nanoparticles

Growing antimicrobial resistance is considered a potential threat for human health security by health organizations, such as the WHO, CDC and FDA, pointing to MRSA as an example. New antibacterial drugs and complex derivatives are needed to combat the development of bacterial resistance. Six new copper and cobalt complexes of azole derivatives were synthesized and isolated as air-stable solids and characterized by melting point analyses, elemental analyses, thermogravimetric analyses (TGA), and infrared and ultraviolet/visible spectroscopy. The analyses and spectral data showed that the complexes had 1:1 (M:L) stoichiometries and tetrahedral geometries, the latter being supported by DFT calculations. The antibacterial activities of the metal complexes by themselves and combined with silver nanoparticles (AgNPs; 2 μg mL−1) were assessed in vitro by broth microdilution assays against eight bacterial strains of clinical relevance. The results showed that the complexes alone exhibited moderate antibacterial activities. However, when the metal complexes were combined with AgNPs, their antibacterial activities increased (up to 10-fold in the case of complex 5), while human cell viabilities were maintained. The minimum inhibitory concentration (MIC50) values were in the range of 25–500 μg mL−1. This study thus presents novel approaches for the design of materials for fighting bacterial resistance. The use of azole complexes combined with AgNPs provides a new alternative against bacterial infections, especially when current treatments are associated with the rapid development of antibiotic resistance.

Seborrheic dermatitis: predisposing factors and ITS2 secondary structure for Malassezia phylogenic analysis.

Seborrheic dermatitis (SD) is a chronic, widespread skin condition, which is considered a multifactorial disease influenced, in part, by Malassezia spp. opportunistic activities, as well as various endogenous and exogenous factors. Malassezia species are lipophilic, lipid-dependent yeasts that are members of the normal mycobiota of the human skin. Their isolation from SD lesions varies around the world and the study of the relationship among factors such as gender, age, immunosuppressive condition of the patient and SD development, can lead to a better understanding of this disease. To elucidate the association of age and gender with the development of SD and to precisely determine the Malassezia species involved in the disease, samples were obtained from 134 individuals, including individuals without lesions, human immunodeficiency virus positive patients, individuals with seborrheic dermatitis, and HIV patients with seborrheic dermatitis. Malassezia spp. were identified by phenotypic and genotypic methods and a phylogenetic analysis was performed using Bayesian inference. This study revealed that age and gender are not predisposing factors for SD development, and that the most frequent species of Malassezia related to SD development among the Colombian population is M. restricta. We also report the isolation of M. yamatoensis for the first time in Colombia, and propose an ITS2 secondary structure from Malassezia taxa that can be used for precise identification and to establish more robust phylogenetic relationships.

Exophiala psychrophila: a new agent of chromoblastomycosis

Chromoblastomycosis is a chronic cutaneous and subcutaneous mycosis, is caused by dematiaceous fungi, the most frequently implicated are Fonsecaea, Phialophora, Cladophialophora, Rhinocladiella and Exophiala. We report a woman who was treated before with mycological cure, but she experience a relapse requiring treatment again. Direct microscopic examination and skin biopsy with culture were necessary to identify a Exophiala psychrophila, and for our knowledge this is the first case reported.

Lack of correlation of ECV and outcome in an in vivo murine model of systemic fusariosis

The efficacy of liposomal amphotericin B and voriconazole was evaluated against the systemic infection by Fusarium oxysporum species complex or Fusarium keratoplasticum. Although MIC values were within the epidemiological cutoff values (ECVs) recently stablished for Fusarium spp., no efficacy was obtained, indicating that ECVs for Fusarium are not relevant for in vivo efficacy.