Cidália Pina-Vaz

Antifungal activity of Thymus oils and their major compounds

The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. Essential oils have been used empirically. The essential oils of Thymus (Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina) have often been used in folk medicine. The aim of the present study was to evaluate objectively the antifungal activity of Thymus oils according to classical bacteriological methodologies − determination of the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) − as well as flow cytometric evaluation. The effect of essential oils upon germ tube formation, an important virulence factor, was also studied. The mechanism of action was studied by flow cytometry, after staining with propidium iodide. The chemical composition of the essential oils was investigated by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The antifungal activity of the major components (carvacrol, thymol, p‐cymene and 1,8‐cineole) and also possible interactions between them were also investigated. The essential oils of T. vulgaris and T. zygis showed similar antifungal activity, which was greater than T. mastichina. MIC and MLC values were similar for all the compounds tested. At MIC values of the essential oils, propidium iodide rapidly penetrated the majority of the yeast cells, indicating that the fungicidal effect resulted primarily from an extensive lesion of the cell membrane. Concentrations below the MIC values significantly inhibited germ tube formation. This study describes the potent antifungal activity of the essential oils of Thymus on Candida spp., warranting future therapeutical trials on mucocutaneous candidosis.

New Microsatellite Multiplex PCR for Candida albicans Strain Typing Reveals Microevolutionary Changes

ABSTRACT Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.

Prevalence, Distribution, and Antifungal Susceptibility Profiles of Candida parapsilosis, C. orthopsilosis, and C. metapsilosis in a Tertiary Care Hospital

ABSTRACT Candida parapsilosis, an emergent agent of nosocomial infections, was previously made up of a complex of three genetically distinct groups (groups I, II, and III). Recently, the C. parapsilosis groups have been renamed as distinct species: C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. In Portugal, no data pertaining to the distribution and antifungal susceptibility of these Candida species are yet available. In the present report, we describe the incidence and distribution of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis among 175 clinical and environmental isolates previously identified by conventional methods as C. parapsilosis. We also evaluated the in vitro susceptibilities of the isolates to fluconazole, voriconazole, posaconazole, amphotericin B, and two echinocandins, caspofungin and anidulafungin. Of the 175 isolates tested, 160 (91.4%) were identified as C. parapsilosis sensu stricto, 4 (2.3%) were identified as C. orthopsilosis, and 5 (2.9%) were identified as C. metapsilosis. Six isolates corresponded to species other than the C. parapsilosis group. Interestingly, all isolates from blood cultures corresponded to C. parapsilosis sensu stricto. Evaluation of the antifungal susceptibility profile showed that only nine (5.6%) C. parapsilosis sensu stricto strains were susceptible-dose dependent or resistant to fluconazole, and a single strain displayed a multiazole-resistant phenotype; two (1.3%) C. parapsilosis sensu stricto strains were amphotericin B resistant. All C. orthopsilosis and C. metapsilosis isolates were susceptible to azoles and amphotericin B. A high number of strains were nonsusceptible to the echinocandins (caspofungin and anidulafungin).

Highly Polymorphic Microsatellite for Identification of Candida albicans Strains

ABSTRACT The polymorphism of a new microsatellite locus (CAI) was investigated in a total of 114 Candida albicans strains, including 73 independent clinical isolates, multiple isolates from the same patient, isolates from several episodes of recurrent vulvovaginal infections, and two reference strains. PCR genotyping was performed automatically, using a fluorescence-labeled primer, and in the 73 independent isolates, 26 alleles and 44 different genotypes were identified, resulting in a discriminatory power of 0.97. CAI was revealed to be species specific and showed a low mutation rate, since no amplification product was obtained when testing other pathogenic Candida species and no genotype differences were observed when testing over 300 generations. When applying this microsatellite to the identification of strains isolated from recurrent vulvovaginal infections in eight patients, it was found that 13 out of 15 episodes were due to the same strain. When multiple isolates, obtained from the same patient and plated simultaneously, were typed for CAI, the same genotype was found in each case, confirming that the infecting population was clonal. Moreover, the same genotype appeared in isolates from the rectum and the vagina, revealing that the former could be a reservoir of potentially pathogenic strains. This new microsatellite proves to be a valuable tool to differentiate C. albicans strains. Furthermore, when compared to other molecular genotyping techniques, CAI proved to be very simple, highly efficient, and reproducible, being suitable for low-quantity and very-degraded samples and for application in large-scale epidemiological studies.

Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species.

Ibuprofen, a non-steroidal anti-inflammatory drug, exhibited antimicrobial activity against Candida albicans and non-albicans strains. At 10 mg/ml, ibuprofen showed a rapid cidal activity against exponential growth phase C. albicans, accompanied by rapid and extensive leakage of intracellular K+, permeation to propidium iodide, lysis of spheroplasts and severe membrane ultrastructural alterations. These results indicate that the killing of Candida cells is due to direct damage to the cytoplasmic membrane. At 5 mg/ml, ibuprofen inhibited growth; however, it did not kill the yeasts and did not directly affect the cytoplasmic membrane. Evaluation of yeast metabolic vitality with the fluorescent probe FUN-1 showed that growth inhibition induced by the fungistatic drug concentration was due to metabolic alterations. The combination of ibuprofen with fluconazole resulted in synergic activity with eight of the 12 Candida strains studied, including four of the five fluconazole-resistant strains. The MICs of fluconazole for the fluconazole-resistant strains decreased 2-128-fold when the drug was associated with ibuprofen. When in combination with fluconazole, MICs for ibuprofen decreased by up to 64-fold for all the 12 strains studied. These results point to the practicability of using ibuprofen, alone or in combination with azoles, in the treatment of candidosis, particularly when applied topically, taking advantage of the drugs antifungal and anti-inflammatory properties.

Antifungal activity of local anesthetics against Candida species.

OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity. METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts. RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes. A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively. CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane.

Anti-Candida activity of essential oils.

Anti-Candida activity of essential oils has been widely studied and as a consequence they are being investigated as possible alternatives or complementary therapeutic agents for candidosis. We reviewed the most studied essential oils concerning chemical composition and in vitro/in vivo studies under the perspective of their possible clinical use.

Comparison of Two Probes for Testing Susceptibilities of Pathogenic Yeasts to Voriconazole, Itraconazole, and Caspofungin by Flow Cytometry

ABSTRACT A cytometric approach to determine the susceptibilities of Candida spp. and Cryptococcus neoformans to voriconazole, itraconazole, and caspofungin is described. A total of 63 clinical isolates with different susceptibility patterns were exposed for 1, 2, 4, and 6 h to serial concentrations of each antifungal agent, followed by staining with two fluorescent probes: propidium iodide (PI) and FUN-1. FUN-1 was able to identify the susceptibility patterns of the assayed strains to the three agents after 1 h. PI penetrated a maximum of 50% of the cells treated with PI, at the highest concentration of caspofungin, 16 μg/ml, after 6 h of incubation (this percentage varied with the strain and was drug concentration and time of incubation dependent) and did not stain cells treated with high concentrations of either azole after 6 h. The use of FUN-1 appears to be an excellent fast and reliable alternative to the classical dilution method for determining the susceptibility of Candida spp. and C. neoformans to these three antifungal agents.

Adhesion, biofilm formation, cell surface hydrophobicity, and antifungal planktonic susceptibility: relationship among Candida spp.

We have performed the characterization of the adhesion profile, biofilm formation, cell surface hydrophobicity (CSH) and antifungal susceptibility of 184 Candida clinical isolates obtained from different human reservoirs. Adhesion was quantified using a flow cytometric assay and biofilm formation was evaluated using two methodologies: XTT and crystal violet assay. CSH was quantified with the microbial adhesion to hydrocarbons test while planktonic susceptibility was assessed accordingly the CLSI protocol for yeast M27-A3 S4. Yeast cells of non-albicans species exhibit increased ability to adhere and form biofilm. However, the correlation between adhesion and biofilm formation varied according to species and also with the methodology used for biofilm assessment. No association was found between strains site of isolation or planktonic antifungal susceptibility and adhesion or biofilm formation. Finally CSH seemed to be a good predictor for biofilm formation but not for adhesion. Despite the marked variability registered intra and inter species, C. tropicalis and C. parapsilosis were the species exhibiting high adhesion profile. C. tropicalis, C. guilliermondii, and C. krusei revealed higher biofilm formation values in terms of biomass. C. parapsilosis was the species with lower biofilm metabolic activity.

Candida albicans CUG Mistranslation Is a Mechanism To Create Cell Surface Variation

ABSTRACT In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface β-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions. IMPORTANCE The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule β-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution. The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule β-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution.