Lívia S. Ramos

Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil

OBJECTIVES The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

Protease and phospholipase activities of Candida spp. isolated from cutaneous candidiasis

BACKGROUND Cases of superficial and invasive mycoses caused by emerging species of Candida have been increasingly reported over the last thirty years. The production of hydrolytic enzymes plays a central role in the fungal infective process. In Candida infections the secretion of both proteases and phospholipases are well-known virulence attributes. AIMS To determine the protease and phospholipase production from 58 human clinical isolates of Candida obtained from individuals with cutaneous candidiasis seen in the Human and Veterinary Diagnostic Mycology Sector from Universidade Federal Fluminense (UFF), Brazil, from November 2008 to August 2009. METHODS Fungal identification was performed using biochemical tests. Proteolytic activity was detected on agar plates containing bovine serum albumin, and phospholipase production was determined on egg-yolk plates. RESULTS The Candida species isolated were Candida parapsilosis (27.59%), Candida famata (18.96%), Candida albicans (15.52%), Candida haemulonii (12.06%), Candida ciferri (8.62%), Candida guilliermondii (6.90%), Candida tropicalis (5.17%) and Candida lipolytica (5.17%). All isolates of C. albicans produced both protease and phospholipase. As regards the isolates of non-C. albicans Candida species, 53.06% and 4.08% were able to produce protease and phospholipase, respectively. For example, the majority of isolates of C. parapsilosis (15/16) produced protease, while 40% of C. ciferri isolates (2/5) were phospholipase producers. This study shows, for the first time, that C. ciferri and C. haemulonii strains were able to produce protease. CONCLUSIONS Collectively, our results showed that different species of Candida isolated from cutaneous lesions were able to produce proteases and/or phospholipases, which are multifunctional molecules directly involved in the infectious process of these fungi.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Fungal Biofilm - A Real Obstacle against an Efficient Therapy: Lessons from Candida.

The past decades have witnessed a dramatic increase in invasive fungal infections, especially caused by different species belonging to the Candida genus. Nowadays, even after many improvements in several medical procedures, Candida infections (candidiasis) still account for an unacceptable high rate of morbimortality in hospital settings. Corroborating this statement, fungal biofilms formed on both abiotic and living surfaces are responsible for an important medical and economic burden, since biofilm lifestyle confers numerous advantages to the pathogens, including high tolerance to environmental stresses such as antimicrobials and host immune responses. Aggravating this scenario, the currently used antifungal drugs have mostly been developed to target exponentially growing fungal cells and are poorly or not effective against biofilm structures. So, the challenges to inhibit biofilm formation (e.g., blocking the fungal adhesion and its fully development due to the changes of physicochemical properties of the inert substrates by covering or impregnating them with antimicrobial compounds, for example, silver nanoparticles) and/or to disarticulate mature biofilm architecture (e.g., by using compounds capable in destabilizing, weakening or destroying the extracellular matrix components, including inhibitors of quorum sensing signals, hydrolytic enzymes, surfactants, chelator agents and biocides) are stimulating researchers around the world to search novel strategies and new chemotherapeutic options to control fungal biofilm. In this context, the present review summarizes some promising approaches and/or strategies that could improve our ability to prevent or eradicate fungal biofilms in medical settings, focusing on the lessons learned with Candida model.

Different classes of hydrolytic enzymes produced by multidrug-resistant yeasts comprising the Candida haemulonii complex

The production of enzymes in clinical isolates of Candida haemulonii (Ch, n = 5), Candida duobushaemulonii (Cd, n = 4) and Candida haemulonii var. vulnera (Chv, n = 3) was identified by agar plate. Aspartic protease, phytase, caseinolytic and hemolytic activities were detected in all the isolates. A distinct scenario was evidenced regarding the production of lipases. In this way, 80%, 50% and 100% of Ch, Cd and Chv, respectively, were phospholipase producers. Regarding esterase activity, 100%, 50% and 66.7% of Ch, Cd and Chv, respectively, were positive isolates. Esterase activity was significantly higher in isolates recovered from cutaneous candidiasis compared with those recovered from body fluids.

Antifungal Potential of Copper(II), Manganese(II) and Silver(I) 1,10-Phenanthroline Chelates Against Multidrug-Resistant Fungal Species Forming the Candida haemulonii Complex: Impact on the Planktonic and Biofilm Lifestyles

Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii, which form the C. haemulonii complex, are emerging etiologic agents of fungal infections known to be resistant to the most commonly used antifungals. The well-established anti-Candida potential of metal complexes containing 1,10-phenanthroline (phen) ligands encouraged us to evaluate different copper(II), manganese(II), and silver(I) phen chelates for their ability to inhibit planktonic growth and biofilm of C. haemulonii species complex. Two novel coordination complexes, {[Cu(3,6,9-tdda)(phen)2].3H2O.EtOH}n and [Ag2(3,6,9-tdda)(phen)4].EtOH (3,6,9-tddaH2 = 3,6,9-trioxaundecanedioic acid), were synthesized in a similar fashion to the other, previously documented, sixteen copper(II), manganese(II), and silver(I) chelates employed herein. Three isolates of each C. haemulonii species complex were used and the effect of the metal chelates on viability was determined utilizing the CLSI standard protocol and on biofilm-growing cells using the XTT assay. Cytotoxicity of the chelates was evaluated by the MTT assay, employing lung epithelial cells. The majority of the metal chelates were capable of interfering with the viability of planktonic-growing cells of all the fungal isolates. The silver complexes were the most effective drugs (overall geometric mean of the minimum inhibitory concentration (GM-MIC) ranged from 0.26 to 2.16 μM), followed by the manganese (overall GM-MIC ranged from 0.87 to 10.71 μM) and copper (overall GM-MIC ranged from 3.37 to >72 μM) chelates. The manganese chelates (CC50 values ranged from 234.51 to >512 μM) were the least toxic to the mammalian cells, followed by the silver (CC50 values ranged from 2.07 to 13.63 μM) and copper (CC50 values ranged from 0.53 to 3.86 μM) compounds. When tested against mature biofilms, the chelates were less active, with MICs ranging from 2- to 33-fold higher levels when compared to the planktonic MIC counterparts. Importantly, manganese(II), copper(II), and silver(I) phen chelates are relatively cheap and easy to synthesize and they offer significant antifungal chemotherapeutic potential for the treatment of highly resistant pathogens.

Virulence of Candida haemulonii complex in Galleria mellonella and efficacy of classical antifungal drugs: a comparative study with other clinically relevant non-albicans Candida species

&NA; Candida haemulonii complex has emerged as notorious yeasts causing invasive infections with high rates of treatment failures. Since there is a particular interest in the development of non‐mammalian host models to study microbial virulence, with the aim to evade the ethical impact of animal tests, herein we compared the virulence of C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera with non‐albicans Candida species (C. tropicalis, C. krusei and C. lusitaniae) on Galleria mellonella and the efficacy of antifungal drugs. All these fungi induced a dose‐dependent effect on larvae killing, a decrease in hemocyte density and fungi were phagocytozed by hemocytes in equal proportions. Fungal inoculation caused early larvae melanization after some minutes of injection, followed by an augmented pigmentation after 24 h. Differences among species virulence can be explained, in part, by differences in growth rate and production of hydrolytic enzymes. First‐line antifungals were tested with equivalent therapeutic doses and MIC profile in vitro was correlated with in vivo antifungal efficacy. Additionally, fungal burden increased in infected larvae along time and only caspofungin reduced the number of CFUs of C. haemulonii species complex. So, G. mellonella offers a simple and feasible model to study C. haemulonii complex virulence and drug efficacy.

Trichosporon asahii secretes a 30-kDa aspartic peptidase

Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase.

What are the advantages of living in a community? A microbial biofilm perspective!

Biofilm formation is the preferred mode of growth lifestyle for many microorganisms, including bacterial and fungal human pathogens. Biofilm is a strong and dynamic structure that confers a broad range of advantages to its members, such as adhesion/cohesion capabilities, mechanical properties, nutritional sources, metabolite exchange platform, cellular communication, protection and resistance to drugs (e.g., antimicrobials, antiseptics, and disinfectants), environmental stresses (e.g., dehydration and ultraviolet light), host immune attacks (e.g., antibodies, complement system, antimicrobial peptides, and phagocytes), and shear forces. Microbial biofilms cause problems in the hospital environment, generating high healthcare costs and prolonged patient stay, which can result in further secondary microbial infections and various health complications. Consequently, both public and private investments must be made to ensure better patient management, as well as to find novel therapeutic strategies to circumvent the resistance and resilience profiles arising from biofilm-associated microbial infections. In this work, we present a general overview of microbial biofilm formation and its relevance within the biomedical context.

Relationship between the Antifungal Susceptibility Profile and the Production of Virulence-Related Hydrolytic Enzymes in Brazilian Clinical Strains of Candida glabrata

Candida glabrata is a facultative intracellular opportunistic fungal pathogen in human infections. Several virulence-associated attributes are involved in its pathogenesis, host-pathogen interactions, modulation of host immune defenses, and regulation of antifungal drug resistance. This study evaluated the in vitro antifungal susceptibility profile to five antifungal agents, the production of seven hydrolytic enzymes related to virulence, and the relationship between these phenotypes in 91 clinical strains of C. glabrata. All C. glabrata strains were susceptible to flucytosine. However, some of these strains showed resistance to amphotericin B (9.9%), fluconazole (15.4%), itraconazole (5.5%), or micafungin (15.4%). Overall, C. glabrata strains were good producers of catalase, aspartic protease, esterase, phytase, and hemolysin. However, caseinase and phospholipase in vitro activities were not detected. Statistically significant correlations were identified between micafungin minimum inhibitory concentration (MIC) and esterase production, between fluconazole and micafungin MIC and hemolytic activity, and between amphotericin B MIC and phytase production. These results contribute to clarify some of the C. glabrata mechanisms of pathogenicity. Moreover, the association between some virulence attributes and the regulation of antifungal resistance encourage the development of new therapeutic strategies involving virulence mechanisms as potential targets for effective antifungal drug development for the treatment of C. glabrata infections.