Species diversity, antifungal susceptibility and phenotypic and genotypic characterisation of Exophiala spp. infecting patients in different medical centres in Brazil.

The Exophiala genus is responsible for many superficial and invasive infections resulting from black fungi. Identification of Exophiala at the species level is based on morphological observations complemented by molecular tests. The aim of this study was to identify 23 clinical isolates of Exophiala spp. and evaluate the antifungal susceptibility to seven different agents. Molecular identification was based on an analysis of ITS region of rDNA using genomic databases. The micromorphology was evaluated by microculture and scanning electron microscopy. The susceptibility tests were performed using the antifungal agents 5-fluorocytosine (5-FC), amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), caspofungin (CFG) and terbinafine (TRB). The ITS analysis identified 100% of the following isolates as: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1) and E. mesophila (1). The antifungal susceptibility tests showed that the triazoles compounds were in vitro the most active agents against Exophiala. ITS sequencing enabled the accurate identification of the 23 tested isolates. The triazoles, particularly itraconazole and posaconazole, exhibited MIC values lower than AMB, CAS and 5-FC. Although the guidelines do not indicate AMB for treatment against Exophiala spp., this study showed activity for all of the tested species, except E. mesophila.

Unraveling the role of high-intensity resistance training on left ventricle proteome: Is there a shift towards maladaptation?

High-intensity resistance training (RT) induces adaptations that improve physiological function. However, high intensity, volume and/or frequency may lead to injury and other health issues such as adverse cardiac effects. The aim of this study was to evaluate the effect of RT on left ventricle proteome, and to identify the pathways involved on the harmful adaptations induced by this protocol. Male Wistar rats were randomized into 2 groups: Trained (T) and Sedentary (S). Animals from T group were trained for 6weeks, and then all the animals were sacrificed and left ventricle was isolated for analysis. We identified 955 proteins, and 93 proteins were considered; 36 were expressed exclusively in T group, and 4 in S group. Based on quantitative analysis, 42 proteins were found overexpressed and 11 underexpressed in T group compared with S group. Using the Gene Ontology to relate the biological processes in which these proteins are involved, we conclude that RT protocol promotes changes similar to those found in the initial phase of heart failure, but we also observed a concomitant increased expression of protective proteins, suggesting the activation of pathways to avoid major damages on left ventricle and delay the onset of pathological hypertrophy. STATEMENT OF SIGNIFICANCE OF THE STUDY: Our study shows that high-intensity RT protocol changes left ventricle proteome, modifying metabolic profile of heart tissue and inducing the expression of proteins that acts against cardiac injury. We hypothesize that these adaptations occur to prevent the onset of cardiac dysfunction. Despite highly significant, it remains to be determined whether these adaptations are sufficient to further keep left ventricle function and exert cardioprotection, and whether this panel will be shifted towards maladaptation, and heart failure.

Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B.

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

Interaction of the rattlesnake toxin crotamine with model membranes.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. A common gene ancestry and structural similarity with the antimicrobial ß-defensins (identical disulfide bond pattern and highly positive net charge) suggested potential antimicrobial activities for this snake toxin. Although crotamine demonstrated low activity against both Gram-positive and Gram-negative bacteria, a pronounced antifungal activity was observed against Candida spp., Trichosporon spp., and Cryptococcus neoformans. Crotamine's selective antimicrobial properties, with no observable hemolytic activity, stimulated us to evaluate the potential applications of this polypeptide as an antiyeast or candicidal agent for medical and industrial application. Aiming to understand the mechanism(s) of action underlying crotamine antimicrobial activity and its selectivity for fungi, we present herein studies using membrane model systems (i.e., large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs), with different phospholipid compositions. We show here that crotamine presents a higher lytic activity on negatively charged membranes compared with neutral membranes, with or without cholesterol or ergosterol content. The vesicle burst was not preceded by membrane permeabilization as is generally observed for pore forming peptides. Although such a property of disrupting lipid membranes is very important to combat multiresistant fungi, no inhibitory activity was observed for crotamine against biofilms formed by several Candida spp. strains, except for a limited effect against C. krusei biofilm.

Breakthrough candidemia due to multidrug-resistant Candida glabrata during prophylaxis with a low dose of micafungin.

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-ß-D-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Molecular identification, antifungal susceptibility profile, and biofilm formation of clinical and environmental Rhodotorula species isolates.

Rhodotorula species are emergent fungal pathogens capable of causing invasive infections, primarily fungemia. They are particularly problematic in immunosuppressed patients when using a central venous catheter. In this study, we evaluated the species distribution of 51 clinical and 8 environmental Rhodotorula species isolates using the ID32C system and internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing and biofilm formation capability using a crystal violet staining assay were performed. Using ITS sequencing as the gold standard, the clinical isolates were identified as follows: 44 R. mucilaginosa isolates, 2 R. glutinis isolates, 2 R. minuta isolates, 2 R. dairenensis isolates, and 1 Rhodosporidium fluviale isolate. The environmental isolates included 7 R. mucilaginosa isolates and 1 R. slooffiae isolate. Using the ID32C system, along with a nitrate assimilation test, only 90.3% of the isolates tested were correctly identified. In the biofilm formation assay, R. mucilaginosa and R. minuta exhibited greater biofilm formation ability compared to the other Rhodotorula species; the clinical isolates of R. mucilaginosa showed greater biofilm formation compared to the environmental isolates (P = 0.04). Amphotericin B showed good in vitro activity (MIC ≤ 1 µg/ml) against planktonic cells, whereas voriconazole and posaconazole showed poor activity (MIC(50)/MIC(90), 2/4 µg/ml). Caspofungin and fluconazole MICs were consistently high for all isolates tested (≥64 µg/ml and ≥ 4 µg/ml, respectively). In this study, we emphasized the importance of molecular methods to correctly identify Rhodotorula species isolates and non-R. mucilaginosa species in particular. The antifungal susceptibility profile reinforces amphotericin B as the antifungal drug of choice for the treatment of Rhodotorula infections. To our knowledge, this is the first study evaluating putative differences in the ability of biofilm formation among different Rhodotorula species.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine.

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the ß-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 µg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 µg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 µg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 µg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR.

Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the "gold standard" for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.

In vitro susceptibility of a large collection of Candida Strains against fluconazole and voriconazole by using the CLSI disk diffusion assay.

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC(®) image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period.

Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex.

Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).

Changes in cell wall synthesis and ultrastructure during paradoxical growth effect of caspofungin on four different Candida species.

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ß-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ß-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

The occurrence of Aeromonas spp. in the bottled mineral water, well water and tap water from the municipal supplies

The aim of this work was to study the occurrence of Aeromonas sp in the bottled mineral water, well water and tap water from the municipal supplies. Positive samples were found for Aeromonas spp. 12.7 percent from the mineral water, 8.3 percent from the artesian water and 6.5 percent from the tap water. The recovery of Aeromonas spp. was significantly higher in the bottled mineral and artesian water than in the tap water from municipal supplies. The occurrence of the Aeromonas spp. did not correlate significantly with the contamination indicator bacteria (i.e. total coliforms) in the artesian water samples. However, a significant correlation was found between Aeromonas spp. and total coliforms in the both mineral water and tap water samples. The presence or absence of a correlation between the indicator bacteria and Aeromonas could reflect the occasional appearance of the pathogen in the drinking water and the different rates of survival and recovery of these agents compared with those fecal indicators. The finding that 41.6, 14.8 and 9.0 percent of the artesian water, bottled mineral water and tap water, respectively, sampled in the current study failed to meet the Brazilian standard for total coliforms in the drinking water should therefore be of concern.

A porcentagem de amostras positivas para Aeromonas foi de 12.7 por cento para água mineral, 8.3 por cento para água de poço artesiano e 6.5 por cento para água do sistema público de abastecimento. O isolamento de Aeromonas spp. foi significativamente maior em água mineral e água de poço artesiano do que em água do sistema público. A ocorrência de Aeromonas spp. não teve correlação significativa com os indicadores de contaminação tradicionalmente utilizados (coliformes totais) em amostras de água de poço artesiano. No entanto, esta correlação foi positiva e significativa em água mineral e água do sistema público. A presença ou ausência de correlação entre bactérias indicadoras e a presença de Aeromonas pode refletir o aparecimento ocasional de patógenos em águas para consumo humano e as diferentes taxas de sobrevivência e isolamento destes agentes comparados com os indicadores fecais de contaminação. A constatação de que 41.6 por cento, 14.8 por cento e 9.0 por cento respectivamente amostras de água de poço, água mineral e água do abastecimento público utilizadas neste estudo apresentaram índices de coliformes maiores do que os aceitáveis pela legislação brasileira é um fato preocupante. Estes números mostram a necessidade de melhoria nos sistemas de monitoramento para a indústria de águas minerais e o sistema público de abastecimento. As cepas isoladas pertencentes ao gênero Aeromonas foram identificadas ao nível de espécie como A. hydrophila e A. jandaei. A significância do grande número de isolamentos de espécies de Aeromonas em saúde pública ainda não está clara. É necessário o estudo dos efeitos de cepas específicas utilizando modelos animais de infecção. Estes resultados podem contribuir para a reavaliação dos critérios empregados para a análise da qualidade microbiológica da água e a definição de limites de densidades para o gênero Aeromonas em águas destinadas ao consumo humano.

In vitro evaluation of putative virulence attributes of oral isolates of Candida spp. obtained from elderly healthy individuals.

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

Characteristics of biofilm formation by Candida tropicalis and antifungal resistance.

Candida tropicalis is a common species related to nosocomial candidemia and candiduria. Most Candida spp. infections are associated with biofilm formation on implanted medical devices or on host epithelial cell surfaces. Sessile cells display phenotypic traits dramatically different from those of their free-living, planktonic counterparts, such as increased resistance to antimicrobial agents and to host defenses. The characteristics of C. tropicalis biofilm formation in vitro are described. By an XTT-reduction assay, an increase in metabolic activity was observed up to 24 h of biofilm formation, and this activity showed a linear relationship with sessile cell density. Scanning electron microscopy was used to further characterize C. tropicalis biofilms. The initial adherence of yeast cells was followed by germination, microcolony formation, filamentation and maturation at 24-48 h. Mature biofilms consisted of a dense network of yeast cells and filamentous forms of C. tropicalis. Increased resistance of sessile cells against fluconazole and amphotericin B was also demonstrated. Real-time reverse transcription-PCR quantification showed that sessile cells overexpressed ERG11 (coding for lanosterol 14 alpha-demethylase) and MDR1 (coding for an efflux protein belonging to the major facilitator superfamily). These mechanisms may contribute to the fluconazole resistance of the C. tropicalis biofilm.

Utilização de um meio cromogênico e da técnica de semi-nested PCR para identificação de espécies de Candida / Use of chromogenic medium and semi-nested PCR-based assay to identify Candida species

Nesse trabalho, o meio cromogênico CHROMagar Candida e a técnica de semi-nested PCR (sn-PCR) foram comparados quanto a sua capacidade de identificar a espécie de 52 isolados clínicos de Candida sp. Com o emprego do meio cromogênico, 39 (75%) isolados foram identificados presuntivamente como C. albicans (n = 22), C. glabrata (n = 9), C. tropicalis (n = 5) e C. krusei (n = 3). Treze isolados (25%) não puderam ser identificados. Por meio da técnica de sn-PCR, que se baseia na amplificação de uma região do cluster gênico do RNA ribossomal (5.8S – 28S), 43 (83%) isolados foram identificados como C.albicans (n = 24), C. glabrata (n = 11), C. tropicalis (n = 5), C. parapsilosis (n = 3) e nove isolados não puderam ser identificados em nível de espécie. Entre os 52 isolados analisados, 34 (65,4%) apresentaram resultados concordantes pelos dois métodos, 12 (23,1%) apresentaram resultados discrepantes, e 6 (11,5%) não puderam ser identificadas por nenhuma das duas metodologias. Os resultados indicam que ambas as técnicas apresentam limitações, mas o método de sn-PCR é mais adequado que o meio cromogênico para a identificação de espécies do gênero Candida, devido ao menor número de isolados que não puderam ser identificados.