Genomic Diversity of Burkholderia pseudomallei in Ceara, Brazil.

Burkholderia pseudomallei is a Gram-negative bacterium that causes the sapronotic disease melioidosis. An outbreak in 2003 in the state of Ceara, Brazil, resulted in subsequent surveillance and environmental sampling which led to the recognition of B. pseudomallei as an endemic pathogen in that area. From 2003 to 2015, 24 clinical and 12 environmental isolates were collected across Ceara along with one from the state of Alagoas. Using next-generation sequencing, multilocus sequence typing, and single nucleotide polymorphism analysis, we characterized the genomic diversity of this collection to better understand the population structure of B. pseudomallei associated with Ceara. We found that the isolates in this collection form a distinct subclade compared to other examples from the Western Hemisphere. Substantial genetic diversity among the clinical and environmental isolates was observed, with 14 sequence types (STs) identified among the 37 isolates. Of the 31,594 core single-nucleotide polymorphisms (SNPs) identified, a high proportion (59%) were due to recombination. Because recombination events do not follow a molecular clock, the observation of high occurrence underscores the importance of identifying and removing recombination SNPs prior to evolutionary reconstructions and inferences in public health responses to B. pseudomallei outbreaks. Our results suggest long-term B. pseudomallei prevalence in this recently recognized region of melioidosis endemicity.IMPORTANCEB. pseudomallei causes significant morbidity and mortality, but its geographic prevalence and genetic diversity are not well characterized, especially in the Western Hemisphere. A better understanding of the genetic relationships among clinical and environmental isolates will improve knowledge of the population structure of this bacterium as well as the ability to conduct epidemiological investigations of cases of melioidosis.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification.

In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

An alternative method for the analysis of melanin production in Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato.

Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the assessment of melanin production and its quantification may contribute to the understanding of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neoformans sensu lato and C. gattii sensu lato. Eight C. neoformans sensu lato and three C. gattii sensu lato, identified through URA5 methodology, Candida parapsilosis ATCC 22019 (negative control) and one Hortaea werneckii (positive control) were inoculated on minimal medium agar with or without L-DOPA, in duplicate, and incubated at 35°C, for 7 days. Pictures were taken from the third to the seventh day, under standardised conditions in a photographic chamber. Then, photographs were analysed using grayscale images. All Cryptococcus spp. strains produced melanin after growth on minimal medium agar containing L-DOPA. C. parapsilosis ATCC 22019 did not produce melanin on medium containing L-DOPA, while H. werneckii presented the strongest pigmentation. This new method allows the indirect analysis of melanin production through pixel quantification in grayscale images, enabling the study of substances that can modulate melanin production.

A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production.

Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9) with 4.1% (m/m) sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45-37.90 and 155.84-260.29 µM, respectively. In addition, Mo-CBP2 (18.90 µM) increased the cell membrane permeabilization and reactive oxygen species production in C. albicans and promoted degradation of circular plasmid DNA (pUC18) from Escherichia coli. The data presented in this study highlight the potential use of Mo-CBP2 as an anticandidal agent, based on its ability to inhibit Candida spp. growth with apparently low toxicity on mammalian cells.

Azole resistance in Candida spp. isolated from Catú Lake, Ceará, Brazil: an efflux-pump-mediated mechanism

Abstract Since, there is no study reporting the mechanism of azole resistance among yeasts isolated from aquatic environments; the present study aims to investigate the occurrence of antifungal resistance among yeasts isolated from an aquatic environment, and assess the efflux-pump activity of the azole-resistant strains to better understand the mechanism of resistance for this group of drugs. For this purpose, monthly water and sediment samples were collected from Catú Lake, Ceará, Brazil, from March 2011 to February 2012. The obtained yeasts were identified based on morphological and biochemical characteristics. Of the 46 isolates, 37 were Candida spp., 4 were Trichosporon asahii, 3 were Cryptococcus laurentii, 1 Rhodotorula mucilaginosa, and 1 was Kodamaea ohmeri. These isolates were subjected to broth microdilution assay with amphotericin B, itraconazole, and fluconazole, according to the methodology standardized by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of amphotericin B, itraconazole, and fluconazole were 0.03125–2 µg/mL, 0.0625 to ≥16 µg/mL, and 0.5 to ≥64 µg/mL, respectively, and 13 resistant azole-resistant Candida isolates were detected. A reduction in the azole MICs leading to the phenotypical reversal of the azole resistance was observed upon addition of efflux-pump inhibitors. These findings suggest that the azole resistance among environmental Candida spp. is most likely associated with the overexpression of efflux-pumps.

Azole resistance in Candida spp. isolated from Catú Lake, Ceará, Brazil: an efflux-pump-mediated mechanism.

Since, there is no study reporting the mechanism of azole resistance among yeasts isolated from aquatic environments; the present study aims to investigate the occurrence of antifungal resistance among yeasts isolated from an aquatic environment, and assess the efflux-pump activity of the azole-resistant strains to better understand the mechanism of resistance for this group of drugs. For this purpose, monthly water and sediment samples were collected from Catú Lake, Ceará, Brazil, from March 2011 to February 2012. The obtained yeasts were identified based on morphological and biochemical characteristics. Of the 46 isolates, 37 were Candida spp., 4 were Trichosporon asahii, 3 were Cryptococcus laurentii, 1 Rhodotorula mucilaginosa, and 1 was Kodamaea ohmeri. These isolates were subjected to broth microdilution assay with amphotericin B, itraconazole, and fluconazole, according to the methodology standardized by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of amphotericin B, itraconazole, and fluconazole were 0.03125-2µg/mL, 0.0625 to ≥16µg/mL, and 0.5 to ≥64µg/mL, respectively, and 13 resistant azole-resistant Candida isolates were detected. A reduction in the azole MICs leading to the phenotypical reversal of the azole resistance was observed upon addition of efflux-pump inhibitors. These findings suggest that the azole resistance among environmental Candida spp. is most likely associated with the overexpression of efflux-pumps.

Exogenous tyrosol inhibits planktonic cells and biofilms of Candida species and enhances their susceptibility to antifungals.

Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth.

ß-Lactam antibiotics and vancomycin inhibit the growth of planktonic and biofilm Candida spp.: an additional benefit of antibiotic-lock therapy?

The aim of this study was to evaluate the effects of cefepime, meropenem, piperacillin/tazobactam (TZP) and vancomycin on strains of Candida albicans and Candida tropicalis in planktonic and biofilm forms. Twenty azole-derivative-resistant strains of C. albicans (n=10) and C. tropicalis (n=10) were tested. The susceptibility of planktonic Candida spp. to the antibacterial agents was investigated by broth microdilution. The XTT reduction assay was performed to evaluate the viability of growing and mature biofilms following exposure to these drugs. Minimum inhibitory concentrations (MICs) ranged from 0.5 mg/mL to 2 mg/mL for cefepime, TZP and vancomycin and from 0.5 mg/mL to 1 mg/mL for meropenem and the drugs also caused statistically significant reductions in biofilm cellular activity both in growing and mature biofilm. Since all of the tested drugs are commonly used in patients with hospital-acquired infections and in those with catheter-related infections under antibiotic-lock therapy, it may be possible to obtain an additional benefit from antibiotic-lock therapy with these drugs, namely the control of Candida biofilm formation.

Glucose and lactose as cryoprotectants for fungal strains immobilised in sodium alginate: an emphasis on the conservation of the zygomycetes Rhizopus and Mucor.

This research aimed at investigating the cryoprotectant action of glucose and lactose on strains of Malassezia spp. and zygomycetes immobilised in sodium alginate. Twelve strains of Malassezia spp. (nine M. furfur, two M. globosa and one M. sympodialis) and 12 zygomycetes (five Rhizopus oryzae and seven Mucor hiemales) were immobilised in sodium alginate, within plastic beads, maintained in appropriate media containing glucose and lactose at concentrations of 9% and 23% and preserved at temperatures of -20 and -80 °C. Strain viability was evaluated from 15 to 270 days of storage, through the observation of macro-micromorphologic characteristics. The Malassezia spp. strains were only viable until 90 days of storage, whereas for zygomycetes, viable strains were observed until after 270 days of storage at -80 °C, in the media containing 23% glucose or lactose. The use of 23% glucose or lactose at -80 °C in a sodium alginate cell immobilisation system is efficient for cryopreserving zygomycetes. This research creates perspectives for the use of glucose and lactose in sodium alginate cell immobilisation systems for the preservation of fungi with low viability.

Minimum inhibitory concentrations of amphotericin B, azoles and caspofungin against Candida species are reduced by farnesol.

The objective of this study was to evaluate the antifungal activity of farnesol and its interaction with traditional antifungals against drug-resistant strains of Candida species. To do so, we studied the minimum in vitro inhibitory concentration (MIC) of amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), caspofungin (CAS) and farnesol against 45 isolates of Candida spp., i.e., 24 C. albicans, 16 C. parapsilosis and 5 C. tropicalis through the use of the broth microdilution method. Then, the isolates were tested with the combination of farnesol plus drugs to which they were previously found to be resistant. Additionally, the strains were pre-incubated at sub-inhibitory farnesol concentrations and their antifungal susceptibilities were re-evaluated. We found the MIC values for farnesol varied from 4.68-150 µM for Candida spp., with 19 isolates having a MIC > 1 mg/l, 18 a MIC ≥ 64 mg/l, 35 having a MIC ≥ 1 mg/l and 6 isolates a MIC ≥ 2 mg/l or were resistant to AMB, FLC, ITC and CAS, respectively. Significant MIC reductions were observed when farnesol and antifungal drugs were combined (P < 0.05) and when Candida strains were incubated with farnesol (P < 0.05). We conclude that the in vitro effects of farnesol improved the activity of traditional antifungals to which the Candida spp. isolates were resistant. These results support further investigation of the role of farnesol in the balance of the sterol biosynthetic pathway and how it interferes with cell viability.

Evaluation of the genetic diversity of Histoplasma capsulatum var. capsulatum isolates from north-eastern Brazil.

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceará, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceará, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1b and 1c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1-5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceará have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.

Clinical-epidemiological features of 13 cases of melioidosis in Brazil.

The aim of this work was to catalog the clinical and ecoepidemiological characteristics of melioidosis in Brazil. The clinical-epidemiological features of melioidosis in Ceará are similar to those in other regions where the disease is endemic. These findings support the inclusion of this Brazilian state as part of the zone of endemicity for melioidosis.

Histoplasmosis in HIV-positive patients in Ceará, Brazil: clinical-laboratory aspects and in vitro antifungal susceptibility of Histoplasma capsulatum isolates.

This study contains a descriptive analysis of histoplasmosis in AIDS patients between 2006 and 2010 in the state of Ceará, Brazil. Additionally, the in vitro susceptibility of Histoplasma capsulatum isolates obtained during this period was assessed. We report 208 cases of patients with histoplasmosis and AIDS, describing the epidemiological, clinical, laboratory and therapeutic aspects. The in vitro antifungal susceptibility test was carried out by the microdilution method, according to Clinical and Laboratory Standards Institute, with H. capsulatum in the filamentous and yeast phases, against the antifungals amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. In 38.9% of the cases, histoplasmosis was the first indicator of AIDS and in 85.8% of the patients the CD4 cell count was lower than 100 cells/mm(3). The lactate dehydrogenase levels were high in all the patients evaluated, with impairment of hepatic and renal function and evolution to death in 42.3% of the cases. The in vitro susceptibility profile demonstrated there was no antifungal resistance among the isolates evaluated. There was a significant increase in the number of histoplasmosis cases in HIV-positive patients during the period surveyed in the state of Ceará, northeastern Brazil, but no antifungal resistance among the recovered isolates of H. capsulatum.

Yeast microbiota of raptors: a possible tool for environmental monitoring.

Twenty-two raptors from a rehabilitation centre were evaluated for the presence of yeasts prior to returning them to the wild, and the recovered Candida isolates were tested for in vitro antifungal susceptibility and phospholipase production. Samples were collected from the crop/lower esophagus and cloaca. In vitro antifungal susceptibility and phospholipase production of 21 Candida strains were assessed through broth microdilution and growth on egg yolk agar respectively. Twenty-seven isolates, belonging to seven species, were recovered from 16 tested birds, with C. albicans and C. famata as the most prevalent species. Three out of 21 isolates (2 C. albicans and 1 C. tropicalis) were simultaneously resistant to fluconazole and itraconazole. As for phospholipase production, 8 (8/21) isolates (6 C. albicans, 1 C. famata and 1 C. parapsilosis) showed enzymatic activity. The most relevant finding in this study was the isolation of resistant Candida spp. from wild raptors that had never been submitted to antifungal therapy, which suggests exposure to environmental contaminants. Based on this, we propose the assessment of Candida spp. from the gastrointestinal tract of raptors as a tool for environmental monitoring.

Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp.

In the present study, it was sought to compare yeast microbiota of wild and captive Macrobrachium amazonicum and evaluate the antifungal susceptibility and production of virulence factors by the recovered isolates of Candida spp. Additionally, cultivation water was monitored for the presence of fungi. Overall, 26 yeast isolates belonging to three genera and seven species were obtained, out of which 24 were Candida spp., with Candida famata as the most prevalent species for both wild and captive prawns. From cultivation water, 28 isolates of filamentous fungi were obtained, with Penicillium spp., Cladosporium spp. and Aspergillus spp. as the most frequent genera. Eight out of 24 Candida spp. isolates were resistant to azole derivatives, out of which four were recovered from wild-harvested prawns. As for production of virulence factors, three (12.5%) and eight (33.3%) isolates presented phospholipase and protease activity, respectively. This is the first comparative study between wild and captive prawns and the first report on yeast microbiota of M. amazonicum. The most relevant finding was the high percentage of resistant Candida spp., including from wild individuals, which suggests the occurrence of an environmental imbalance in the area where these prawns were captured.

Alkylphenol Activity against Candida spp. and Microsporum canis: A Focus on the Antifungal Activity of Thymol, Eugenol and O-Methyl Derivatives.

In recent years there has been an increasing search for new antifungal compounds due to the side effects of conventional antifungal drugs and fungal resistance. The aims of this study were to test in vitro the activity of thymol, eugenol, estragole and anethole and some O-methyl-derivatives (methylthymol and methyleugenol) against Candida spp. and Microsporum canis. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC). The minimum fungicidal concentrations (MFC) for both Candida spp. and M. canis were found by subculturing each fungal suspension on potato dextrose agar. Thymol, methylthymol, eugenol, methyl-eugenol, anethole, estragole and griseofulvin respectively, presented the following MIC values against M. canis: 4.8-9.7; 78-150; 39; 78-150; 78-150; 19-39 µg/mL and 0.006-2.5 mg/mL. The MFC values for all compounds ranged from 9.7 to 31 µg/mL. Concerning Candida spp, thymol, methylthymol, eugenol, methyleugenol, anethole, estragole and amphotericin, respectively, showed the following MIC values: 39; 620-1250; 150-620; 310-620; 620; 620-1250 and 0.25-2.0 mg/mL. The MFC values varied from 78 to 2500 µg/mL. All tested compounds thus showed in vitro antifungal activity against Candida spp. and M. canis. Therefore, further studies should be carried out to confirm the usefulness of these alkylphenols in vivo.

Isolation of pathogenic yeasts in the air from hospital environments in the city of Fortaleza, northeast Brazil.

This paper reports the results of environmental surveillance of yeasts in specific areas of two tertiary local hospitals. From March 2007 to February 2008, samples from the air of two public hospitals were collected on a monthly basis. The samples were collected through passive sedimentation method (day and night exposure) of Petri dishes. A total of 240 air samples from 10 hospital environments were analyzed. These environments presented similar contamination levels, from which 80 fungi isolates were isolated: Candida parapsilosis (n = 34), Rhodotorula spp. (19), Trichosporon asahii (11), C. tropicalis (8), C. albicans (4), C. glabrata (1), C. guilliermondii (1), C. krusei (1) and Saccharomyces spp. (1). Regarding the presence of yeasts and climatic conditions, there were 40 strains (50%) in semi-critical areas (natural ventilation) and critical areas (air conditioned). Considering the presence of microorganisms with pathogenic potential, environmental monitoring is necessary to prevent possible hospital infections.