Leaf-cutting ants: an unexpected microenvironment holding human opportunistic black fungi

Fungus-growing ants of the genus Atta are known for their leaf-cutting habit, a lifestyle they have maintained since their 50-million-year-old co-evolution with a mutualistic fungus, cultivated as food. Recent studies have highlighted that, in addition to the mutualistic fungus, nests of ants harbor a great diversity of microbial communities. Such microorganisms include the dematiaceous fungi, which are characterized by their melanized cell walls. In order to contribute to the knowledge of fungal ecology, as well as opportunistic strains that may be dispersed by these social insects, we isolated and identified fungi carried by gynes of Atta capiguara and Atta laevigata, collected from colonies located in Fazenda Santana, Botucatu (São Paulo, Brazil). The isolation was carried out using the oil flotation technique, which is suitable for the growth of black fungi. Inoculated plates were incubated at 25 and 35 °C until black cultures were visible (20–45 days). Isolates were identified based on microscopic and molecular characteristics. Some isolated genera were: Cladophialophora, Cladosporium, Exophiala, Ochroconis, Phaeococcomyces, Phialophora and Penidiella. Hyaline species were also found. The results obtained from this work showed that leaf-cutting gynes may contribute to the dispersal of opportunistic dematiaceous fungi. It is suggested that more attention should be paid to this still unexplored subject.     

Rare Invasive Fungal Infections: Epidemiology, Diagnosis and Management

Rare yeast and filamentous fungi belonging to hyalohyphomycetes (e.g., Scedosporium, Fusarium), zygomycetes and dematiaceous (e.g., Alternaria, Bipolaris) are implicated in human infections ranging from colonization and localized infections in immunocompetent individuals to fungemias and disseminated diseases in immunocompromised patients and accounting <10 % of all isolated fungal pathogens. The diagnosis of yeast, Fusarium and Scedosporium infections is based on blood cultures and of filamentous fungal infections on histopathology, direct microscopy and culture of infected tissues. The panfungal marker 1,3-b-D glucan test as well as cross reaction with antigen tests for other fungi can be used; whereas, PCR assays have been developed for direct detection of these fungi in blood and in tissues. Amphotericin B is the drug of choice for most rare yeast infections except for Trichosporon infections where voriconazole is used. The management of the other infections includes surgery combined with antifungal therapy mainly with amphotericin B for zygomycetes, voriconazole or amphotericin B for hyalohyphomycetes, and itraconazole or amphotericin B for dematiaceous fungi.     

Phaeohyphomycosis Caused by <Emphasis Type="Italic">Rhytidhysteron rufulum</Emphasis> and Review of Literature

Phaeohyphomycosis is caused by a heterogeneous group of mycelial dematiaceous (phaeoid) fungi, which produce melanin pigment. This condition is often confused with chromoblastomycosis. Rhytidhysteron is a dematiaceous fungus, which has been recently found to be causing human infections. Till date only three cases of infection with Rhytidhysteron rufulum have been reported in the literature. All three cases have been from North India. Hereby, we present another two cases where Rhytidhysteron was isolated. Both the patients belonged to Chandigarh (India) and presented with subcutaneous lesions. The isolates were confirmed by ITS sequencing. Both the patients were immunocompetent and gave no history of trauma or any other predisposing factor. Phaeohyphomycosis are often missed due to lack of knowledge regarding the fungi causing the infections and there is need for clinical, pathological and microbiological correlation for effective diagnosis and treatment in these cases.     

Diversity of endophytic fungi in <Emphasis Type="Italic">Eucalyptus microcorys</Emphasis> assessed by complementary isolation methods

Brazil is the world’s largest producer country of eucalyptus. Although widely applied in the charcoal industry, no studies have focused on the microorganisms associated with Eucalyptus microcorys. Here, we evaluated the composition and structure of endophytic fungal communities in leaves of E. microcorys through two isolation techniques. A total of 120 fresh leaves were collected in a year-long survey at an eucalyptus plantation in the State of São Paulo (Brazil). Endophytic fungi were isolated by particle filtration (PF) and direct leaf fragment plating (LP) in two media: modified dicloran and synthetic nutrient agar, both supplemented with rose bengal and chloramphenicol. The isolates were grouped into morphospecies and identified by morphology and DNA sequencing. We recovered a total of 709 isolates, representing 59 taxa. All taxa found are reported as endophytic for the first time for E. microcorys. Castanediella eucalypticola and Neophaeomoniella eucalypti are new occurrences reported for Brazil. The LP technique recovered a higher number of taxa and isolates than the PF. However, the PF technique retrieved a higher species/isolate ratio than the LP method, 0.12 and 0.09, respectively. Fungal diversity assessed by diversity metrics did not significantly differ between isolation methods. Both techniques recovered a high number of unique taxa, demonstrating that neither method would individually represent the species richness from E. microcorys. The use of LP and PF provided a greater number of observed taxa and consequently new occurrence of species for Brazil.     

White rot Basidiomycetes isolated from Chiloé National Park in Los Lagos region, Chile

Wood decomposition is an important component in forest ecosystems but information about the diversity of fungi causing decay is lacking. This is especially true for the temperate rain forests in Chile. These investigations show results of a biodiversity study of white-rot fungi in wood obtained from Chiloé National Park in Los Lagos region, Chile. Culturing from white-rotted wood followed by sequencing of the complete internal transcribed spacer region of the ribosomal DNA (rDNA) or partial large subunit region of the rDNA, identified 12 different species in the Basidiomycota. All of these fungi were characterized as white rot fungi and were identified with a BLAST match of 97 % or greater to sequences in the GenBank database. Fungi obtained were species of Phlebia, Mycoacia, Hyphodontia, Bjerkandera, Phanerochaete, Stereum, Trametes, and Ceriporiopsis. This report identifies for the first time in Chile the species Ceriporiopsis subvermispora, Hyphodontia radula, Phlebia radiata, Phanerochaete affinis, Peniophora cinerea, Stereum gausapatum, Phlebia setulosa and Phanerochaete sordida. Scanning electron microscopy was used to characterize the type of decay caused by the fungi that were isolated and a combination of selective lignin degraders and simultaneous white rot fungi were found. Fungi that cause a selective degradation of lignin are of interest for bioprocessing technologies that require modification or degradation of lignin without cellulose removal.     

Analysis of Black Fungal Biofilms Occurring at Domestic Water Taps (II): Potential Routes of Entry

Formation of tenacious and massive black biofilms was occasionally observed at the water–air interphase of water taps and in associated habitats at several locations in Germany. Exophiala lecanii-corni was proven to be the dominant component of these biofilms. Water utility companies were interested to understand by which route fungi building these black biofilms enter their habitat at affected sites in domestic sanitary. A wide variety of fungi is known to be common in wet indoor environments, as well as in the drinking water resources. Two possible routes of entry are therefore considered as follows: (a) distribution by the drinking water system or (b) a retrograde route of colonisation. Previous compositional analysis revealed that the black constituents of biofilms primarily belong to the herpotrichiellaceous black yeast and relatives. Therefore, a systematic search for black fungi in the drinking water system was performed using Sabouraud’s glucose agar medium with chloramphenicol and erythritol–chloramphenicol agar as isolation media. Cadophora malorum was the dominant fungus in the investigated drinking water systems, and samples taken from the house connections (n = 50; 74 %, <200 cfu/L), followed by a so far undescribed Alternaria sp. (28 %; <10 cfu/L) and E. castellanii (26 %; <10 cfu/L). Of note, C. malorum was not present in any previously analysed biofilm. Since E. lecanii-corni was not found in any water sample from the distribution system tested, but represented the most abundant species in dark biofilms previously analysed, a retrograde route of contamination in case of E. lecanii-corni can be assumed.     

Fungal endophytes from cactus Cereus jamacaru in Brazilian tropical dry forest: a first study

Endophytic fungi live within the healthy tissues of plants and can promote host species tolerance to different environmental stresses. However, most studies have been of plants in humid environments, and there are few reports of the benefits of such associations for plants of extreme environments. Our aims were to identify endophytic fungi using morphological taxonomy, to explore richness and estimate species frequency in a cactus, C. jamacaru, in Brazilian tropical dry forest (Caatinga). We thus identified 59 taxa, corresponding to 69.7 % of the total number of isolates; the other 30.3 % were sterile mycelia. Cladosporium cladosporioides and Fusarium oxysporum were the species most commonly isolated, followed by Acremonium implicatum, Aureobasidium pullulans, Trichoderma viride, Chrysonilia sitophila, and Aspergillus flavus. Forty-seven species are recorded for the first time as endophytic fungi of cacti, and 18 others as endophytes for Brazil; this suggests that C. jamacaru harbors a highly diverse fungal community as measured by a diversity index. However, species accumulation curves suggest that our study still underestimates endophyte diversity because it does not provide an exhaustive sample. To our knowledge, this is the first report of endophytic fungi from C. jamacaru in tropical dry forests.     

Diversity of coelomycetous fungi in human infections: A 10-y experience of two European reference centres

The coelomycetous fungi are difficult to properly identify from their phenotypic characterization and their role as etiologic agents of human infections is not clear. We studied the species distribution of these fungi among clinical isolates that had been collected and stored over a ten-year period in two European reference laboratories (France and Spain). We identified phenotypically and molecularly 97 isolates by sequencing the D1-D2 fragment of the 28S nrRNA (LSU) gene and we provided the in vitro antifungal susceptibility pattern of seven antifungals against 46 isolates. Species of the orders Pleosporales and Glomerellales were present in both collections, and Botryosphaeriales and Diaporthales only in the French one. The most prevalent species were Medicopsis romeroi, Neocucurbitaria keratinophila, Neocucurbitaria unguis-hominis and Paraconiothyrium cyclothyrioides, which had been recovered primarily from superficial tissues. The Didymellaceae was the most common family represented, with 27 isolates distributed into five genera. Most of the isolates tested were susceptible to antifungals, and only the geometric mean (GM) and minimal inhibitory concentration (MIC) values of itraconazole and caspofungin had higher values. This study provides a good picture of the great diversity of coelomycetous fungi in the European clinical context, and the basis for future studies on this interesting but neglected group of fungi.     

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4?×?10⁴ to 2.1?×?10⁶?CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100 %), followed by Penicillium chrysogenum (62 %), Aspergillus niger (54 %), and Aspergillus flavus (44 %). Citrinin was present in 100 % samples (0.23–20.65 mg/kg), aflatoxin in 92 % samples (0.61–77.33 μg/kg) and Ochratoxin-A in 100 % samples (0.23–2.48 μg/kg); 100 % citrinin and 76.09 % aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.     

Black Yeast Biota in the Mangrove, in Search of the Origin of the Lethargic Crab Disease (LCD)

Knowledge of natural ecology is essential for a better understanding of pathogenicity and opportunism in black yeast-like fungi. Although etiological agents of diseases caused by these fungi are supposed to originate from the environment, their isolation from nature is difficult. This is probably due to their oligotrophic nature, low competitive ability, and, overall, insufficient data on their natural habitat. We obtained environmental samples from mangrove areas where mortalities by lethargic crab disease (LCD) are reported and areas without disease recorded. Isolation of chaetothyrialean black yeasts and relatives was performed using a highly selective protocol. Species-specific primers were used to determine if these isolates represented Exophiala cancerae or Fonsecaea brasiliensis, two proven agents of LCD, in order to test hypotheses about the origin of the disease. Isolates, identified by morphology as Fonsecaea- or Exophiala-like, were tested specific diagnostic markers for the fungi associated with LCD. Although several black fungi were isolated, the main causative agent of the LCD, E. cancerae, was not found. Molecular markers for F. brasiliensis revealed 10 positive bands for isolates from biofilms on mangrove leaves, branches, and aerial roots, of which four were confirmed by ITS sequencing. The absence of E. cancerae in environmental samples suggests that the species is dependent on the crab, as a genuine pathogen, different from F. brasiliensis, which is probably not dependent on the host species, U. cordatus. However, we did not attempt isolation from the marine water, which may represent the pathway of dispersion of the black yeast species between neighbor mangroves.     

Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra

This multidisciplinary research combines knowledge in molecular biology with fungal morphology, aiming at the identification of infecting fungi from historical documents on the Archive of the University of Coimbra. The identification of infecting fungi on several bibliographic documents and support materials was based both on ribosomal DNA loci amplification and sequencing, and morphological identification, using macro- and microscopical traits. A high fungal diversity was found in all types of support: parchment, laid-paper and wood-pulp paper. Fourteen fungal genera were isolated, identified, and kept in culture. The most frequent were Cladosporium, Penicillium and Aspergillus, and other less frequent genera, such as Alternaria, Botrytis, Chaetomium, Chromelosporium, Epicoccum, Phlebiopsys and Toxicocladosporium were also present. Within these genera, 20 different species were identified, from which 15 were found only in a single support type. Cladosporium cladosporioides and Penicillium chrysogenum were the only species present in all support types.     

Mitochondrial DNA Diversity of Orchid Bee Euglossa fimbriata (Hymenoptera: Apidae) Populations Assessed by PCR-RFLP

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085–0.289) and nucleotide diversity (0.000–0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (?_st = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (?_st = 0.161–0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.     

First aeromycological study in an avocado agroecosystem in Mexico

Avocados (Persea americana Mill.) are economically and nutritionally valuable despite their susceptibility to several fungal diseases. This study was conducted in an agroecosystem of avocado trees in México. The air samples were collected every week by gravimetric methods at a height of 2 m from the ground level. Fungal colonies were isolated and transferred onto PDA and identified using morphological methods. During air sampling, different meteorological variables were measured. The symptoms of avocado diseases were determined by visual observations. To the best of our knowledge, this is the first study on aeromycological characterization in an avocado agroecosystem. Thirty-two airborne fungal genera were identified; Fusarium (97.2 %) and Colletotrichum (94.4 %) were the most common fungal pathogens present in the atmosphere of avocado. In addition, seven genera of important phytopathogenic fungi of other crops (Alternaria, Capnodium, Pestalotia, Stemphylium, Rhizopus, Curvularia, and Phyllachora) were isolated. The maximum concentration of total fungi was observed in June (358 CFU m^?3) and the minimum in September (83 CFU m^?3). The total fungal concentrations were significantly negatively correlated with the temperature. The symptoms of five diseases of avocado, viz. anthracnose, scab, spot, canker trunk, and vascular wilt, were identified in the area study. It was observed that the symptoms of all the above-mentioned diseases were present in June; and in the same month, the fungal concentrations were highest.     

Hepatocellular carcinoma and liver cirrhosis TP53 mutation analysis reflects a moderate dietary exposure to aflatoxins in Espírito Santo State, Brazil

The close relationship between aflatoxins and 249^ser TP53 gene mutation (AGG to AGT, Arg to Ser) in hepatocellular carcinoma (HCC) makes this mutation an indirect indicator of dietary contamination with this toxin. We have examined the prevalence of codon 249 TP53 mutation in 41 HCC and 74 liver cirrhosis (without HCC) cases diagnosed at the HUCAM University Hospital in Vitoria, Espírito Santo State, Brazil. DNA was extracted from paraffin sections and from plasma. The mutation was detected by DNA amplification, followed by restriction endonuclease digestion and confirmed by direct sequencing. DNA restriction showed 249^ser mutation in 16 HCC and 13 liver cirrhosis, but sequencing confirmed mutations in only 6 HCC and 1 liver cirrhosis. In addition, sequencing revealed 4 patients with mutations at codon 250 (250^ser and 250^leu) in HCC cases. The prevalence of TP53 mutation was 10/41 (24.3 %) in HCC and 1/74 (1.4 %) in liver cirrhosis. No relationship between the presence of mutations and the etiology of HCC was observed. TP53 exon 7 mutations, which are related to aflatoxins exposure, were found at 14.6 % (249^ser), 7.3 % (250^leu) and 2.4 % (250^ser) in 41 cases of HCC and 1.4 % in 74 liver cirrhosis (without HCC) cases, suggesting a moderate dietary exposure to aflatoxins in the Espírito Santo State, Brazil.     

Biophysical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.     

Phaeohyphomycoses in a Free-Ranging Loggerhead Turtle (Caretta caretta) from Southern Brazil

This report describes the occurrence of mycotic infection in a loggerhead turtle, Caretta caretta, found on Mostardas beach in the state of Rio Grande do Sul, Southern Brazil. The specimen was observed alive, emaciated, and died the following day. A necropsy was performed soon after death and tissue samples routinely processed for histopathological and molecular evaluation. Significant pathological alterations included multifocal to coalescing, 0.5–4 cm in diameter nodules were observed throughout the peritoneum and kidneys that revealed caseous, grayish content when sectioned; histopathological evaluation revealed severe peritonitis and nephritis associated with intralesional fungi. Fungal PCR that targeted the internal transcribed spacer region of fungi revealed three different species of fungi: Cladosporium cladosporioides and Alternata arborescens within the kidneys while Ampelomyces sp. was identified within peritoneal granulomas. C. cladosporioides and A. arborescens are melanized fungi that produce phaeohyphomycosis in a wide range of species. However, the importance of the identification of the mycoparasite Ampelomyces sp. DNA within the peritoneal granulomas remains unclear.     

Occurrence and fumonisin B<Subscript>2</Subscript> producing potential of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> in Brazil nuts

Bertholletia excelsa is the tree that produces Brazil nuts which have vast economic importance in the Amazon region and as an export commodity. The aim of this study was to assess the presence of Aspergillus section Nigri in Brazil nut samples at different stages of its production chain and to verify the toxigenic potential for fumonisin B₂ (FB₂) production of these isolates along with the presence of this mycotoxin in Brazil nut samples. The fungal infection ranged from 0 to 80% at the different stages of the harvest and processing chain and the water activity of the nuts from 0.273 to 0.994. A total of 1052 A. section Nigri strains were isolated from Brazil nuts and 200 strains were tested for their ability to produce FB₂: 41 strains (20.5%) were FB₂ producers with concentrations ranging from 0.09 to 37.25 mg/kg; 2 strains (1%) showed traces of FB₂, less than the detection limit (0.08 mg/kg); and 157 (78.5%) were not FB₂ producers. Although several samples showed high contamination by A. section Nigri, no sample was contaminated by FB₂.     

Factors That Influence Microbial Contamination of Fluids Associated with Hemodialysis Machines

Studies were conducted on the microbiological quality of fluids associated with different types of dialysis systems located in six dialysis centers and 14 homes. Included were (i) single-pass systems employing either parallel flow (Kiil or Gambro) or capillary cartridge dialyzers and (ii) recirculating single-pass and batch recirculating systems using coil dialyzers. Microbiological assays were performed on the water used to prepare dialysis fluid, the concentrated dialysate, and either pre- and postdialyzer dialysate (single-pass systems) or the dialysate contained in storage reservoirs and recirculating cannisters (recirculating systems). The levels of microbial contamination consisting of gram-negative bacteria were directly related to the type of dialysis system, method of water treatment, distribution system, and in some instances, the type of dialyzer. Recirculating single-pass and batch recirculating systems consistently contained significantly higher levels of contamination than single-pass systems. These results were directly related to the design of recirculating systems which permits carbon- and nitrogen-containing waste products dialyzed from the patient to accumulate, be used as nutrients by microorganisms, and subsequently allow for 2- to 4-log increases in contamination levels during a dialysis treatment. In contrast, levels of contamination in single-pass machines were related more to the quality of the water used to prepare dialysis fluid and the adequacy of cleaning and disinfection procedures than to the design of the system.     

In Vitro Susceptibility of Environmental Isolates of Exophiala dermatitidis to Five Antifungal Drugs

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.