Caracterização de isolados de Corynespora cassiicola e avaliação da sensibilidade in vitro a fungicidas

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2015.

Synthesis, Antimicrobial and Antiproliferative Activity of Novel Silver(I) Tris(pyrazolyl)methanesulfonateand 1,3,5-Triaza-7-phosphadamantane Complexes

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)-(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), [Ag(PTA)][BF4] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh3 = triphenylphosphane, PCy3 = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane) have been synthesized and fully characterized by elemental analyses, H-1, C-13, and P-31 NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N-3-facially coordinating mode, while in 2 and 5 a N2O-coordination is found, with the SO3 group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA1S9), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA EB system on addition of Ag complexes shows that the fluorescence quenching of DNA EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375) with an activity often higher than that of AgNO3, which has been used as a control, following the same order of activity inhibition on DNA, i.e., 3 > 2 > 1 > 5 > AgNO3 >> 4.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Biorremediação de solos contaminados com produtos petrolíferos

Mestrado em Engenharia Química. Ramo Tecnologias de Protecção Ambiental.

Aspergillus flavus contamination in two Portuguese wastewater treatment plants

Filamentous fungi from genus Aspergillus were previously detected in wastewater treatment plants (WWTP) as being Aspergillus flavus (A. flavus), an important toxigenic fungus producing aflatoxins. This study aimed to determine occupational exposure adverse effects due to fungal contamination produced by A. flavus complex in two Portuguese WWTP using conventional and molecular methodologies. Air samples from two WWTP were collected at 1 m height through impaction method. Surface samples were collected by swabbing surfaces of the same indoor sites. After counting A. flavus and identification, detection of aflatoxin production was ensured through inoculation of seven inoculates in coconut-milk agar. Plates were examined under long-wave ultraviolet (UV; 365 nm) illumination to search for the presence of fluorescence in the growing colonies. To apply molecular methods, air samples were also collected using the impinger method. Samples were collected and collection liquid was subsequently used for DNA extraction. Molecular identification of A. flavus was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR detection system (Corbett). Among the Aspergillus genus, the species that were more abundant in air samples from both WWTP were Aspergillus versicolor (38%), Aspergillus candidus (29.1%), and Aspergillus sydowii (12.7%). However, the most commonly species found on surfaces were A. flavus (47.3%), Aspergillus fumigatus (34.4%), and Aspergillus sydowii (10.8%). Aspergillus flavus isolates that were inoculated in coconut agar medium were not identified as toxigenic strains and were not detected by RT-PCR in any of the analyzed samples from both plants. Data in this study indicate the need for monitoring fungal contamination in this setting. Although toxigenic strains were not detected from A. flavus complex, one cannot disregard the eventual presence and potential toxicity of aflatoxins.

Influence of mixtures of acenaphthylene and benzo[a] anthracene on their degradation by Pleurotus ostreatus in sandy soil

Purpose Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds commonly found as soil contaminants. Fungal degradation is considered as an environmentally friendly and cost-effective approach to remove PAHs from soil. Acenaphthylene (Ace) and Benzo[a]anthracene (BaA) are two PAHs that can coexist in soils; however, the influence of the presence of each other on their biodegradation has not been studied. The biodegradation of Ace and BaA, alone and in mixtures, by the white rot fungus Pleurotus ostreatus was studied in a sandy soil. Materials and methods Experimental microcosms containing soil spiked with different concentrations of Ace and BaAwere inoculated with P. ostreatus. Initial (t 0) and final (after 15 days of incubation) soil concentrations of Ace and BaA were determined after extraction of the PAHs. Results and discussion P. ostreatus was able to degrade 57.7% of the Ace in soil spiked at 30 mg kg−1 dry soil and 65.8% of Ace in soil spiked at 60 mg kg−1 dry soil. The degradation efficiency of BaA by P. ostreatus was 86.7 and 77.4% in soil spiked with Ace at 30 and 60 mg kg−1 dry soil, respectively. After 15 days of incubation, there were no significant differences in Ace concentration between soil spiked with Ace and soil spiked with Ace + BaA, irrespective of the initial soil concentration of both PAHs. There were also no differences in BaA concentration between soil spiked with BaA and soil spiked with BaA + Ace. Conclusions The results indicate that the fungal degradation of Ace and BaA was not influenced by the presence of each other’s PAH in sandy soil. Bioremediation of soils contaminated with Ace and BaA using P. ostreatus is a promising approach to eliminate these PAHs from the environment.

Shannon entropy analysis of the genome code

This paper studies the chromosome information of twenty five species, namely, mammals, fishes, birds, insects, nematodes, fungus, and one plant. A quantifying scheme inspired in the state space representation of dynamical systems is formulated. Based on this algorithm, the information of each chromosome is converted into a bidimensional distribution. The plots are then analyzed and characterized by means of Shannon entropy. The large volume of information is integrated by averaging the lengths and entropy quantities of each species. The results can be easily visualized revealing quantitative global genomic information.

Histopathology of cutaneous and mucosal lesions in human paracoccidioidomycosis

Biopsies from cutaneous and mucosal lesions from 40 patients with active paracoccidioidomycosis, were studied histopathologically. All cases exhibited chronic granulomatous inflammation and 38 also presented suppuration; this picture corresponded to the mixed mycotic granuloma (MMG). Pseudoepitheliomatous hyperplasia and the transepidermic (or epithelial) elimination of the parasite, were observed in all cases. In paracoccidioidomycosis elimination takes place through formation of progressive edema, accompained by exocytosis. The edema gives rise to spongiosis, microvesicles and microabscesses which not only contain the fungus but also, various cellular elements. Cells in charge of the phagocytic process were essentialy Langhans giant cells; PMN's, epithelioid and foreign body giant cells were poor phagocytes. An additional finding was the presence of fibrosis in most biopsies.

Radiometric detection of metabolic activity of Paracoccidioides brasiliensis and its susceptibility to amphotericin B and diethylstilbestrol

Paracoccidioidomycosis (South American blastomycosis) is a systemic disease, strikingly more frequent in males, caused by the dimorphic fungus Paracoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on this fungus "in vitro". The Y form of the yeast, grown in liquid Sabouraud medium was inoculated into sterile reaction vials containing the 6B aerobic medium along with 2.0 μCi of 14C-substrates. Control vials, prepared in the same way, contained autoclaved fungi. To study the effects of amphotericin B (AB) (0.1 and 10 μg/ml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 μg/ml) extra controls with live fungi and no drug were used. All vials were incubated at 35°C and metabolism measured daily with a Bactec instrument. 14CO2 production by P. brasiliensis was slow and could be followed for as long as 50 days. AB at 10mg/ml and DSB at 5 μg/ml inhibited the metabolism and had a cidal effect on this fungus. The results with DSB might explain the low incidence of the disease in females. This technique shows promise for studying metabolic pathways, investi gating more convenient 14C-substrates to expedite radiometric detection and for monitoring the effects of other drugs and factors on the metabolism of P. brasiliensis "in vitro".

Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia

FEMS Yeast Research, Vol. 8, Nº 3

Inapparent lung involvement in patients with the subacute juvenile type of paracoccidioidomycosis

Three patients with the diagnosis of subacute juvenile paracoccidioidomycosis who, at the time of their first visit, had no signs or symptoms of lung involvement, were studied. Initially the diagnosis was confirmed by the observation of P. brasiliensis in biopsy material obtained from clinically involved lymphadenopathies. The lung X-rays done in all patients, did not reveal pathologic changes, although it was possible to observe and isolate the fungus from sputum samples obtained from the three patients. This fact reinforces the pulmonary genesis of the mycosis and proofs the existence of a pulmonary primary infection, even in patients with the juvenile manifestations, in whom the lung component is obscured by the predominant lymph node involvement.

Chlamydospore formation by Paracoccidioides brasiliensis mycelial form

To investigate the role of some adverse environmental conditions in chlamy-dospore formation by the mycelial form of P. brasiliensis, we cultured four P. brasiliensis isolates (18, Bt4, 1183, Pb9) at 25°C within solid agar medium either rich or poor in nutrients. Isolates 18 and 1183 were also cultured under anaerobiosis in a nitrogen atmosphere. Isolate 18 produced great number of terminal and intercalary chlamydospore after 7-10 days of culture in a medium poor in nutrients (2% agar with 0.1% dextrose and polypepton). The three other isolates also produced chlamydospores under the same conditions, but in lower numbers. Chlamydospore production by isolate 18 was abolished when the fungus was cultured in two agar media rich in nutrients (brain heart infusion and potato dextrose agar). Anaerobic incubation of isolate 18 under an atmosphere of N2 showed small mycelial outgrowth with numerous chlamydospores. At the electron microscopical level, the chlamydospores showed one or various nuclei and numerous mitochondria, indicating great potential for further development. Accordingly, chlamydospores produced multiple budding after only 24 h incubation at 35°C. The results demonstrate that under adverse environmental conditions P. brasiliensis mycelial form produces chlamydospores within a short period of time.

In vitro action of some disinfectants on Paracoccidioides brasiliensis yeast forms

The fungicidal action of sodium hypochlorite (0.3, 1, 2.5, 5 and 10%); formaldehyde (2, 5, and 10%); and ethyl alcohol (70%) on yeast forms of Paracoccidioides brasiliensis Pb 18 and a newly-isolated Goiana strain was described. Contact between the fungus and the disinfectants was maintained for 1, 2, 24, 48 and 72 hours at room temperature. Viability was evaluated by the fluorescein diacetate-ethidium bromide treatment, culture in solid and liquid media (36ºC and 26ºC); yeast to mycelial germination at room temperature; and radiometric study of metabolic activity. All concentrations of disinfectants were found to be effective in inactivating Pb 18 and Goiana strains, except for the 1-hour contact with 2% formaldehyde, in which fluorescein diacetate-ethidium bromide treatment was found to reveal 40 and 27% of viable cells, respectively. The yeast to mycelial germination method was considered to reveal faster and similar results as compared to culture in solid and liquid media.

Possibility of in-hospital infection by Cryptococcus neoformans in patients with AIDS

The objective of the present work was to carry out a survey of soil samples taken from different areas of a hospital of infectious disease located in the city of Cordoba, where three AIDS patients were hospitalized during different periods in the same ward. The three of them returned with meningeal cryptococcosis between three or five months after having been discharged. Cryptococcus neoformans was isolated in 8/10 samples collected outside the hospital, near the pigeon house. The samples collected from the AIDS patients ward and its surroundings were negative. These findings suggest that the patients may have been infected by the fungus during their first stay in hospital.

Experimental paracoccidioidomycosis in hamster: transmission electron microscopy of inoculation site lesion

Interaction between Paracoccidioides brasiliensis (Pb) and inflammatory cells in hamster testis was studied sequentially by transmission electron microscopy. In early lesions (six hours after inoculation), polymorphonuclear neutrophils (PMNs) were the major and mononuclear cells and eosinophils were the minor constituents of the inflammatory cells. PMNs were later replaced by mononuclear cells. Viable Pb cells were phagocytosed or surrounded by inflammatory cells. Preserved Pb cells usually had broad host-parasite interphases, whereas dying ones had narrow interphases. The outer layer of the fungus wall was sometimes broken by PMN in some focal points, broken pieces being peeled off and phagocytosed. Small Pb cells were uninuclear, and were often related to broad interphase. Large Pb cells were multinucleated with irregularly shaped wall, and sometimes had lomasome and/or myelin like structures. Different interaction patterns of Pb with inflammatory cells may be due to functionally different host cell flow to the inoculation site or due to the age of Pb cells or both.