Antibiótico antifungico produzido por um estreptomiceto da região de Araraquara

With the significant increase in the incidence of invasive fungal infections during the last decade, mainly in patients with cancer, AIDS and other hospitalized patients who stay for long periods in intensive care units, there is an urgent need to screen for new antifungal agents possessing some advantages over known ones. This article reports a search in the field for a microorganism producing antibacterial and antifungal substances. Strains from soil samples collected in the region of Araraquara, Brazil, were isolated and analyzed for their antimicrobial potential against standard microorganisms (fungi Candida albicans and Aspergillus oryzae and bacteria Staphylococcus aureus and Escherichia coli). Out of the 64 strains isolated, 34 produced detectable antimicrobial activity. The streptomycete strain Ar4014 was chosen for further study, owing to its good antimicrobial activity against Candida albicans. Two of the fermentation media tested, 608-K and 602-B, were found to be best for the production and extraction of the antibiotic from Ar4014. After chromatographic separation of the crude extract on a silica column, the active fractions obtained showed UV-VIS absorption peaks characteristic of normal pentaenic antibiotics. The antibiotic was provisionally designated Ara 4014-75.

Effect of the association of nystatin with a tissue conditioner on its ultimate tensile strength

Purpose: This study evaluated the ultimate tensile strength of a tissue conditioner without nystatin incorporation (GI - control group) and the same tissue conditioner modified by the addition of nystatin in two concentrations: GII - 500,000 International Units (U) and GIII - 1,000,000 U, in which each milligram of the medicament corresponded to 6079 U. Materials and Methods: Dumbbell-shaped specimens (N = 7) with a central cross-sectional area of 33 × 6 × 3 mm were produced for the three experimental groups. After polymerization following manufacturer's instructions, specimens were immersed in distilled water at 37°C for either 24 hours or 7 days and then tested in tension in the MTS 810 at 40 mm/minute. Data were analyzed by two-way ANOVA followed by Tukey's test, at 95% level of confidence. Results: The means (force-grams (gf) ± standard deviation) of the ultimate tensile strength were: GI - 634.29 ± 122.80; GII - 561.92 ± 133.56; and GIII - 547.30 ± 73.47 for 24-hour storage, and GI - 536.68 ± 54.71; GII - 467.50 ± 143.51; and GIII - 500.62 ± 159.76 for 7-day storage. There were no statistically significant differences among the three experimental groups (p > 0.05). The ultimate tensile strength means of all experimental groups after 7 days were significantly lower than those observed after 24 hours (p = 0.04). Conclusions: The results of this study suggest that the addition of nystatin into the tissue conditioner investigated in concentrations below 1,000,000 U did not affect its ultimate tensile strength. Copyright © 2006 by The American College of Prosthodontists.

Chorismate synthase: An attractive target for drug development against orphan diseases

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors. © 2007 Bentham Science Publishers Ltd.

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of βαβαββ-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future. © 2007 Bentham Science Publishers Ltd.

Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca 2+ levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca 2+ with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca 2+ accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca 2+ homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca 2+ channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca 2+/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca 2+ homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Estudo de metodologia analítica para a determinação do cetoconazol em formulações farmacêuticas

Ketoconazole is a synthetic broad-spectrum oral and topical antifungal drug derived from imidazole, effective in the treatment of superficial mycoses and systemic infections. In this study we have tested several methods to analyze ketoconazole in various pharmaceutical products containing this drug, employing techniques such as UV and IR spectrophotometry and thermal analysis. The results showed that UV spectrophotometry is a fast, practical and economical method and indicated that other methods, such as IR spectrophotometry and thermal analysis, could be good alternative methods for ketoconazole analysis in certain pharmaceutical forms.

Species distribution and susceptibility profile of Candida species in a Brazilian public tertiary hospital

Background. Species identification and antifungal susceptibility tests were carried out on 212 Candida isolates obtained from bloodstream infections, urinary tract infections and dialysis-associated peritonitis, from cases attended at a Brazilian public tertiary hospital from January 1998 to January 2005. Findings. Candida albicans represented 33% of the isolates, Candida parapsilosis 31.1%, Candida tropicalis 17.9%,Candida glabrata 11.8%, and others species 6.2%. In blood culture, C. parapsilosis was the most frequently encountered species (48%). The resistance levels to the antifungal azoles were relatively low for the several species, except for C. tropicalis and C. glabrata. Amphotericin B resistance was observed in 1 isolate of C. parapsilosis. Conclusions. The species distribution and antifungal susceptibility herein observed presented several epidemiological features common to other tertiary hospitals in Latin American countries. It also exhibited some peculiarity, such as a very high frequency of C. parapsilosis both in bloodstream infections and dialysis-associated peritonitis. C. albicans also occurred in an important number of case infections, in all evaluated clinical sources. C. glabrata presented a high proportion of resistant isolates. The data emphasize the necessity to carry out the correct species identification accompanied by the susceptibility tests in all tertiary hospitals. © 2010 Bagagli et al; licensee BioMed Central Ltd.

Mutagenicidade de duas espécies do gěnero Alchornea avaliadas através de ensaios com Salmonella microssomo e teste do micronúcleo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Utilização de extratos de plantas medicinais e óleo de Eucaliptus no controle in vitro de Microsporum canis

INTRODUCTION: Microsporum canis is the most common cause of canine and feline dermatophytosis and thus has an important zoonotic role. OBJECTIVES: the aim of this study was to determine the antifungal action of medicinal plant extracts and of eucalyptus oil against pathogenic fungus Microsporum canis. METHODS: the extracts were prepared by mixing 300 g of previously washed leaves with 450 mL of distilled water. Then the material was triturated, filtered, sterilized and conserved at 10 + 2 oC. Fifteen milliliters of sterilized medium Sabouraud dextrose (Difco) at a temperature of 55 + 1 oC was added in Petri dishes containing the extracts in one, two, three, four and five mm concentrations. The fungus was inoculated once the medium was solidified. The inoculated dishes were maintained in B.O.D. incubator at 36 ± 0,5 oC until the fungus developed in the controls. RESULTS: the extracts from Punica granatum, Mangifera indica and Eucalyptus spp reduced the growth of fungus, but the extracts from Cymgopogom nardus, Tagetes minuta, Ruta graviolens, Cyperus rotundus, Annona moricata and Calendula spp leaves and flowers boosted the growth of fungus. The other extracts and the eucalyptus oil neither show any fungicidal action nor encourage mycelium growth. CONCLUSIONS: the use of most tested extracts and eucalyptus oil is not suitable for the treatment of Microsporum canis dermatophytosis due to lack of inhibitory effects. The extracts from Cymgopogom nardus, Tagetes minuta, Ruta graviolens, Cyperus rotundus, Annona moricata and from of Calendula spp leaves and flowers help the development of the fungus making clear that phytotherapy should be properly used, otherwise it can worsen the problem. However; extracts from Mangifera indica, Punica granatum and Eucalyptus spp. can be used as fungistatic.

Advances in sickle cell disease treatment: From drug discovery until the patient monitoring

Sickle Cell Disease (SCD) is one of the most prevalent hematological diseases in the world. Despite the immense progress in molecular knowledge about SCD in last years few therapeutical sources are currently available. Nowadays the treatment is performed mainly with drugs such as hydroxyurea or other fetal hemoglobin inducers and chelating agents. This review summarizes current knowledge about the treatment and the advancements in drug design in order to discover more effective and safe drugs. Patient monitoring methods in SCD are also discussed. © 2011 Bentham Science Publishers Ltd.

Avaliação do potencial antagônico de leveduras, visando biocontrole de deterioração por penicillium expansum

Considerable losses during apple fruit storage occur due to microbiological diseases, mainly caused by Penicillium expansum, which in addition to fruit pulp deterioration produces patulin, a mycotoxin with carcinogenic and teratogenic activity. Biological control of post-harvest disease by antagonist yeasts focused on killer toxins is an appreciable alternative to the chemical fungicides, due to the low possibility of toxic residues demonstrated during fermentative processes. Twenty out of 44 yeasts (16 isolated from fruits, 10 from corn silage and 18 from laboratory anthill), showed antagonism against spores of P. expansum. The assay in solid medium pointed the strongest nutrient competition antagonism by D. hansenii strain C1 (31 mm inhibition diameter), while D. hansenii strain C7 (15 mm) showed higher antibiosis and parasitism pattern. In the following step the extracellular activity was tested performing the assay with culture supernatant in Yeast Medium agar, where C. guilliermondii P3 was more effective against conidia germination (inhibition rate of 58.15%) while P. ohmeri showed better inhibition on micelial growth (66.17%). The antibiosis showed by both yeasts could suggest probable mechanism associated with killer phenomenon, once both strains were killer positive against sensitive reference strains (S. cerevisiae NCYC 1006 and P. kluyveri CAY-15). In order to enhance the production of antifungal substance, these yeasts were cultivated with P. expansum, but the difference between culture supernatant obtained from yeasts cultivated alone and with mould was not significant (P > 0.05). The results demonstrated that the yeasts application constitute a promising tool, enhancing the biological control of P. expansum in post-harvest diseases of apple fruit.

Concentração letal LC50 do extrato aquoso de folhas de terminalia catappa em guaru, phalloceros caudimaculatus

This trial was conducted in order to determine the 50% lethal concentration (LC (I) 50-96h) of the aqueous extract of dried leaves of Terminalia catappa, a plant with antifungal, antibacterial and antiparasitic activity. Due to the increasing use of herbal medicines in aquaculture, its use is an option for controlling diseases in fish. Toxicity tests are important before recommending any treatment, since some products have therapeutic concentration close to lethal. To conduct the study used 135 fish species Guarus (Phalloceros caudimaculatus), exposed to increasing concentrations of: 0.0, 50.0, 100.0, 150.0, 200.0, 250.0, 300.0 mL of stock solution per liter of water. The results were calculated by the method Trimmed Spearman Karbo, demonstrating that the 50% lethal concentration (LC (I) 50-96h) estimated was 208.52 mL / L, with lower limit of 187.79 mL / L and higher 231.54 mL / L. Observed changes in behavior of the test organisms at concentrations above 250 mL / L decrease in the levels of dissolved oxygen and pH. Concentrations below 250 mL /L result in mortality rate near zero, so the aqueous extract shown low toxicity.

Influence of TNF-α blockers on the oral prevalence of opportunistic microorganisms in ankylosing spondylitis patients

Objectives: To compare the oral prevalence and antimicrobial susceptibility of Candida spp., staphylococci, enterobacteriaceae, and pseudomonas spp.from ankylosing spondylitis (AS) patients receiving conventional and anti-TNF-α therapy. Methods: The study included 70 AS patients, diagnosed according to the modified New York criteria (1984). The volunteers were divided into 2 groups: a biological group (AS BioG) (n=35) (on anti-TNF-α therapy) and a conventional group (AS ConvG) (n=35). The control group (ContG) (n=70) was made up of healthy individuals matched for age, gender, and oral conditions. After clinical examination, oral rinse samples were collected and plated in specific culture media. The number of colony-forming units per milliliter (cfu/ml) was obtained, and isolates were identified using the API system. Antimicrobial susceptibility tests were performed according to the NCCLS guidelines. Prevalence and counts of microorganisms were statistically compared between the 3 groups, using the Mann-Whitney and Chi-square tests. Significance level was set at 5%. Results: In both the AS BioG and the AS ConvG, staphylococci counts were higher than that in the ContG (p<0.0001). Candida albicans and staphylococcus epidermidis were the most commonly found species in all the groups. Serratia marcescens and klebsiella oxytoca were more prevalent in the AS BioG and the AS ConvG, respectively. Two Candida isolates (2.8%) from the AS BioG and 5 (10.8%) from the AS ConvG were resistant to amphotericin B and 5-fluorocytosine. A low percentage of staphylococci isolates was resistant to amoxicillin, ciprofloxacin, and doxycycline. Conclusion: Higher counts of staphylococci were observed in both AS groups, regardless of the current therapy, age, sex, and oral conditions. Anti-TNF-α therapy could not be correlated with increased counts of microorganisms. © Copyright CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 2012.

Disseminated Amphotericin-Resistant Fusariosis in Acute Leukemia Patients: Report of Two Cases

Disseminated fusariosis has emerged as a significant, usually fatal infection in immunocompromised hosts despite antifungal treatment. We describe here two patients with acute leukemia who developed disseminated amphotericin-resistant fusariosis, and review of six studies of cases series in the literature. Two Fusarium solani strains were isolated from blood and skin cultures of one patient, and one strain from the blood culture of the second patient. Both patients died despite antifungal treatment. Strains were identified by sequencing of ITS1 and ITS4 regions. Random amplified polymorphic DNA analysis of the three F. solani isolates showed a low degree of similarity. Screening for Fusarium spp. contaminants within our facility was negative. Using the CLSI M-38-A2 broth dilution method and E tests®, we found that the MICs were low for voriconazole (0. 12 and 0. 5 mg/L, respectively), unexpectedly high for amphotericin B (≥8 and ≥32 μg/mL, respectively) and itraconazole (≥16 mg/ml). Patients with leukemia or persistent neutropenia should be assessed for disseminated fungal infections, including biopsy and skin cultures. Antifungal susceptibility tests are important due to the possibility of the strains being amphotericin resistant. Treatments must be aggressive, with high doses of antifungals or combined therapy. © 2012 Springer Science+Business Media Dordrecht.