Repositório RCAAP

An aerobic Gram positive spore-forming bacterium was isolated from cellulose pulp mill effluent. This microorganism, identified as Bacillus sp. and named IS13, was able to rapidly degrade the organic chlorinated compound 4,5,6-trichloroguaiacol (4,5,6-TCG) from a culture containing 50 mg/l, which corresponds to about 3x104 times the concentration found in the original effluent. The biodegradation of this compound, usually found in cellulose pulp mill effluents, was evaluated by spectrophotometry and gas chromatography analysis. During 4,5,6-TCG decreasing, the lack of by-products had shown by such analysis lead to verify the possibility of either adsorption or absorption of 4,5,6-TCG by the cells, instead of real biodegradation. There were no traces of 4,5,6-TCG after lysozyme and SDS cell disruption. Vigorous extraction was applied before spectrophotometry analysis and there was no release of residual 4,5,6-TCG. Plasmid isolation was attempted by using different protocols. The best results were reached by CTAB method, but no plasmid DNA was found in Bacillus sp. IS13. The results suggest that genes located at the bacterial chromosome might mediate the high decrease of 4,5,6-TCG. The importance of this work is that, in being a natural ocurring microorganism, Bacillus sp. IS13, can be used as inoculum in plant effluents to best organochlorinated compounds biodegradation.

Candida spp was isolated from 59 (68.60%) out of eighty six samples of oral mucosa of AIDS patients. The identification, based or the production of a germ tube and chlamydospores, and on the assimilation and fermentation of carbohydrates, revealed 52 strains (88.13%) of C. albicans, 4 (6.77%) of C. tropicalis and 3 (5.08%) of C. krusei. The susceptibility of these strains to amphotericin B, flucytosine, itraconazole, fluconazole and ketoconazole was determined using the agar dilution method. Comparing the minimum inhibitory concentration values found in the susceptibility test with the serum levels achieved by these drugs, only 8.47% and 5.08% of the yeasts strains proved to be resistant to amphotericin B and flucitosyne, respectively. A high frequency of strains resistant to azole derivatives (25.42%, to itraconazole, 45.76%, to ketoconazole and 66.10% to fluconazole) was observed.

The fatty acid profiles of several fungi of the order Mucorales (Zygomycetes), including Backusella lamprospora (Lendner) Benny and R.K. Benj., Benjaminiella youngii P.M. Kirk, Circinella simplex van Tieghem, Cunninghamella blakesleeana Lendner, Mortierella ramanniana (Möller) Linnem., Mucor circinelloides f. janssenii (Lendner) Schipper, Mycotypha microspora Fenner, Rhizomucor miehei (Cooney and R. Emerson) Schipper and Rhizomucor pusillus (Lindt) Schipper, and of Volutella sp. Fr., from the class Ascomycetes, were qualitatively analysed by gas-liquid chromatography in order to determine the taxonomic value of these chemotaxonomic markers. The fatty acids present in all strains were palmitic (16:0), oleic (18:1), linoleic (18:2) and <FONT FACE="Symbol">g</FONT>-linolenic (18:3) acid, with the exception that the latter was not found in Volutella sp. Chemotaxonomic markers for some species and genera were obtained, including a non-identified fatty acid, FAME8 (minimum and maximum retention times of 27.92 and 28.28 minutes) for Rhizomucor miehei CCT 2236 and Rhizomucor pusillus CCT 4133, and FAME3 (minimum and maximum of 16.53 and 16.61 minutes) for Benjaminiella youngii CCT 4121. The chemotaxonomic marker of the order Mucorales was the fatty acid 18:3<FONT FACE="Symbol">w</FONT>6, confirming previous data from literature. The results of the present study suggest that qualitative fatty acid analysis can be an important chemotaxonomic tool for the classification of fungi assigned to the order Mucorales (Zygomycetes).

A modified method for direct determination of cellulolytic activity using Avicel colored with Remazol Brilliant Blue R (RBBR) in Agar test tubes is discussed. Refinements were introduced in a simple method for quantitation of cellulase activity, based on the release of dye from Avicel-RBBR medium by the enzymatic hydrolysis. Modifications in Avicel-dye preparation were enhanced and a spectrophotometer for direct OD measurement in agar test tubes used. The use of a spectrophotometer improved the precision of the collected data, since absorbance measurements could be done at the maximum wavelenght for RBBR (595 nm).

An experiment under greenhouse conditions was carried out to evaluate the relative contribuition of arbuscular mycorrhizal fungi (AMF) in the process of nitrogen transfer from cowpea to maize plants, using the isotope 15N. Special pots divided in three sections (A, B and C), were constructed and a nylon mesh screen of two diameters: 40µm (which allowed the AMF hyphae to pass but not the plant roots) or 1µm (which acted as a barrier to AM hyphae and plant roots) was inserted between the sections B and C. Section A had 25.5 mg of N/kg using (15NH4)2SO4 as N source. Two cowpea seedlings inoculated with Rhizobium sp. were transplanted with their root systems divided between the sections A and B. Ten days later, 2 seeds of maize were sown into the section C which was inoculated with Glomus etunicatum. Thirty-five days after transplanting, the maize plants were harvested. AMF inoculation increased dry weight and 15N and P content of maize plant shoots. Direct transfer of 15N via AMF hyphae was 21.2%; indirect transfer of 15N mediated by AMF mycelium network, was 9.6%, and indirect transfer not mediated by AM mycelium network , was 69.2%.

High temperatures can affect the survival, establishment and symbiotic properties of Rhizobium strains. Bean nodulating Rhizobium strains are considered particularly sensitive because on this strains genetic recombinations and/or deletions occur frequently, thus compromising the use of these bacteria as inoculants. In this study R. tropici and R. leguminosarum bv. phaseoli strains isolated from Cerrado soils were exposed to thermal stress and the strains’ growth, survival and symbiotic relationships as well as alterations in their genotypic and phenotypic characteristics were analyzed. After successive thermal shocks at 45ºC for four hours, survival capacity appeared to be strain-specific, independent of thermo-tolerance and was more apparent in R. tropici strains. Certain R. leguminosarum bv. phaseoli strains had significant alterations in plant dry weight and DNA patterns obtained by AP-PCR method. R. tropici strains (with the exception of FJ2.21) were more stable than R. leguminosarum bv. phaseoli strains because no significant phenotypic alterations were observed following thermal treatments and they maintained their original genotypic pattern after inoculation in plants.

A strain of Aspergillus niger isolated from soil samples showed great capacity to produce extracellular inulinase. Although the enzyme has been synthesized in presence of monosaccharides, sucrose and sugar cane molasse, the productivity was significantly higher (p&lt;0.05) when the microorganism was inoculated in media formulated with dahlia extract and pure inulin, as carbon sources. With regard to the nitrogen source, the best results were obtained with casein and other sources of proteic nitrogen, comparatively to the mineral nitrogen. However, statistic significance (p&lt;0.01) only was found between the productivity obtained in the medium prepared with casein and ammonium sulphate. The optimum pH of the purified enzyme for inulin hydrolysis was found between 4.0 and 4.5 and the optimun temperature at 60oC. When treated by 30 minutes in this temperature no loss of activity was observed. The enzyme showed capacity to hydrolyse sucrose, raffinose and inulin from which it liberated only fructose units showing, therefore, an exo-action mechanism. Acting on inulins from several sources, the enzyme showed larger hydrolysis speed on the polissaccharide from chicory (Cichorium intibus), comparatively, to the inulins from dahlia (Dahlia pinnata) and Jerusalem artichoke (Helianthus tuberosus) roots.

The bacteriostatic activity of 2’,4’,2-trihydroxychalcone; 2’,4’,3-trihydroxychalcone and 2’,4’,4-trihydroxychalcone, prepared by condensation of 2,4-dihydroxyacetophenone and benzaldehyde substituted, against Staphylococcus aureus ATCC 25923 was assayed by agar plate method. The three compounds presented important inhibition halos. In order to elucidate structure-activity relationships, the minimal inhibitory concentrations against S. aureus were determined by the broth dilution method and the results obtained were compared to that of 2',4'-dihydroxychalcone. The sequence observed was: MIC 2’,4’,3-(OH)3 &gt; MIC 2’,4’-(OH)2 &gt; MIC 2’,4’,4-(OH)3 &gt; &gt; MIC 2’,4’,2-(OH)3. These results showed that the introduction of an electron donating group (-OH) in the aromatic B-ring causes an increase in bioactivity, and that the intensity of action depends on the position of the OH substitute.

Among three strains of Pycnoporus sanguineus, MIP 89007 produced more cinnabarin than MIP 95001 and MIP 95002. The antimicrobial activity of cinnabarin was tested against 11 species of bacteria isolated from food. Bacillus cereus and Leuconostoc plantarum were the most sensitive to cinnabarin, being inhibited by 0.0625 mg/ml. Klebsiella pneumoniae was the least sensitive (&gt;4.0 mg/ml).

The major microbial problem in the petroleum refining industry is contamination of stored products, which can lead to loss of product quality, formation of sludge and deterioration of pipework and storage tanks, both in the refinery and at the end-user. Three major classes of fuel are discussed in this article - gasoline, aviation kerosene and diesel, corresponding to increasingly heavy petroleum fractions. The fuel that presents the most serious microbiological problems is diesel. The many microorganisms that have been isolated from hydrocarbon fuel systems are listed. The conditions required for microbial growth and the methods used to monitor and to control this activity are discussed. The effects of various fuel additives, including biocides, are considered.

The effects of bovine S protein (vitronectin) on phagocytosis of Streptococcus dysgalactiae strains isolated from cattle with mastitis were investigated. Phagocytized streptococci were determined by a fluorometric microassay using glass adherent polymorphonuclear neutrophils (PMN). Preincubation of S. dysgalactiae with bovine S protein significantly increased their phagocytosis by PMN. Bovine S protein had no effect on phagocytic killing of non-S protein binding S. pyogenes cultures. Enzymatic digestion of the bovine S protein binding sites on S. dysgalactiae with pronase resulted in a significative reduction of the effects of S protein on phagocytosis. It could thus be concluded that in addition to its role as a promoter of cellular adhesion and complement inhibitor, bovine S protein may also influence the phagocytosis of S. dysgalactiae during inflammatory processes.

This article describes the main aspects of bovine herpesvirus type-5 (BHV-5) neurologic infection and disease in rabbits, a candidate animal model for studying BHV-5 neuropathogenesis. Intranasal inoculation of weanling rabbits with a Brazilian BHV-5 isolate produced neurological disease and death in 78.8% (26/33) of the animals. Neurological signs started as early as 5 days post-inoculation and lasted from 10-12 hours up to several days. Most animals evolved to a moribund state or death within 24 (69.2%) to 48 hours (88.5%). Neurological disease was characterized by excitability or depression, tremors, bruxism, walking or running in circles, backward arching of the head and body, incoordination, backward and sideways falling, paddling, profound depression and death. Moderate levels of infectivity were detected in several areas of the brain, most consistently in the ventro-lateral hemisphere (in 16 out of 20 animals), anterior cerebrum (15/20), midbrain (11/20), dorso-lateral hemisphere (10/20) and pons (12/26). Infectious virus was also recovered from the olfactory bulb (9/20), medulla oblongata (10/26), cerebellum (7/20), posterior cerebrum (5/20) and trigeminal ganglia (4/20). No gross lesions were observed. Microscopic lesions were mild and consisted of non-suppurative meningitis, mononuclear perivascular cuffing and focal gliosis. These changes were observed most consistently in the ventro-lateral hemisphere and anterior cerebrum. Passive immunity partially protected rabbits from BHV-5-induced encephalitis. Rabbits born to immunized dams showed a significative delay in the onset of clinical disease and reduced morbidity and mortality rates compared to rabbits born to unvaccinated dams. These results demonstrate that BHV-5-induced neurological disease can consistently be reproduced in rabbits and point towards the use of this species as an animal model to study BHV-5 neuropathogenesis.

One-hundred-five (105) clinical isolates of mycoplasma from caprine origin and one isolate from ovine were surveyed for plasmids, which were present in thirty-three (31%) of them. These mycoplasmas originated from 13 herds. Ten of them were symptomatic for mycoplasmal disease (mastitis, polyarthritis, septicemia) and three herds were asymptomatic, i.e., clinically normal. Twenty-eight isolates were Mycoplasma mycoides subspecies mycoides LC (large colony or caprine biotype), four were Mycoplasma capricolum subsp. capricolum and one was Mycoplasma cottewii. The isolated plasmids were linearized by EcoRI, EcoRV, EcoRI and EcoRV or BamHI and EcoRV, and were of five sizes (1.1, 1.6, 1.7, 1.8, and 1.9 Kbp). Based on restriction enzyme digestion and size of the linearized supercoiled extrachromosomal DNA, five plasmid types were recovered (p1II, p2III, p2V, p3I, and p4IV). The small size of these DNA elements probably exclude replicative forms of DNA virus, which are equal or larger than 8.0 Kbp.

Bovine Leukemia Virus (BLV) is the etiologic agent of Enzootic Bovine Leukosis, a retrovirus exogenous to the bovine species. Once infected, there is no detectable viraemia but instead there is a strong and persistent immunological response to BLV structural proteins, essentially the gp51 envelope glycoprotein and the mayor core protein p24. We describe the test procedure of an indirect ELISA (I-ELISA) using polyclonal reagents for the detection of antibodies to BLV. For comparison, the sera were simultaneously tested by agar gel immunodiffussion (AGID) test, which is currently used as diagnostic standard for BLV infection. The antigen applied does not require a high degree of purification and the data from the analysis of the negative sera showed that the establishment of a cut-off level corresponding to 3 times the standard deviation (SD) above the mean for the negative control set of sera provided acceptable specificity, reducing the risk of false positives results. A comparison of the results obtained by AGID test and I-ELISA showed that considering a total of 465 serum samples, all of the 234 samples (50%) that were positive by AGID were positive to the I-ELISA. Of 225 serum samples which yielded negative results in the AGID test, 69 (15%) were found to be positive by the I-ELISA and 156 (33%) were negative by both techniques. Few sera (2%) that were non-specific by AGID were defined as negative or positive by I-ELISA.

The isolation of genetic markers, like drug resistance and auxotrophy, is a laborious but important step in genetic research. The isolation of auxotrophic mutants of Trichoderma harzianum using the filtration enrichment technique was more effective than using the total isolation technique. Most of 12 auxotrophic mutants exhibited similar growth rate and higher sporulation when compared with the wild type, but only two mutants (TWS-410 and TW5-523) could grow in 500µg/L of benomyl.

The lysis-centrifugation system (Isolator TM) is recognized as the standard method for recovery of Candida spp. from blood. In this study, the effect of the concentration process of this system was compared with conventional methods of blood culture using liquid and biphasic media. The tests were performed in vitro using Candida albicans in counts of 100, 10, 1, and 0.5 cells/ml of blood. Cultures onto chocolate agar were performed with the sediment obtained after centrifugation of IsolatorTM tube and with liquid and biphasic media after their incubation for 24 h at 35oC. Gram stain prepared from the conventional methods were also evaluated in the first 24 h. It was possible to detect Candida albicans in blood, regardless both the number of cells or methodology. As blastoconidia were observed in Gram stains at the same time that growth was noted, time for diagnosis was also not different for the compared methods. Therefore, we suggest that the process of concentration is not the single important factor responsible for the recovery rates of Candida albicans from blood by the IsolatorTM system.

Caffeine catabolism and a xanthine oxidase involved in the alkaloid breakdown were studied in Pseudomonas putida L, a strain displaying high ability to grow on this substrate. Cells cultured with unlabelled caffeine and 14C labeled caffeine and xanthine showed that this alkaloid was broken-down via theobromine/paraxanthine -&gt; 7-methylxanthine -&gt; xanthine -&gt; uric acid -&gt; allantoin -&gt; allantoic acid. Methyluric acids were formed from the oxidation of theobromine, paraxanthine and 7-methylxanthine, although no bacterial growth was observed on these compounds, indicating that this might be due to a wide substrate specificity of xanthine oxidase. This was confirmed by activity staining in PAGE where activity was observed with theophylline and 3-methylxanthine, which are not involved in the alkaloid breakdown. A single band of activity was detected without addition of NAD+, showing an oxidase form of the enzyme. The enzyme optimum temperature and pH were 30oC and 7.0, respectively. The determined Km was 169 µM, and the pI 3.1 - 4.0. The molecular weight determined by side by side comparison of activity staining of the enzyme in PAGE and PAGE of BSA was 192 kDa, which was coincident with the sum (198.4 kDa) of three subunits (71, 65.6 and 61.8 kDa) of the purified protein.

In order to characterize fusion products from yeast protoplasts and their segregants, with important features to the wine making industry, electrophoretic karyotyping and RAPD (Random Amplified Polymorphic DNA) were utilized. Electrophoretic karyotyping was performed by the CHEF ("contour-clamped homogeneous electric field electrophoresis") method, which allowed the detection of chromosomal band complementation in fusion products and the presence of patterns of both parental and intermediary strains in segregants. By utilizing two primers, an amplification pattern of DNA fragments was obtained. While fusion products (diploid) showed a pattern of complementary bands, segregants showed bands of either parental strains or even intermediary bands

Fifty-six strains of Basidiomycetes, including native Brazilian fungi isolated from different ecosystems and edible mushrooms, were screened for production of exopolysaccharides and biomass in submerged culture. Agaricus sp. (CCB 280) and Oudemansiella canarii (Jungh.) Hohn (CCB 179) were the highest exopolysaccharide producers (6.01 and 3.54 g dry w./l respectively) after 7 days of incubation. The best producer of biomass was Schizophyllum commune Fr.:Fr. (CCB 473) with 16.68 g dry w./l in 14 days of incubation. When the culture filtrate was submitted to freezing prior to polysaccharide precipitation, a gelatinous fraction was formed.

One hundred and thirty seven samples of peanuts and peanut containing foods were collected in markets in the State of São Paulo, Brazil, between January 1995 an December 1997. Most of the samples were collected by the Inspection Service of São Paulo Secretary of Health. The foods included raw peanuts, peanut candies ("paçoca" and "pé de moleque"), peanut butter, fried/roasted salted peanuts, "torrone", chocolate coated peanuts and salt-coated peanuts. The samples were analyzed for aflatoxins using a thin-layer chromatographic method. About 45% of the samples were positive for aflatoxins and 27% exceeded the limits of the Brazilian legislation (30.0 µg.kg-1 for aflatoxins B1+G1). The aflatoxins were confirmed by derivatization with trifluoroacetic acid. The 90th percentile was 110.0 in 1995, 60.0 in 1996 and 118.0 µg.kg-1 in 1997. The aflatoxins concentration in the raw peanut samples ranged from 5.0 to 382.0 µg.kg-1 and 27.1% were above the legal limits. Contamination in peanut candies was above the limit in 32.8% of the samples and the aflatoxins levels ranged from 6.0 to 494.0 µg.kg-1. Contamination of salty peanuts was less frequent, around 10% of the samples and the toxin levels were usually below 10 µg.kg-1. The maximum level of contamination, 536.0 µg.kg-1, was found in a sample of peanut with a salty coat ("amendoim japonês"). Results of previous studies in peanuts and peanut products in the city of São Paulo from 1980-1987 had 68.75% of the samples with levels greater than the limit 30.0 µg.kg-1 and the 90th percentile ranged from 42.0 to 333.0 µg.kg-1. In 1994, 36.0% of the samples showed results above the limit and the 90th percentile was 489 µg.kg-1. The results show that aflatoxins contamination in peanuts is decreasing but it is still a serious problem in Brazil, a country where the climate, the agricultural practices and storage conditions favour fungal growth.