Antifungal susceptibility of yeasts isolated from anthropogenic watershed.

Yeasts are unicellular fungi widespread in the environment, and studies suggest that there is a positive correlation between yeast and polluted aquatic environments. The aim of this study was to analyze the diversity and resistance of yeasts isolated from water samples collected along the Arroio Dilúvio in Porto Alegre to antifungals. Yeast isolates from the Arroio Dilúvio were subjected to susceptibility assays against antifungals using the minimum inhibitory concentration (MIC) test, and amplification of the ITS1-5.8S-IT2 region; in addition, the ITS-5.8S region was sequenced for some of the isolates. The amplification product was subjected to PCR-RFLP, and the restriction profile allowed the construction of a dendrogram. Susceptibility tests showed a high prevalence of resistance to azole antifungals, where 16.8% of the isolates had a resistance profile to amphotericin B. The sequence analysis allowed the identification of Candida species, including potentially pathogenic species, and species of the Debaryomyces spp. The resistance to antifungals in yeasts isolated from Arroio Dilúvio reinforces the importance of studies of environmental microbiota, and indicates that environmental degradation influences the phenotype displayed.

Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost.

During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C) and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1). For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1) for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

Genetic Background of ß-Lactamases in Enterobacteriaceae Isolates from Environmental Samples.

The prevalence of ß-lactamase-producing Enterobacteriaceae has increased worldwide. Although antibiotic-resistant bacteria are usually associated with hospitals, there are a growing number of reports of resistant bacteria in other environments. Concern about resistant microorganisms outside the hospital setting highlights the need to investigate mechanisms of antibiotic resistance in isolates collected from the environment. The present study evaluated the resistance mechanism to ß-lactam antibiotics in 40 isolates from hospital sewage and surface water from the Dilúvio Stream, Porto Alegre City, Southern Brazil. The multiplex PCR technique was used to detect several resistance genes of ß-lactamases: extended-spectrum ß-lactamases (ESBLs), carbapenemases, and ß-lactamase AmpC. After genes, detection amplicons were sequenced to confirm their identification. The clonal relationship was established by DNA macrorestriction using the XbaI enzyme, followed by pulsed-field gel electrophoresis (PFGE). The results indicated that resistance genes were present in 85% of the isolates. The most prevalent genes encoded narrow-spectrum ß-lactamase, such as TEM-1 and SHV-1 with 70% of the strains, followed by carbapenemase KPC and GES (45%), ESBL types SHV-5 and CTX-M-8 (27.5%), and AmpC (ACT-1/MIR-1) (2.5%). Twelve isolates contained only one resistance gene, 14 contained two, and eight isolates had three resistance genes. PFGE indicated a clonal relationship among K. pneumoniae isolates. It was not possible to establish a clonal relationship between Enterobacter sp. isolates. The results highlight the potential of these resistance genes to spread in the polluted environment and to present a health risk to communities. This report is the first description of these resistance genes present in environmental samples other than a hospital in the city of Porto Alegre/RS.

Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost

ABSTRACT During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C) and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1). For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1) for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

Phenotypic Tests for the Detection of ß-Lactamase-Producing Enterobacteriaceae Isolated from Different Environments.

Some bacteria from the Enterobacteriaceae family are showing a significant capability to disseminate ß-lactams resistance mechanisms among them, and these same mechanisms can be carried out from the hospital environment to superficial water. The aim of this study was to evaluate different phenotypic methods for the detection ß-lactamases production by enterobacteria isolated from the anthropogenic environment: hospital wastewater and from a stream that cross the city of Porto Alegre. The applied tests were the modified Hodge test (MHT) and phenotypic tests with the following inhibitors: carbapenemase-phenylboronic acid (APB), metallo-ß-lactamase-EDTA, AmpC ß-lactamase-cloxacillin, and the confirmatory test for extended-spectrum ß-lactamase (ESBL)-clavulanic acid. For this evaluation, 131 isolates were initially subjected to antibiogram using the following antimicrobials: cefotaxime (30 µg), cefpodoxime (10 µg), ceftazidime (30 µg), ertapenem (10 µg), meropenem (10 µg), and aztreonam (30 µg). After this first screening, 62 isolates showed a profile resistance for at least one antimicrobial. These isolates were subjected to all phenotypic tests. Of those, 40 isolates were positive for at least one phenotypic test. In MHT test, one isolate was positive and five were with inconclusive results. The results achieved with the inhibitors are as follows: APB 25/40 positive strains; EDTA 8/40 positive strains; and with CLOXA 2/40 positive strains. ESBL production was observed for 34/40 strains. This assessment shows a high level of bacteria which can produce enzymes that inactivate ß-lactams present in the different environment like the stream waters and from the hospital settings.

Genetic diversity among monoconidial and polyconidial isolates of Bipolaris sorokiniana.

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat-growing regions of the world. This fungus shows a high genetic diversity and morphological and physiologic variability. In this study, 19 polysporic and 57 monosporic isolates of B. sorokiniana were characterized using universal rice primers-URP-PCR. The results obtained when the dendrogram was constructed with all the data produced with the amplification products showed very distinct clusters. However, the similarity among the isolates was low where 37 and 26.3 % of the monosporic and polysporic isolates, respectively, showed similarity above 70 %. All primers amplified multiple DNA fragments of polysporic as well as the monosporic isolates. Isolates fingerprints were constructed based on binary characters revealed by the three primers. An amplified fragment of approximately 750 bp was observed among 40 % of the isolates, when primer URP-1F was used. When primers URP-4R and URP-2R were used, a fragment of 450 and 400 bp was present in 31.5 and 29 % of the isolates, respectively. It was expected a higher similarity among the isolates since the monosporic cultures were originated from the polysporic. The dendrogram did not enable the separation of B. sorokiniana isolates by their geographic origin. This low correlation suggests that gene transfer may have occurred by parasexual combination in this fungus population. However, in spite of the research efforts for that end, it has not been possible to establish patterns that characterize the profile of B. sorokiniana.

Anti-phytopathogen potential of endophytic actinobacteria isolated from tomato plants (Lycopersicon esculentum) in southern Brazil, and characterization of Streptomyces sp. R18(6), a potential biocontrol agent.

Tomato plants (Lycopersicon esculentum) are highly susceptible to phytopathogen attack. The resulting intensive application of pesticides on tomato crops can affect the environment and health of humans and animals. The objective of this study was to select potential biocontrol agents among actinobacteria from tomato plants, in a search for alternative phytopathogen control. We evaluated 70 endophytic actinobacteria isolated from tomato plants in southern Brazil, testing their antimicrobial activity, siderophore production, indoleacetic acid production, and phosphate solubility. The actinomycete isolate with the highest antimicrobial potential was selected using the agar-well diffusion method, in order to optimize conditions for the production of compounds with antimicrobial activity. For this study, six growth media (starch casein-SC, ISP2, Bennett's, Sahin, Czapek-Dox, and TSB), three temperatures (25 degrees C, 30 degrees C, and 35 degrees C) and different pH were tested. Of the actinobacteria tested, 88.6% showed antimicrobial activity against at least one phytopathogen, 72.1% showed a positive reaction for indoleacetic acid production, 86.8% produced siderophores and 16.2% showed a positive reaction for phosphate solubility. Isolate R18(6) was selected due to its antagonistic activity against all phytopathogenic microorganisms tested in this study. The best conditions for production were observed in the SC medium, at 30 degrees C and pH 7.0. The isolate R18(6) showed close biochemical and genetic similarity to Streptomyces pluricolorescens.

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD).

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.