Sueli Terezinha Van der Sand

Intraspecific genetic diversity of Drechslera tritici-repentis as detected by random amplified polymorphic DNA analysis

The phytopathogenic fungus Drechslera tritici-repentis causes tan spot, an important disease of wheat in the southern Brazilian state of Rio Grande do Sul. Twelve D. tritici-repentis isolates were obtained from wheat seeds from different locations in the state. Their colony morphology on potato dextrose agar and polymorphisms in genomic DNA by the random amplified polymorphic DNA (RAPD) method were investigated. For the RAPD method, 23 primers were tested of which nine were selected for use in the study of D. tritici-repentis polymorphisms. The degree of similarity between isolates was calculated using a simple matching coefficient and dendrograms constructed by the unweighted pair-group method with arithmetical averages (UPGMA). The morphological and RAPD analyses showed intraspecific polymorphisms within the isolates, but it was not possible to establish a relationship between these polymorphisms and the geographical regions from where the host seeds were collected.

Temperatura de degradação de resíduos em processo de compostagem e qualidade microbiológica do composto final

The composting technique is a promising alternative for the treatment of solid organic residues, along with the sludge produced in sewage treatment plants. The final compost might show fertilizer properties, which can be used to recover poor soils. The high temperatures that the composting process can achieve are responsible for the reduction of the pathogenic microbial population, helminth eggs and virus present in the beginning of the process. This behavior assures the microbial quality of the compost according to the 375/2006 Resolution of the CONAMA. The aim of this study was to evaluate the influence of temperature on Escherichia coli, Salmonella sp., helminth egg and enteric virus presence in the final product from a composting process. For this matter colimetric assays were performed together with the seedling of the samples in different culture media, the assay for helminth eggs visualization under the microscope and the identification of enteric virus. The results showed an oscillation on E. coli and heterotrophic bacteria counts, even after the thermophilic phase. On the other hand, Salmonella sp., helminth eggs and enteric virus were not found in the final compost.

Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil

The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulase-positive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18%) showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.

Genetic variability of Bipolaris sorokiniana isolates using URP-PCR

Spot blotch disease, caused by Bipolaris sorokiniana, is one of the major diseases of wheat and is responsible for large losses of wheat crops worldwide. We used polymerase chain reaction (PCR) with universal rice primers (URP) for molecular characterization of 60 monosporic B. sorokiniana isolates from Brazil and other countries, and evaluated the diversity of the samples. PCR amplification generated 232 different DNA fragments ranging in size from 100 to 2018 bp. The primers URP-4R, URP-2R, and URP-1F generated greater numbers of amplified fragments (36, 30, and 25, respectively) from the single-spore isolates, and some diversity was observed among the isolates generated using these primers. The primers URP-2F, URP-6R, URP-17R, URP-30F, and URP-38F produced a pattern of monomorphic fragments and 73% of the isolates showed an average of 44 different DNA-amplified fragments. Primer URP-2F generated a 578-bp fragment that was common to 83.7% of the isolates; primer URP-6R generated a 548-bp fragment and primer URP-38F generated a 650bp product that was common to 89.1% and 80% of the isolates, respectively. The URP-PCR primers provided important information about the genetic profiles of the monosporic cultures, which showed intraspecific variability among the monoconidial isolates and among the monosporic cultures that originated from the same polysporic strain. Our results indicate that URPs are sensitive and give reproducible results for assaying the genetic variability of B. sorokiniana.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

INTRODUCTION This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

Evaluation of antimicrobial activity of the endophytic actinomycete R18(6) against multiresistant Gram-negative bacteria

Endophytic actinomycetes are promising sources of antimicrobial substances. This study evaluates the activity of metabolites produced by the endophytic actinomycete R18(6) against Gram-negative bacteria multiresistant to antimicrobials. R18(6) isolate was grown in submerged cultures under different conditions: carbon source, temperature, pH and incubation time to optimize antimicrobials production. The actinomycete grown in base medium supplemented with 1% glucose, pH 6.5 and incubation at 30 ºC for 96 h with shaking at 100 rpm, exhibited the highest activity against the used Gram-negative bacteria. Minimum inhibitory concentration (MIC) of the crude extract produced by the microorganism varied between 1/32 and 1/256. It had bactericide or bacteriostatic activity, depending on the Gram-negative organism. The active extract was stable at high temperatures, and unstable in medium containing proteolytic enzymes. Micromorphology of R18(6) was investigated by optical and scan microscopy, revealing that it was morphologically similar to the genusStreptomyces.

Genetic Background of β-Lactamases in Enterobacteriaceae Isolates from Environmental Samples

The prevalence of β-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 β-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 β-lactamases: extended-spectrum β-lactamases (ESBLs), carbapenemases, and β-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 β-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.

Production of lipolytic enzymes by bacteria isolated from biological effluent treatment systems

This work aimed to evaluate the production of lipolytic complexes, produced by microorganisms isolated from a biological treatment system of effluents from a hotel. To select the best lipolytic microorganism for use in biotechnological processes, we tested 45 bacterial isolates recovered from the raw effluent of the hotels restaurant waste tank. Lipase production was assayed in culture medium supplemented with olive oil and rhodamine B, incubated at 25 °C and 30 °C for 24 h - 48 h. Results showed 22 isolates lipase producers. All isolates were inoculated on medium without yeast extract to select the ones with highest enzyme yields. Out of these, nine isolates showed high lipase activity. The strain with the larger halo was assayed in submerged culture using an orbital shaker and a bioreactor, with three different substrates (olive oil, grape seed oil, and canola oil). Isolate G40 identified as Acinetobacter baylyi was selected to run the production assays because it showed the best result in the solid medium. In the bioreactor, maximum lipase production was obtained after 12 h of culture with the three substrates evaluated: 0,358 U/mL.min-1 in olive oil, 0,352 U/mL.min-1 with grapeseed oil, and 0,348 U/mL.min-1 with canola oil.

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.

Freqüência de biovares de ralstonia solanacearum em diferentes cultivares e épocas de cultivo de batata no rio grande do sul

The occurrence of biovars I and/or II of Ralstonia solanacearum in a potato (Solanum tuberosum) field has direct consequences for the success of the measures adopted to control bacterial wilt. Biovars differ regarding aggressiveness, latency and survival. An experiment was conducted in a naturally infested potato field in two seasons to find (1) the incidence of biovars I and II, (2) the relationship between biovar and planting season, and (3) the relationship between biovar and potato cultivar. Bacterial isolates from potato cultivars Achat, Baronesa, Elvira, Macaca, Monte Bonito, and Trapeira were identified as biovar I or II through PCR, using T3A and T5A primers. Both biovars I and II were found in the naturally infested field. Among 73 strains, 5.5 and 94.5 % were identified as biovar I and II, respectively. Biovar II was found in the spring and in the fall, regardless of the cultivar, but biovar I only in the spring season and on symptomless plants of Achat and Macaca cultivars. The highest population of biovar I on these two cultivars may be evidence for a possible relationship between biovar and cultivar.