Microbiology, Imunology and Parasitology

Actinomycetes are Gram-positive bacteria widely distributed in the environment and known for the diverse biologically act ive molecules they produce. This study assesses the activity of compounds produced by 40 actinomycete isolates against Enterococcus spp. Staphylococcus aureus and Staphylococcus epidermidis clinical isoalates. The susceptibility profile of the samples was assessed using the agar disk diffusion method. Antimicrobial activity of actinomycetes was evaluated according to the double layer method. Here, two isolates (50 and 8S) exhibited activity against 90% of clinical Staphylococcus spp. and Enterococcus spp. These isolates were grown in starch casein broth at 30˚C for 7 days with constant shaking. After, the culture obtained was filtered to obtain a crude extract, whose antibiotic activity was assessed using the well diffusion technique. In this assay, isolate 50 presented higher activities than that exhibited by isolate 8S. The isolate 50 has been used in different assays looking for production of metabolite optimization.

Virulent phenotype of Shigella is directly related to the presence of pINV plasmid of 210 to 230 kb. IpaBCD proteins encoded by the plasmid direct the bacterial invasion into the host epithelial cells. InvE protein positively regulates the expression of virulence genes ipaBCD. The polymerase chain reaction was used to amplify a 362 base-pair fragment specific for the invE gene in sewage samples. Sewage samples were used in the assays because invasive bacteria like Shigella can used this way to infect human. The amplification products were digested with the restriction enzyme BglI and the resulting fragments confirmed the specificity of the amplification. A detection limit of 10 5 CFU/mL of Shigella was obtained for cultured bacteria and also for sewage samples seeded with the pathogen. Sewage samples were seeded with S. dysenteriae in enrichment culture and incubated for 8h at 37ºC. After incubation DNA was extracted and the PCR assay ensued. A minimal concentration of 10 2 CFU/mL was enough for the detection of the microorganisms after the enrichment of the sample.

Musca domestica is a major problem in poultry production where management has relied mostly on pesticides, which eventually have led to the development of widespread resistance in insect populations and environmental contamination. In this study, we explored the use of entomopathogenic fungi for the control of houseflies in Brazil. A total of 10 isolates of different fungal pathogens were tested on the larval stages of M. domestica, which were originally derived from a commercial aviary. Among the test fungi, Metarhizium anisopliae (sensu stricto) CG46 and CG30 were the most virulent isolates, causing up to 60% mortality at day 10, but only CG46 was selected for a field trial as it attained higher sporulation on larval cadavers. A commercial product based on the M. anisopliae isolate ESALQ-1037 (Metarril † ) was also field-tested using standard treatment procedures. After spraying both fungi in the aviary manure, there was a significant reduction (twofold lower) in adult density of M. domestica captured per trap compared to the control shed. Therefore, our results indicate that M. anisopliae is a promising biological control agent for M. domestica larvae, but new strategies for fungal application and appropriate formulation need to be developed to improve its effectiveness in poultry environments.

Entomopathogenic fungi are important controllers of pest-insects populations in agricultural production systems and in natural environment. These fungi have enzymatic machinery which involve since the recognition and adherence of spores in their hosts culminating with infection and death of these insects. The main objective of this study was to analyzed extracellular enzyme production of the fungi strains Beauveria bassiana, Metarhizium anisopliae and Paecilomyces sp when cultured on substrates. These fungi were grown in minimal media containing specific substrates for the analysis of different enzymes such as amylases, cellulases, esterases, lipases, proteases (gelatin and caseinase), pectinases and cuticles of Musca domestica larvae and adults. All the assays were performed with and without the presence of dextrose in the culture media. The quantification of enzyme activity was performed by the ratio of halo / colony (H/C) and the results subjected to variance analysis level of 5% (ANOVA) followed by post-Tukey test. All strains were positive for lipase and also they showed a high significant enzyme production for gelatin at concentrations of 4 and 1%. B. bassiana and Paecilomyces sp. were positive for amylase, pectinase and caseinase, and only Paecilomyces sp. showed cellulase activity.

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the b-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and b-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10°C, 28°C, 37°C and 45°C and glucose was observed for E. faecalis and E. faecium.

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.

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.

Net blotch, caused by the phytopathogen Drechslera teres, is a common disease of barley (Hordeum vulgare L) and is responsible for large economic losses in some barley growing areas. In this study the morphology and genetic variability of eight D. teres isolates from different regions of the Brazilian state of Rio Grande do Sul were investigated. Colony morphology was studied on potato-dextrose-agar (PDA) and genetic variability investigated using the random amplified polymorphic-DNA (RAPD) technique. 27 commercially available primers were tested of which 16 were selected for use in polymorphic analysis due to their good resolution and reproducibility. Similarity coefficients were used to construct dendrograms based on colony morphology and RAPD data showing the relationship between the eight isolates studied. Colony morphology showed variability between the isolates while RAPD assays showed high similarity coefficients, but grouping of the isolates according to the geographic origins of the seeds from which they were isolated was not possible.

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, CzapekeDox, and TSB), three temperatures (25 C, 30 C, and 35 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 C and pH 7.0. The isolate R18(6) showed close biochemical and genetic similarity to Streptomyces pluricolorescens.

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.

Introduction: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. Methods: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. Results: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). Conclusions: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

Acanthamoeba is a protist potential pathogen, capable of causing a blinding keratitis in contact lens wearers and disseminated infection, leading to granulomatous amebic encephalitis in immunocompromised individuals. This amoeba is a ubiquitous organism that has been isolated from various domestic water systems, such as cooling towers and hospital water networks. The objective of this work was to investigate the presence of Acanthamoeba in mineral water bottles marketed in Porto Alegre, southern Brazil. Positive samples were further classified at the genotype level after sequencing the ASA.S1 region of 18S rDNA gene. Six of the eight isolates belonged to T5 genotype, one to T4 genotype, and one was T11. Several genotypes have been reported worldwide as causative of pathologies in humans, including genotypes T4, T5 and T11. Overall, the widespread distribution of potentially pathogenic Acanthamoeba strains in the studied source demands more awareness within the public and health professionals, because this pathogen is emerging as a risk for human health worldwide.

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% coagulasepositive (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.