A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

Aims: Detection of Xylella fastidiosa in citrus plants and insect vectors.Methods and Results: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay.Conclusions: the use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively.Significance and Impact of the study: the employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.

A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.

Occurrence of fungi in water used at a haemodialysis centre

Aims: The aim of this study was to identify and determine the diversity, occurrence and distribution of fungi in water used at a haemodialysis centre.Methods and Results: Samples in the hydraulic circuit for the distribution of the water, dialysate samples and samples of sterilization solution from dialysers were collected over a 3-month period, and 500 ml of each sample was filtered through membranes. All together 116 isolates of fungi were recovered from 89% of all water samples collected inside the haemodialysis unit, with prevalence of moulds in tap water samples and of yeasts in dialysate samples. Fusarium spp. was the most abundant genus found, whereas Candida parapsilosis was the predominant yeast species.Conclusions: This study demonstrated that various fungi were present in the water system. These data suggest the inclusion of the detection and quantification of fungi in the water of haemodialysis.Significance and Impact of the Study: The recovery of fungi from aqueous haemodialysis environments implies a potential risk for haemodialysis patients and indicates the need for continuous maintenance and monitoring. Further studies on fungi in haemodialysis water systems are required to investigate the organism ability to persist, their role in biofilm formation and their clinical significance.

Virulence characteristics and epidemiology of Yersinia enterocolitica and Yersiniae other than Y-pseudotuberculosis and Y-pestis isolated from water and sewage

Aims: To determine the species, bio-sero-phagetypes, antimicrobial drug resistance and also the pathogenic potential of 144 strains of Yersinia spp. isolated from water sources and sewage in Brazil.Methods and Results: the 144 Yersinia strains were characterized biochemically, serologically and had their antibiotic resistance and phenotypic virulence markers determined by microbiological and serological standard techniques. The Y. enterocolitica strains related to human diseases were also tested for the presence of virulence genes, by the PCR technique. The isolates were classified as Y. enterocolitica, Y. intermedia, Y. frederiksenii, Y. kristensenii and Yersinia biochemically atypical. The 144 isolates belonged to various bio-serogroups. Half of the strains showed resistance to three or more drugs. The Y. enterocolitica strains related to human diseases exhibited phenotypic virulence characteristics and virulence genes.Conclusions: Water from various sources and sewage are contaminated with Yersinia spp. in Brasil. Among these bacteria, virulent strains of Y. enterocolitica were found, with biotypes and serogroups related to human diseases.Significance and Impact of the Study: This is the first documented description of the occurrence of pathogenic Y. enterocolitica in water sources and sewage in Brazil. The occurrence of virulence strains of Y. enterocolitica shows that the environment is a potential source of human infection by this species in this country.

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambe Stream (State of Parana, Brazil)

The incidence of Vibrio cholerae, Aeromonas spp, and Plesiomonas shigelloides was determined in Rater samples from Cambe Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve. Aer. hydrophila, 12 Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V, cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp and V. cholerae, while none of the Plt. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp, and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of Candida albicans adhesion and biofilm formation on a denture base acrylic resin containing silver nanoparticles

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

Anti-poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real-time PCR

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

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

Keratinolytic activity of Streptomyces sp isolated of poultry processing plant

The keratin is not degraded by common enzyme, keratinases have the ability to degrade native keratin and others insoluble enzymes. In the present work was Studied keratinase produced by Streptomyces sp LMI-1 isolated from industrial plant of poultry processing. The enzyme degraded 87% of feathers after 120 h, it was stimulated by Ba(2+) and inhibited by Ca(2+), Mn(2+), EDTA and Hg(+). The optimum pH and temperature for the enzyme was 8.5 and 60 degrees C, respectively. The enzyme was stable after 2 hours at 50 degrees C. The culture broth analysis by thin layer chromatography showed presence of amino acids serine, methionine, proline, tyrosine and leucine after 72 hours of incubation. The microorganism showed potential for use in industrial process because of higher enzyme production and feathers degradation.

Biosurfactant synthesis by Pseudomonas aeruginosa LBI isolated from a hydrocarbon-contaminated site

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

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of the beta-glucanolytic enzyme complex of Trichoderma harzianum Rifai for the production of gluco-oligosaccharide fragments by enzymatic hydrolysis of 1,3;1,6-beta-D-glucans

Botryosphaeran, a new exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the beta-glucanolytic complex from Trichoderma harzianum Rifai. beta-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP >= 4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes' specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability

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