Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings

Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Effectiveness of Microwave Sterilization on Three Hard Chairside Reline Resins

Purpose: The aim of this study was to evaluate the effectiveness of microwave irradiation sterilization on hard chairside reline resins. Materials and Methods: Specimens of three reline resins (Kooliner, Tokuso Rebase, and Ufi Gel Hard) were fabricated and subjected to ethylene oxide sterilization. The specimens were then individually inoculated (107 cfu/mL) with Tryptic Soy Broth media containing one of the tested microorganisms (C albicans, S aureus, B subtilis, and P aeruginosa). After 48 hours at 37°C, the samples were vortexed for 1 minute and allowed to stand for 9 minutes, followed by a short vortex to resuspend any organisms present. After inoculation, 40 specimens of each material were immersed in 200 mL of water and subjected to microwave irradiation at 650 W for 6 minutes. Forty non-irradiated specimens were used as positive controls. Replicate specimens (25 μL) of suspension were plated at dilutions of 10-3 to 10-6 on plates of selective media appropriate for each organism. All plates were incubated at 37°C for 48 hours. After incubation, colonies were counted, and the data were statistically analyzed by the Kruskal-Wallis test. Twelve specimens of each material were prepared for SEM. Results: All immersed specimens showed consistent sterilization of all the individual organisms after microwave irradiation. SEM examination indicated an alteration in cell morphology after microwave irradiation. Conclusion: Microwave sterilization for 6 minutes at 650 W proved to be effective for the sterilization of hard chairside reline resins.

Monitoramento sanitário de um sistema integrado de tratamento de águas residuárias da suinocultura

Objective. To assess the potential for contamination of wastewaters from pig farming. Methods. Wastewaters from pig farming were stored in a tank. After 0, 30, 60, 90, and 120 days of hydraulic retention, they were added to lysimeters filled with argillaceous, sandy, or medium soil. Finally, these lysimeters were submitted to simulations of either a rainy season or a dry season. The number of colony-forming units (CFUs) of total coliforms, fecal coliforms, and fecal streptococci was measured in the effluents of the storage tank (for the various periods of hydraulic retention), in the percolate from the lysimeters, and in the three types of soil. The microbiological analyses were carried out using the membrane filter technique. The pH analyses were done potentiometrically. Results. For the three microorganisms, the largest decrease in bacterial counts in the storage tanks occurred with 90 or 120 days of retention. There was a marked decrease in the bacterial count in the percolates of the three soils. For the three soil types the greatest reduction in bacterial counts was found in medium soil, due to its acidity (pH < 7.0). Hydraulic retention was not sufficient to ensure the sanitary adequacy of the wastewaters and their use for irrigation, given that fecal coliform values were above 1 000 CFU per 100 mL. Therefore, adding the residues to the soil was considered a second stage of treatment. Conclusions. The retention of wastewaters followed by adding them to soil was effective in minimizing the contaminating effect of pig farming residues. The storage time for wastewaters from pig farming could be decreased from 120 to 90 days.

Comparação entre o sistema Autoścan-4 e métodos convencionais na identificação de bacilos Gram-negativos não fermentadores, isolados de materiais clínicos de pacientes em Presidente Prudente - SP

Ninety eight strains of glucose-nonfermenting Gram-negative bacilli were analyzed and isolated from several clinical materials of 95 patients admitted at the Dr. Domingos Leonardo Cerávolo University Hospital and three from outpatients. All of them were assisted in the Laboratory of Clinical Analysis of Unoeste University, Presidente Prudente, SP, from the period of October of 1999 to April of 2001. In this work, the level of agreement between the semi-automated commercial system AutoScan-4 and the conventional system for the identification of those bacteria were studied comparatively. There was agreement in 81 (82.7%), showing that both methodologies are useful for identification; partial agreement in six strains (6.1%) and disagreement in 11 (11.2%). The comercial system did not identify nine (9.2%) of the strains and reported them as very rare biotypes.

Screening for yeast with antibacterial properties from an ethanol distillery

A general screening for the expression of antibacterial activity and non-flocculating type of yeast strains from must and fermented broth of alcohol distilleries was performed. From 60 strains only Saccharomyces sp. M26 presented a inhibitory halo in Lactobacillus fermentum culture and significant reduction in the culture turbidity (71%) and specific growth rate (56%) when compared to the control. Freezing did not affect the antibacterial activity of the Saccharomyces sp. M26 extract and heating at 90°C for 20 min completely destroyed this activity. It is expected the decrease of lactic acid bacteria growth in the S. cerevisiae alcoholic fermentation should allow for better control of these bacteria in the process. © 2003 Elsevier Ltd. All rights reserved.

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cryl genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.

Hypochlorous acid inhibition by acetoacetate: Implications on neutrophil functions

Type-1 diabetic patients experience hyperketonemia caused by an increase in fatty acid metabolism. Thus, the aim of this study was to measure the effect of ketone bodies as suppressors of oxidizing species produced by stimulated neutrophils. Both acetoacetate and 3-hydroxybutyrate have suppressive effect on the respiratory burst measured by luminol-enhanced chemiluminescence. Through measurements of hypochlorous acid production, using neutrophils or the myeloperoxidase/H2O2/Cl- system, it was found that acetoacetate but not 3-hydroxybutyrate is able to inhibit the generation of this antimicrobial oxidant. The superoxide anion scavenging properties were confirmed by ferricytochrome C reduction and lucigenin-enhanced chemiluminescence assays. However, ketone bodies did not alter the rate of oxygen uptake by stimulated neutrophils, measured with an oxygen electrode. A strong inhibition of the expression of the cytokine IL-8 by cultured neutrophils was also observed; this is discussed with reference to the antioxidant-like property of acetoacetate. © 2004 Pharmaceutical Society of Japan.

Determination of Cu, Ni and Pb in Aqueous Medium by FAAS after Pre-Concentration on 2-Aminothiazole Modified Silica Gel

This work describes the synthesis and characterization of 2-aminothiazole modified silica gel (SiAT), and the results of a study of the adsorption and pre-concentration (in batch and using a flow-injection system coupled to an absorption atomic spectrometer) of Cu(II), Ni(II) and Pb(II) in aqueous medium. The adsorption capacities for each metal ions in mmol g -1 were: Cu(II)= 1.18, Ni(II)= 1.15 and Pb(II)= 1.10. The results obtained in the flow experiments showed a recovery of practically 100% of the metal ions adsorbed in a mini-column packed with 100 mg of SiAT, using 100 μL of 2.0 mol L -1 HCl solution as eluent. The sorption-desorption of the metal ions made possible the application of a flow-injection system for the pre-concentration and quantification by FAAS of metal ions at trace level in natural water samples digested and not digest by an oxidizing UV photolysis.

Conformation and lytic activity of eumenine mastoparan: A new antimicrobial peptide from wasp venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Padrão de sensibilidade aos antimicrobianos de bacilos gram-negativos não fermentadores isolados de materiais clínicos de pacientes em Presidente Prudente - SP

The aim of this study was the assessment of isolation frequency and antimicrobial susceptibility pattern of nonfermenting Gram-negative bacilli. Ninety eight strains of nonfermenting Gram-negative bacilli, isolated from several clinical materials of patients admited at the Dr. Domingos Leonardo Cerávolo University Hospital and at Dr. Odilo Antunes Siqueira State Hospital, as well as from every outpatient; assisted at Laboratory of Clinical Analysis of Unoeste University, Presidente Prudente, São Paulo, in the period of October 1999 to April 2001 were analyzed. The most frequent species were Pseudomonas aeruginosa (65.3%) and Acinetobacter baumannii (23.5%). The frequency of the other isolated species was smaller than 2.5%. In the antimicrobial susceptibility tests, the two species more prevalent showed high resistance. The antibiotic most active in vitro was the imipenem, with 79.6% in microdiluition method, and 76.6% in diffusion method, for Pseudomonas aeruginosa strains and 100.0% in both microdiluition and diffusion methods, for Acinetobacter baumannii. The cephalosporins of third generation, the ciprofloxacin and the aminoglycosides, presented percentage of susceptibility varying from 22.4 to 69.7%. These results bring implications to the emergency use of the antimicrobial agents in the treatment of patients with severe infection.

Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Dispersão de bioaerossóis por aeradores em uma estação de tratamento de esgoto sanitário

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

Identificação de Enterobacter spp. através de técnicas convencionais e do sistema semi-automatizado Microscan (Autoscan-4)

A hundred and ninety-five (195) strains of Enterobacter spp., isolated from diverse clinical specimens - urine, feces, cateter, blood, wound, tracheal aspirate, vaginal fluid - were submitted to the conventional identification by biochemical tests, and were also submitted to the identification by panels NegCombo 20 of the system automated MicroScan - AutoScan- 4 (Dade Behring Inc., West Sacramento, CA, USA). The samples were from patients of the Clinical Laboratory from the School of Pharmacy and Biochemistry of UNOESTE, Presidente Prudente, SP, and from patients hospitalized at the University Hospital Domingos Leonardo Cerávolo, UNOESTE. Of the total of strains tested, 191 (97.9%) presented agreement between the two approaches utilized and 4 strains (2.1%) presented identification disagreement, that is, the genus identified was different in each approach. By this study, the conclusion is that both the approaches utilized for the identification presented advantages and disadvantages related to the cost, facility of execution, quickness, reliability and some other characteristics. Even so, our results showed that conventional methods represent a reliable tool for Enterobacter identification.

Aderência e formação do biofilme por Staphylococcus aureus ao polietilenotereftalato (PET) usado como embalagem para acondionamento de medicamentos e cosméticos

The polyethylene terephthalate (PET) is used for package drugs and cosmetics. The aim of this research was examine by scanning electron microscopy (SEM) a Staphylococcus aureus attachment and biofilm formation on a polyethylene terephthalate (PET) surface and hydrophobicity of S. aureus by adherence to hydrocarbons. A suspension of S. aureus was prepared in Mueller-Hinton broth and, coupons of polyethylene terephthalate were incubated for 30 minutes, two, 24 and 48 hours, 15 and 30 days. Afteron the coupons were removed and prepared for scanning electron microscope analysis. The attachment and biofilm formation was observed on the surfaces of PET. The SEM revealed adhesion and biofilm formation on PET surfaces. The hydrophobicity test classified S. aureus as hydrophobic.