Scielo RSS <![CDATA[Brazilian Journal of Microbiology]]> vol. 44 num. 4 lang. en <![CDATA[SciELO Logo]]> <![CDATA[<b>Microbiologically induced deterioration of concrete</b>: <b>a review</b>]]> Microbiologically induced deterioration (MID) causes corrosion of concrete by producing acids (including organic and inorganic acids) that degrade concrete components and thus compromise the integrity of sewer pipelines and other structures, creating significant problems worldwide. Understanding of the fundamental corrosion process and the causal agents will help us develop an appropriate strategy to minimize the costs in repairs. This review presents how microorganisms induce the deterioration of concrete, including the organisms involved and their colonization and succession on concrete, the microbial deterioration mechanism, the approaches of studying MID and safeguards against concrete biodeterioration. In addition, the uninvestigated research area of MID is also proposed. <![CDATA[<b>Biotechnology of polyketides</b>: <b>new breath of life for the novel antibiotic genetic pathways discovery through metagenomics</b>]]> The discovery of secondary metabolites produced by microorganisms (e.g., penicillin in 1928) and the beginning of their industrial application (1940) opened new doors to what has been the main medication source for the treatment of infectious diseases and tumors. In fact, approximately 80 years after the discovery of the first antibiotic compound, and despite all of the warnings about the failure of the "goose that laid the golden egg," the potential of this wealth is still inexorable: simply adjust the focus from "micro" to "nano", that means changing the look from microorganisms to nanograms of DNA. Then, the search for new drugs, driven by genetic engineering combined with metagenomic strategies, shows us a way to bypass the barriers imposed by methodologies limited to isolation and culturing. However, we are far from solving the problem of supplying new molecules that are effective against the plasticity of multi- or pan-drug-resistant pathogens. Although the first advances in genetic engineering date back to 1990, there is still a lack of high-throughput methods to speed up the screening of new genes and design new molecules by recombination of pathways. In addition, it is necessary an increase in the variety of heterologous hosts and improvements throughout the full drug discovery pipeline. Among numerous studies focused on this subject, those on polyketide antibiotics stand out for the large technical-scientific efforts that established novel solutions for the transfer/engineering of major metabolic pathways using transposons and other episomes, overcoming one of the main methodological constraints for the heterologous expression of major pathways. In silico prediction analysis of three-dimensional enzymatic structures and advances in sequencing technologies have expanded access to the metabolic potential of microorganisms. <![CDATA[<b>Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products</b>]]> Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments. <![CDATA[<b>Applications of recombinant <i>Pichia pastoris</i> in the healthcare industry</b>]]> Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry. <![CDATA[<b>Clavulanic acid production by the MMS 150 mutant obtained from wild type <i>Streptomyces clavuligerus</i> ATCC 27064</b>]]> Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant. <![CDATA[<b>Extracellular polysaccharide production by a strain of <i>Pleurotus djamor </i>isolated in the south of Brazil and antitumor activity on Sarcoma 180</b>]]> Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072) and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24) and maximal biomass productivity (Q Xmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells. <![CDATA[<b>Statistical investigation of <i>Kluyveromyces lactis</i> cells permeabilization with ethanol by response surface methodology</b>]]> The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L-1 oNP min-1 g-1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry. <![CDATA[<b>Bioconversion of <i>R-</i>(+)-limonene to perillic acid by the yeast <i>Yarrowia lipolytica</i></b>]]> Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 ºC) in a reaction medium containing 0.5% v/v limonene and 10 gµL of stationary phase cells (dry weight). The best results, corresponding to 564 mgµL of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 ºC for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mgµL, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry. <![CDATA[<b>Extraction and <i>in vitro</i> antioxidant activity of exopolysaccharide by <i>Pleurotus eryngii</i> SI-02</b>]]> The extraction parameters for Pleurotus eryngii SI-02 exopolysaccharide (EPS) produced during submerged culture were optimized using response surface methodology (RSM). The optimum conditions for EPS extraction were predicted to be, precipitation time 20.24 h, ethanol concentration 89.62% and pH 8.17, and EPS production was estimated at 7.27 g/L. The actual yield of EPS under these conditions was 7.21 g/L. The in vitro antioxidant results of the EPS showed that the inhibition effects of EPS at a dosage of 400 mg/L on hydroxyl, superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 59.63 ± 3.72%, 38.69 ± 2.59%, and 66.36 ± 4.42%, respectively, which were 12.74 ± 1.03%, 8.01 ± 0.56%, and 12.19 ± 1.05% higher than that of butylated hydroxytoluene (BHT), respectively. The reducing power of EPS of P. eryngii SI-02 was 0.98 ± 0.05, 60.66 ± 5.14% higher than that of BHT. The results provide a reference for large-scale production of EPS by P. eryngii SI-02 in industrial fermentation and the EPS can be used as a potential antioxidant which enhances adaptive immune responses. <![CDATA[<b>Enhanced production of <i>Aspergillus tamarii</i> lipase for recovery of fat from tannery fleshings</b>]]> The influence of various oil cakes has been investigated for high level production of lipase using Aspergillus tamarii MTCC 5152. By solid state fermentation in wheat bran containing 2.5% w/w gingili oil cake at 70% v/w moisture content the fungus produced a maximal yield of lipase (758 ± 3.61 u/g) after 5 days of incubation using 2% v/w inoculum containing 10(6) spores/mL. Wheat bran and gingili oil cake with supplementation of gingili oil (1.0% w/w), glucose (0.5% w/w) and peptone (0.5% w/w) gives an increased enzyme production of 793 ± 6.56 u/g. The enzyme shows maximum activity at pH 7.0, temperature 50 °C and was stable between the pH 5.0-8.0 and temperature up to 60 °C. Crude lipase (3%) applied to tannery fleshing shows 92% fat solubility. The results demonstrate that fat obtained from tannery fleshing, a by-product of the leather industry has a high potential for biodiesel production and the proteinaceous residue obtained can be used as animal feed. <![CDATA[<b>Cashew apple juice as microbial cultivation medium for non-immunogenic hyaluronic acid production</b>]]> In this work, natural cashew apple juice was used as cultivation medium as an alternative to substitute brain heart infusion medium. The effect of aeration and juice supplementation with yeast extract on the production of hyaluronic acid in batch fermentation was also investigated. Similar levels of cell mass were obtained in inoculum using cashew apple juice supplemented with yeast extract or the conventional brain heart infusion medium. Fermentation in Erlenmeyer flasks produced low biomass and hyaluronic acid concentrations. The hyaluronic acid concentration and viscosity increased from 0.15 g/L and 3.87 cP (no aeration or medium supplementation) to 1.76 g/L and 107 cP, when aeration (2 vvm) and 60 g/L of yeast extract were used. The results suggest the production of low-molecular weight hyaluronic acid oligomers instead of the high molecular weight polymer. <![CDATA[<b>Kinetic characterization of glucose aerodehydrogenase from <i>Aspergillus niger</i> EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme</b>]]> In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL), 36 hours fermentation time, pH 5, 30°C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30°C. The Michaelis-Menton constants (Km, Vmax, Kcat and Kcat/Km) were 20 mM, 45.87 U mL-1, 1118.81 s-1 and 55.94 s-1 mM-1, respectively. The enzyme was found to be thermaly stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness. <![CDATA[<b>Isolation and characterization of novel thermophilic lipase-secreting bacteria</b>]]> The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered. <![CDATA[<b>Characteristics of <i>Saccharomyces cerevisiae </i>yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation</b>]]> Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52" -rough and "PE-02" smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone. <![CDATA[<b>Chemicals and lemon essential oil effect on <i>Alicyclobacillus acidoterrestris</i> viability</b>]]> Alicyclobacillus acidoterrestris is considered to be one of the important target microorganisms in the quality control of acidic canned foods. There is an urgent need to develop a suitable method for inhibiting or controlling the germination and outgrowth of A.acidoterrestris in acidic drinks. The aim of this work was to evaluate the chemicals used in the lemon industry (sodium benzoate, potassium sorbate), and lemon essential oil as a natural compound, against a strain of A.acidoterrestris in MEB medium and in lemon juice concentrate. The results pointed out that sodium benzoate (500-1000-2000 ppm) and lemon essential oil (0.08- 0.12- 0.16%) completely inhibited the germination of A. acidoterrestris spores in MEB medium and LJC for 11 days. Potassium sorbate (600-1200 ppm) was more effective to inhibit the growth of the microbial target in lemon juice than in MEB medium. The effect of sodium benzoate, potassium sorbate and essential oil was sporostatic in MEB and LJC as they did not affect spore viability. <![CDATA[<b>Dynamics of the chemical composition and productivity of composts for the cultivation of <i>Agaricus bisporus</i> strains</b>]]> Two compost formulations based on oat straw (Avena sativa) and brachiaria (Brachiaria sp.) were tested for the cultivation of three Agaricus bisporus strains (ABI-07/06, ABI-05/03, and PB-1). The experimental design was a 2 x 3 factorial scheme (composts x strains) with 6 treatments and 8 repetitions (boxes containing 12 kg of compost). The chemical characterization of the compost (humidity, organic matter, carbon, nitrogen, pH, raw protein, ethereal extract, fibers, ash, cellulose, hemicellulose, and lignin) before and after the cultivation of A. bisporus and the production (basidiomata mass, productivity, and biological efficiency) were evaluated. Data were submitted to variance analysis, and averages were compared by means of the Tukey's test. According to the results obtained, the chemical and production characteristics showed that the best performances for the cultivation of A. bisporus were presented by the compost based on oat and the strain ABI-07/06. <![CDATA[<b>Elimination of coliforms and <i>Salmonella </i>spp. in sheep meat by gamma irradiation treatment</b>]]> This study aimed at evaluating the bacteriological effects of the treatment of sheep meat contaminated with total coliforms, coliforms at 45 °C and Salmonella spp. by using irradiation at doses of 3 kGy and 5 kGy. Thirty sheep meat samples were collected from animals located in Rio de Janeiro State, Brazil, and then grouped in three lots including 10 samples: non-irradiated (control); irradiated with 3 kGy; and irradiated with 5 kGy. Exposure to gamma radiation in a 137Cs source-driven irradiating facility was perfomed at the Nuclear Defense Section of the Brazilian Army Technological Center (CTEx) in Rio de Janeiro. The samples were kept under freezing temperature (-18 °C) until the analyses, which occurred in two and four months after irradiation. The results were interpreted by comparison with the standards of the current legislation and demonstrated that non-irradiated samples were outside the parameters established by law for all groups of bacteria studied. Gamma irradiation was effective in inactivating those microorganisms at both doses tested and the optimal dose was achieved at 3 kGy. The results have shown not only the need for sanitary conditions improvements in slaughter and processing of sheep meat but also the irradiation effectiveness to eliminate coliform bacteria and Salmonella spp. <![CDATA[<b>Presence of enterotoxigenic <i>Staphylococcus aureus </i>in artisan fruit salads in the city of San Luis, Argentina</b>]]> An increase in the consumption of fruit juices and minimally processed fruits salads has been observed in recent years all over the world. In this work, the microbiological quality of artisan fruit salads was analysed. Faecal coliforms, Salmonella spp, Shigella spp, Yersinia enterocolitica and Escherichia coli O157:H7 were not detected; nevertheless, eleven strains of Staphylococcus aureus were isolated. By multiplex PCR, all isolates showed positive results for S. aureus 16S rRNA gene and 63.6% of them were positive for sea gene. Furthermore, PCR sea positive strains were able to produce the corresponding enterotoxin. Finally, the inactivation of these strains in fruit salads by nisin, lysozyme and EDTA, was studied. EDTA produced a total S. aureus growth inhibition after 60 h of incubation at a concentration of 250 mg/L. The presence of S. aureus might indicate inadequate hygiene conditions during salad elaboration; however, the enterotoxigenicity of the strains isolated in this study, highlights the risk of consumers' intoxication. EDTA could be used to inhibit the growth of S. aureus in artisan fruit salads and extend the shelf life of these products. <![CDATA[<b>Antibacterial activity of bacteriocin-like substance P34 on <i>Listeria monocytogenes </i>in chicken sausage</b>]]> The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1) previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products. <![CDATA[<b>Prevalence and quantification of <i>Listeria monocytogenes </i>in beef offal at retail level in Selangor, Malaysia</b>]]> A total of 63 beef offal samples (beef liver = 16; beef lung = 14; beef intestine = 9; beef tripe = 15; beef spleen = 9) from three wet markets (A, B, and C) in Selangor, Malaysia were examined for the prevalence and microbial load of Listeria monocytogenes. A combination of the most probable number and polymerase chain reaction (MPN-PCR) method was employed in this study. It was found that L. monocytogenes detected in 33.33% of the beef offal samples. The prevalence of L. monocytogenes in beef offal purchased from wet markets A, B, and C were 22.73%, 37.50% and 41.18% respectively. The density of L. monocytogenes in all the samples ranged from<3upto&gt; 2,400 MPN/g. The findings in this study indicate that beef offal can be a potential vehicle of foodborne listeriosis. <![CDATA[<b>Virulence profiles of Shiga toxin-producing <i>Escherichia coli </i>and other potentially diarrheagenic <i>E.coli </i>of bovine origin, in Mendoza, Argentina</b>]]> This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing. <![CDATA[<b>Essential oils of thyme and Rosemary in the control of <i>Listeria monocytogenes</i> in raw beef</b>]]> This study was developed in order to evaluate two alternatives for the control of Listeria monocytogenes in raw bovine meat pieces, both based on the use of Thymus vulgaris and Rosmarinus officinalis essential oils (EOs). The antilisterial activity of different concentrations of the EOs was tested in vitro using agar dilution and disk volatilization techniques. In addition, L. monocytogenes was inoculated in meat pieces, which were submerged in edible gelatin coatings containing 2% (v/v) EOs or submitted to the vapor of EOs (0.74 μ L. monocytogenes was quantified after one, 48 and 96 hours of storage (7 °C). In the in vitro tests, the EO of T. vulgaris presented higher activity. The two options used (edible gelatin coating and vapor activity), in spite of exercising effects with differentiated behaviors, presented antibacterial activity against L. monocytogenes inoculated in raw bovine meat (p < 0.05). Greatest antibacterial activity were obtained in the experiment that used edible coatings containing EOs, at 48 hours of storage reductions in bacterial counts between 1.09 and 1.25 Log CFU.g-1 were obtained. In the vapor effect experiment, the EO of T. vulgaris caused the highest reduction in the population of bacteria inoculated in raw bovine meat (p < 0.05), 0.40 Log CFU.g-1 at 96 hours of storage. This study supplied important information regarding new and promising natural alternatives, based on the concept of active packaging, for the control of L. monocytogenes in the meat industry. <![CDATA[<b>The sensitivity of bacterial foodborne pathogens to <i>Croton blanchetianus </i>Baill essential oil</b>]]> The aim of this study was to assess the activity of essential oil extracted from the leaves of C. blanchetianus Baill, popularly known as "marmeleiro", in inhibiting the growth and survival of pathogenic microorganisms in food by determining their survival in vitro and by observing the behaviour of Listeria monocytogenes inoculated into a food model (meat cubes) that was stored at refrigeration temperature (7 ± 1 ºC) for 4 days. The results indicated a bactericidal effect against Aeromonas hydrophila and Listeria monocytogenes and bacteriostatic action against Salmonella Enteritidis. A bacteriostatic effect on meat contaminated with L. monocytogenes was found for all concentrations of essential oils tested. These results showed that essential oil from the leaves of C. blanchetianus Baill represents an alternative source of potentially natural antimicrobial agents that may be used as a food preservative. <![CDATA[<b>Gene detection and toxin production evaluation of hemolysin BL of <i>Bacillus cereus</i> isolated from milk and dairy products marketed in Brazil</b>]]> Bacillus cereusis an ubiquitous, spore-forming bacteria that can survive pasteurization and the majority of the heating processes used in the dairy industry. Besides, it is a pathogen responsible for different types of food poisoning. One type of foodborne disease caused by B.cereusis the diarrheal syndrome, which is caused by the ingestion of vegetative cells producing toxins in the small intestine. One virulence factor for the diarrheal syndrome is the toxin hemolysin BL (HBL), a three-component protein formed by the L1, L2 and B components. In order to evaluate the presence of diarrheal strains isolated from milk and dairy products, 63 B. cereus isolates were obtained from 260 samples of UHT milk, pasteurized milk and powdered milk, sold in commercial establishments and from different brands. The isolates were subjected to the Polymerase Chain Reaction (PCR) for the detection of the encoding genes for the L1, L2 and B components and the toxin production capacity were evaluated with an immunoassay. A total of 23 [36.5%] isolates were identified carrying simultaneously the three tested genes, from which, 20 [86.9%] showed toxigenic capacity. 26 [41.3%] isolates did not carry any of genes tested and the other 14 [22.2%] were positive for one or two of them. The results showed a high toxigenic capacity among the B. cereus isolates able to produce the HBL, indicating a potential risk for consumers. <![CDATA[<b>Molecular characterization of lactobacilli isolated from fermented <i>idli</i> batter</b>]]> Lactic acid bacteria are non pathogenic organism widely distributed in nature typically involved in a large number of spontaneous food fermentation. The purpose of this study was to characterize the bacteriocinogenic lactobacilli from fermented idli batter which can find application in biopreservation and biomedicine. Eight most promising lactobacilli were chosen from twenty two isolates based on their spectrum of activity against other lactic acid bacteria and pathogens. The eight lactobacilli were characterized based on the various classical phenotypic tests, physiological tests and biochemical tests including various carbohydrate utilization profiles. All isolates were homo fermentative, catalase, and gelatin negative. Molecular characterization was performed by RAPD, 16S rRNA analysis, 16S ARDRA, and Multiplex PCR for species identification. RAPD was carried out using the primer R2 and M13. Five different clusters were obtained based on RAPD indicating strain level variation. 16S rRNA analysis showed 99 to 100% homology towards Lactobacillus plantarum. The restriction digestion pattern was similar for all the isolates with the restriction enzyme AluI. The subspecies were identified by performing Multiplex PCR using species specific primer. Among the five clusters, three clusters were clearly identified as Lactobacillus plantarum subsp. plantarum, Lactobacillus pentosus, and Lactobacillus plantarum subsp. argentoratensis. <![CDATA[<b>Halotolerance, ligninase production and herbicide degradation ability of basidiomycetes strains</b>]]> Fungi have been recently recognized as organisms able to grow in presence of high salt concentration with halophilic and halotolerance properties and their ligninolytic enzyme complex have an unspecific action enabling their use to degradation of a number of xenobiotic compounds. In this work, both the effect of salt and polyols on growth of the basidiomycetes strains, on their ability to produce ligninolytic enzyme and diuron degradation were evaluated. Results showed that the presence of NaCl in the culture medium affected fungal specimens in different ways. Seven out of ten tested strains had growth inhibited by salt while Dacryopinax elegans SXS323, Polyporus sp MCA128 and Datronia stereoides MCA167 fungi exhibited higher biomass production in medium containing 0.5 and 0.6 mol.L-1 of NaCl, suggesting to be halotolerant. Polyols such as glycerol and mannitol added into the culture media improved the biomass and ligninases production by D. elegans but the fungus did not reveal consumption of these polyols from media. This fungus degraded diuron in medium control, in presence of NaCl as well as polyols, produced MnP, LiP and laccase. <![CDATA[<b>Expression of enterovirus 71 capsid protein VP1 in <i>Escherichia coli </i>and its clinical application</b>]]> The VPl gene of enterovirus 71 (EV71) was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16), A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25%) from CA16infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD). <![CDATA[<b>Prevalence of virulence genes in strains of <i>Campylobacter jejuni</i> isolated from human, bovine and broiler</b>]]> Campylobacter jejuni isolates of different origins (bovine, broiler meat, human) were screened by polymerase chain reaction for the presence of 4 genes cdtB, cst-II, ggt, and virB11, previously linked to virulence such as adherence, invasion, colonization, molecular mimicry, and cytotoxin production. In addition, the isolates were screened for the presence of the global gene regulator csrA linked to oxidative stress responses, biofilms formation, and cell adhesion. All the C. jejuni isolates were positive for cdtB gene. The csrA gene was detected in 100% and 92% of C. jejuni isolates from human and animal origin and the virB11 gene was detected in 7.3% and 3.6% isolates from chicken and human respectively. All isolates from bovine were negative for the virB11 gene. The isolates showed a wide variation for the presence of the remaining genes. Of the C. jejuni recovered from human 83.6%, and 32.7% were positive for cst-II, and ggt respectively. Out of the isolates from chicken 40% and 5.5% isolates revealed the presence of cst-II, and ggt, respectively. Finally of the C. jejuni isolates from bovine, 97.7% and 22.7% were positive for cst-II, and ggt respectively. We conclude that the genes of this study circulate among humans and animals. These results led us to hypothesize that the isolates associated with enteritis (cdtB positives) are not selected by environmental or host-specific factors. On the other hand, the high frequencies of csrA gene in C. jejuni show that this gene is important for the survival of C. jejuni in animals and humans. <![CDATA[<b>Histopathology of cotton bollworm midgut infected with <i>Helicoverpa armigera</i> cytoplasmic polyhedrosis virus</b>]]> This research was carried out to examine cytopathological effects of Helicoverpa armigera Cytoplasmic polyhedrosis virus (HaCPV) on infected midgut cotton bollworm (Helicoverpa armigera) using transmission and scanning electron microscope. The symptoms on infected host larvae of the host, compared with healthy ones, were getting swollen with milky-white and fragile Histopathological examinations showed infection with HaCPV small polyhedral inclusion bodies (PIB) after 1 or 2 days which were observed in columnar cells of midgut. Virions were partially or completely occupied in a polyhedral matrix to form polyhedral inclusion bodies (PIB) at periphery of virogenic stroma. PIBs were measured 0.5 to 3.5 mm and virions about 46 nm in diameter. Microvilli of infected columnar cells were affected and degenerated immediately prior to rupture of the cell. Some infected columnar cells ruptured to release PIB into the gut lumen 3 days after infection. In addition,PIB were found in goblet cells, 5 or 6 days after infection. Infected goblet cells degenerate to such an extent that only a few of the original microvillus-like cytoplasmic projections and cell organells were left. These cytopathic effects caused in the midgut by HaCPV on cotton bollworm larvae are essentially similar to those have been reported for lepidoperan and dipteran infection by CPV. <![CDATA[<b>Seroprevalence of Hepatitis B and C markers at the population level in the municipality of Caxias do Sul, southern Brazil</b>]]> Chronic viral hepatitis are main public health problems worldwide. Data about the seroprevalence to Hepatitis B and C viruses (HBV e HCV) at the population level are scarce on Brazil and especially for the Southern region of the country. The seroprevalence to HBV and HBC antigens was evaluated on a large portion of the population of the municipality of Caxias do Sul (427,858 inhabitants), Brazil. A total of 60,604 individual serum samples collected from 2008 to 2011 were screened for HBV surface antigen (HBsAg) and for antibodies against HBsAg; anti HCV antibodies were measured by ELISA (Enzyme Linked Immunosorbent Assay). Overall, 1.63% of the individuals were positive for HBsAg and 1.43% showed seropositivity to HCV. From the total, 31,749 samples were analyzed for HBsAg and 28,855 for HCV. For HBsAg, 519 samples showed positive (1.63%) while the results for 37 patients (0.12%) remained inconclusive. For the anti-HCV test, 412 individuals (1.43%) showed positive. From the positive samples for HBsAg 216 (50.6%) were from male individuals whereas for anti-HCV the seroprevalence was slightly higher for females 216 (52.4%). The higher prevalence for both hepatitis viruses were found among individuals at the age group of 40 to 59 years and the lower levels of positivity for both HBV and HBC were among children and teenagers. <![CDATA[<b><i>Cgl-SLT2</i> is required for appressorium formation, sporulation and pathogenicity in <i>Colletotrichum gloeosporioide</i></b>]]> The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity. <![CDATA[<b><i>Mycobacterium tuberculosis </i>belonging to family LAM and sublineage RD<sup>Rio</sup></b>: <b>common strains in Southern Brazil for over 10 years</b>]]> A sublineage of Mycobacterium tuberculosis called RD Rio was described in 2007. Although only recently described, this strain may have been present previously in the population, and its identification in clinical isolates will elucidate bacterial transmission dynamics and host-pathogen interactions. This study evaluated the clonal diversity of the RD Rio sublineage in clinical isolates from Rio Grande-RS obtained between 1998 and 2001. Among the 45 samples analyzed by the MIRU-VNTR method, there were six clusters with two samples each and 33 orphan strains with unique pattern. The strains were distributed across several different lineages including LAM (34.04%), X (14.89%), Haarlem (12.77%), UgandaI (10.64%), S (4.26%), NEW-1 (2.13%) and Cameroon (2.13%); 14.89% of the strains matched to multiple lineages. RD Rio strains were present in 28.9% of the samples and 81.25% of the identified strains belonged to the LAM family. The high clonal diversity observed in this study is a constant feature in this region. The RD Rio sublineage has been in Rio Grande-RS since 1998. The continued monitoring of RD Rio in clinical isolates will enhance the understanding of its epidemiological significance. <![CDATA[<b>Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species</b>]]> Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods. <![CDATA[<b>Investigation of the association between clinical outcome and the cag pathogenicity-island and other virulence genes of <i>Helicobacter pylori</i> isolates from patients with dyspepsia in Eastern Turkey</b>]]> The aims of our work were to determine the presence of the cag pathogenicity-island (cag PAI) and other virulence genes of Helicobacter pylori recovered from patients with gastritis and peptic ulcer, and to investigate the correlation of these virulence genes with clinical outcome. The presence of the cagA, the promoter regions of cagA, cagE, cagT, and the left end of cag-PAI (LEC), cag right junction (cagRJ), the plasticity region open reading frames (ORFs), vacA and oipA genes among 69 H. pylori isolates were determined by polymerase chain reaction. Intact cag PAI was detected in only one (1.4%) isolate. The cagA gene was identified in 52.1% and 76.2% of isolates from patients with dyspepsia (gastritis and peptic ulcer), respectively. The plasticity region ORFs i.e. JHP912 and JHP931 were predominantly detected in isolates from peptic ulcer. Less than 25% of the isolates carried other ORFs. Types I, II and III were the most commonly found among the isolates. None of the isolates possessed type Ib, 1c, IIIb, IV and V motifs. The most commonly vacA genotypes were s1am1a and s1m2 in isolates with peptic ulcer and gastritis, respectively. The results confirmed that the prevalence of oipA (Hp0638) gene was 75% and 85.7% in patients with gastritis and peptic ulcer, respectively. Furthermore, vacA s1am1a positivity was significantly related to peptic ulcer (p < 0.05). <![CDATA[<b>Antifungal activity of extracts from <i>Piper aduncum</i> leaves prepared by different solvents and extraction techniques against dermatophytes <i>Trichophyton rubrum</i> and <i>Trichophyton interdigitale</i></b>]]> The effects of different solvents and extraction techniques upon the phytochemical profile and anti-Trichophyton activity of extracts from Piper aduncum leaves were evaluated. Extract done by maceration method with ethanol has higher content of sesquiterpenes and antifungal activity. This extract may be useful as an alternative treatment for dermatophytosis. <![CDATA[<b>Characterization of mercury-resistant clinical <i>Aeromonas</i> species</b>]]> Mercury-resistant Aeromonas strains isolated from diarrhea were studied. Resistance occurs via mercuric ion reduction but merA and merR genes were only detected in some strains using PCR and dot hybridization. Results indicate a high variability in mer operons in Aeromonas. To our knowledge, this is the first report of mercury-resistant clinical Aeromonas strains. <![CDATA[<b>Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis</b>]]> Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(-)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound. <![CDATA[<b>Interaction of antimicrobial peptide Plantaricin149a and four analogs with lipid bilayers and bacterial membranes</b>]]> The amidated analog of Plantaricin149, an antimicrobial peptide from Lactobacillus plantarum NRIC 149, directly interacts with negatively charged liposomes and bacterial membranes, leading to their lysis. In this study, four Pln149-analogs were synthesized with different hydrophobic groups at their N-terminus with the goal of evaluating the effect of the modifications at this region in the peptide's antimicrobial properties. The interaction of these peptides with membrane models, surface activity, their hemolytic effect on red blood cells, and antibacterial activity against microorganisms were evaluated. The analogs presented similar action of Plantaricin149a; three of them with no hemolytic effect (< 5%) until 0.5 mM, in addition to the induction of a helical element when binding to negative liposomes. The N-terminus difference between the analogs and Plantaricin149a retained the antibacterial effect on S. aureus and P. aeruginosa for all peptides (MIC50 of 19 µM and 155 µM to Plantaricin149a, respectively) but resulted in a different mechanism of action against the microorganisms, that was bactericidal for Plantaricin149a and bacteriostatic for the analogs. This difference was confirmed by a reduction in leakage action for the analogs. The lytic activity of Plantaricin149a is suggested to be a result of the peptide-lipid interactions from the amphipathic helix and the hydrophobic residues at the N-terminus of the antimicrobial peptide. <![CDATA[<b>Extracellular proteases of <i>Halobacillus blutaparonensis </i>strain M9, a new moderately halophilic bacterium</b>]]> Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties. <![CDATA[<b>An oxidant and organic solvent tolerant alkaline lipase by <i>P. aeruginosa</i> mutant</b>: <b>downstream processing and biochemical characterization</b>]]> An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45°C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca2+, Mg2+ and Na+ tend to increase the enzyme activity, whereas, Fe2+ and Mn2+ ions resulted in discreet decrease in the activity. Divalent cations Ca+2 and Mg+2 seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca+2 (5 mM) the optimum temperature shifted from 45°C to 55°C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of Km and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL-1 and 5000 μg.mL-1.min-1, respectively. <![CDATA[<b>Effect of alcohols on filamentation, growth, viability and biofilm development in <i>Candida albicans</i></b>]]> In this study we report the potential of alcohols as morphogenetic regulators in Candida albicans. All the alcohols tested influenced various modes of growth like planktonic as well as biofilm forms. Viability was affected at high concentrations. Among the alcohols, the response of C. albicans to amyl alcohol (pentanol) was noteworthy. Amyl alcohol at a concentration 0.5% which was not inhibitory to growth and viability specifically inhibited morphogenetic switching from yeast to hyphal forms. It also inhibited normal biofilm development favoring yeast dominated biofilms. Based on this study we hypothesize that alcohols produced under anaerobic conditions may not favor biofilm development and support dissemination of yeast cells. Since anaerobic conditions are not found to favor production of quorum sensing molecules like farnesol, the alcohols may play a role in morphogenetic regulation. <![CDATA[<b>Microbiota and anthropic interference on antimicrobial resistance profile of bacteria isolated from Brazilian maned-wolf (<i>Chrysocyon brachyurus</i>)</b>]]> Both the study of Brazilian wild mammal fauna and the conditions that foster the preservation of endangered species, such as Brazilian Maned-wolf (Chrysocyon brachyurus), in wild life are of extreme importance. In order to study the resistance profile of microbiota bacterial colonizing Brazilian Maned-wolf, this work investigated samples from eight male captive and free roaming animals originating from different Brazilian geographical regions. Samples for microbiological purposes were collected with swabs and kept in appropriate transport medium. Using routine microbiological techniques, the isolated bacteria were tested toward antimicrobial drugs by the agar disk diffusion method. Results showed that all samples from wild animals were sensitive toward all drugs tested. Conversely, the resistance profile of bacteria isolated from captive animals varied among strains and animal body site location. Escherichia coli samples from prepuce, anus and ear showed multi-resistance toward at least four drugs, especially against erythromycin and tetracycline, followed by Proteus mirabilis and P. vulgaris strains isolated from anus and ear. Among Gram-positive bacteria, strains of coagulase-negative staphylococci showed multi-resistance mainly toward erythromycin and amoxicillin. The work discusses these findings and suggests that profile of multi-resistance bacteria from captive subjects may be attributed to direct contact with human or through lifestyle factors such as feeding, predation or contact of animals with urban animals such as birds, rodents, and insects from surrounding environments. <![CDATA[<b>Prevalence of antibodies against <i>Toxoplasma gondii</i> and <i>Neospora caninum</i> in dogs</b>]]> Toxoplasmosis and neosporosis are parasitic diseases of global importance. The present study had the objective to determine the influence of age, sex and breed in the prevalence of antibodies against both diseases in dogs from Brotas city, São Paulo State, Brazil. Blood samples of 342 dogs were collected, and the age, sex and breed of each animal were recorded. The serological diagnosis for toxoplasmosis and neosporosis were performed using the immunofluorescent antibody test (IFAT). The Fischer's test was used to calculate the association probability of the variables, with a = 5%. For toxoplasmosis the prevalence of antibodies was 26.9% (CI 95% 22.4-31.8%), and for neosporosis 4.97% (CI 95% 3.1-7.8%). The statistical analysis revealed a higher risk of infection for T. gondii in dogs with more than five years. <![CDATA[<b><i>Methylobacterium</i>-plant interaction genes regulated by plant exudate and quorum sensing molecules</b>]]> Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction. <![CDATA[<b>Effectiveness of halo-tolerant, auxin producing <i>Pseudomonas</i> and <i>Rhizobium</i> strains to improve osmotic stress tolerance in mung bean (<i>Vigna radiata</i> L</b><b>.</b><b>)</b>]]> Halo-tolerant, auxin producing bacteria could be used to induce salt tolerance in plants. A number of Rhizobium and auxin producing rhizobacterial strains were assessed for their ability to tolerate salt stress by conducting osmoadaptation assay. The selected strains were further screened for their ability to induce osmotic stress tolerance in mung bean seedlings under salt-stressed axenic conditions in growth pouch/jar trials. Three most effective strains of Rhizobium and Pseudomonas containing ACC-deaminase were evaluated in combination, for their ability to induce osmotic stress tolerance in mung bean at original, 4, and 6 dS m-1 under axenic conditions. Results showed that sole inoculation of Rhizobium and Pseudomonas strains improved the total dry matter up to 1.4, and 1.9 fold, respectively, while the increase in salt tolerance index was improved up to 1.3 and 2.0 fold by the Rhizobium and Pseudomonas strains, respectively. However, up to 2.2 fold increase in total dry matter and salt tolerance index was observed due to combined inoculation of Rhizobium and Pseudomonas strains. So, combined application of Rhizobium and Pseudomonas strains could be explored as an effective strategy to induce osmotic stress tolerance in mung bean. <![CDATA[<b>Yearly variation of bacterial production in the Arraial do Cabo protection area (Cabo Frio upwelling region)</b>: <b>an evidence of anthropogenic pressure</b>]]> Arraial do Cabo is where upwelling occurs more intensively on the Brazilian coast. Although it is a protection area it suffers anthropogenic pressure such as harbor activities and sporadic sewage emissions. Short-time studies showed a high variability of bacterial production (BP) in this region but none of them evaluated BP during long periods in a large spatial scale including stations under different natural (upwelling and cold fronts) and anthropogenic pressures. During 2006, we sampled surface waters 10 times (5 in upwelling and 5 in subsidence periods) in 8 stations and we measured BP, temperature as well as the concentrations of inorganic nutrients, pigments and particulate organic matter (POM). BP was up to 400 times higher when sewage emissions were observed visually and it had a positive correlation with ammonia concentrations. Therefore, in 2007, we did two samples (each during upwelling and subsidence periods) during sewage emissions in five stations under different anthropogenic pressure and we also measured particles abundance by flow cytometry. The 12 samples in the most impacted area confirmed that BP was highest when ammonia was higher than 2 μM, also reporting the highest concentrations of chlorophyll a and suspended particles. However, considering all measured variables, upwelling was the main disturbing factor but the pressure of fronts should not be neglected since it had consequences in the auto-heterotrophic coupling, increasing the concentrations of non fluorescent particles and POM. Stations clustered in function of natural and anthropogenic pressures degrees and both determined the temporal-spatial variability.