Bacterial soil community in a Brazilian sugarcane field

The assessment of bacterial communities in soil gives insight into microbial behavior under prevailing environmental conditions. In this context, we assessed the composition of soil bacterial communities in a Brazilian sugarcane experimental field. The experimental design encompassed plots containing common sugarcane (variety SP80-1842) and its transgenic form (IMI-1 - imazapyr herbicide resistant). Plants were grown in such field plots in a completely randomized design with three treatments, which addressed the factors transgene and imazapyr herbicide application. Soil samples were taken at three developmental stages during plant growth and analyzed using 16S ribosomal RNA (rRNA)-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries. PCR-DGGE fingerprints obtained for the total bacterial community and specific bacterial groups - Actinobacteria, Alphaproteobacteria and Betaproteobacteria - revealed that the structure of these assemblages did not differ over time and among treatments. Nevertheless, slight differences among 16S rRNA gene clone libraries constructed from each treatment could be observed at particular cut-off levels. Altogether, the libraries encompassed a total of eleven bacterial phyla and the candidate divisions TM7 and OP10. Clone sequences affiliated with the Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria were, in this order, most abundant. Accurate phylogenetic analyses were performed for the phyla Acidobacteria and Verrucomicrobia, revealing the structures of these groups, which are still poorly understood as to their importance for soil functioning and sustainability under agricultural practices.

Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

Endophytic Colonization of Potato (Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte, Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.

Bacterial community in the rhizosphere and rhizoplane of wild type and transgenic eucalyptus

The rhizosphere is a niche exploited by a wide variety of bacteria. The expression of heterologous genes by plants might become a factor affecting the structure of bacterial communities in the rhizosphere. In a greenhouse experiment, the bacterial community associated to transgenic eucalyptus, carrying the Lhcb1-2 genes from pea (responsible for a higher photosynthetic capacity), was evaluated. The culturable bacterial community associated to transgenic and wild type plants were not different in density, and the Amplified Ribosomal DNA Restriction Analysis (ARDRA) typing of 124 strains revealed dominant ribotypes representing the bacterial orders Burkholderiales, Rhizobiales, and Actinomycetales, the families Xanthomonadaceae, and Bacillaceae, and the genus Mycobacterium. Principal Component Analysis based on the fingerprints obtained by culture-independent Denaturing Gradient Gel Electrophoresis analysis revealed that Alphaproteobacteria, Betaproteobacteria and Actinobacteria communities responded differently to plant genotypes. Similar effects for the cultivation of transgenic eucalyptus to those observed when two genotype-distinct wild type plants are compared.

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.

The bacterial diversity in a Brazilian non-disturbed mangrove sediment

The bacterial diversity present in sediments of a well-preserved mangrove in Ilha do Cardoso, located in the extreme south of So Paulo State coastline, Brazil, was assessed using culture-independent molecular approaches (denaturing gradient gel electrophoresis (DGGE) and analysis of 166 sequences from a clone library). The data revealed a bacterial community dominated by Alphaproteobacteria (40.36% of clones), Gammaproteobacteria (19.28% of clones) and Acidobacteria (27.71% of clones), while minor components of the assemblage were affiliated to Betaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The clustering and redundancy analysis (RDA) based on DGGE were used to determine factors that modulate the diversity of bacterial communities in mangroves, such as depth, seasonal fluctuations, and locations over a transect area from the sea to the land. Profiles of specific DGGE gels showed that both dominant (`universal` Bacteria and Alphaproteobacteria) and low-density bacterial communities (Betaproteobacteria and Actinobacteria) are responsive to shifts in environmental factors. The location within the mangrove was determinant for all fractions of the community studied, whereas season was significant for Bacteria, Alphaproteobacteria, and Betaproteobacteria and sample depth determined the diversity of Alphaproteobacteria and Actinobacteria.

Bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria

Copper contaminated areas pose environmental health risk to living organisms. Remediation processes are thus required for both crop production and industrial activities. This study employed bioaugmentation with copper resistant bacteria to improve phytoremediation of vineyard soils and copper mining waste contaminated with high copper concentrations. Oatmeal plant (Avena sativa L) was used for copper phytoextraction. Three copper resistant bacterial isolates from oatmeal rhizosphere (Pseudomonas putida A1 Stenotrophomonas maltophilia A2 and Acinetobacter calcoaceticus A6) were used for the stimulation of copper phytoextraction. Two long-term copper contaminated vineyard soils (Mollisol and Inceptisol) and copper mining waste from Southern Brazil were evaluated. Oatmeal plants substantially extracted copper from vineyard soils and copper mining waste. As much as 1549 mg of Cu kg(-1) dry mass was extracted from plants grown in Inceptisol soil. The vineyard Mollisol copper uptake (55 mg Cu kg(-1) of dry mass) in the shoots was significantly improved upon inoculation of oatmeal plants with isolate A2 (128 mg of Cu kg(-1) of shoot dry mass). Overall oatmeal plant biomass displayed higher potential of copper phytoextraction with inoculation of rhizosphere bacteria in vineyard soil to the extent that 404 and 327 g ha(-1) of copper removal were respectively observed in vineyard Mollisol bioaugmented with isolate A2 (S. maltophilia) and isolate A6 (A. calcoaceticus). Results suggest potential application of bacterial stimulation of phytoaccumulation of copper for biological removal of copper from contaminated areas. (C) 2010 Elsevier Ltd. All rights reserved.

The relationship of flow cytometry results with classical measures of bacterial counts in raw refrigerated milk

Bulk milk was collected from 100 farms throughout the year and analysed after storage for either 24, 48 or 72 h, using flow cytometry. The total bacterial counts obtained by two methods - flow cytometry and standard plate count were compared and the conversion relationship between them was assessed: the results showed no effect of the age of the samples relationship between these two methods.

Bacterial communities and biogeochemical transformations of iron and sulfur in a high saltmarsh soil profile

Microbial community structure in saltmarsh soils is stratified by depth and availability of electron acceptors for respiration. However, the majority of the microbial species that are involved in the biogeochemical transformations of iron (Fe) and sulfur (S) in such environments are not known. Here we examined the structure of bacterial communities in a high saltmarsh soil profile and discuss their potential relationship with the geochemistry of Fe and S. Our data showed that the soil horizons Ag (oxic-suboxic), Bg (suboxic), Cri (anoxic with low concentration of pyrite Fe) and Cr-2 (anoxic with high concentrations of pyrite Fe) have distinct geochemical and microbiological characteristics. In general, total S concentration increased with depth and was correlated with the presence of pyrite Fe. Soluble + exchangable-Fe, pyrite Fe and acid volatile sulfide Fe concentrations also increased with depth, whereas ascorbate extractable-Fe concentrations decreased. The occurrence of reduced forms of Fe in the horizon Ag and oxidized Fe in horizon Cr-2 suggests that the typical redox zonation, common to several marine sediments, does not occur in the saltmarsh soil profile studied. Overall, the bacterial community structure in the horizon Ag and Cr-2 shared low levels of similarity, as compared to their adjacent horizons, Bg and Cr-1, respectively. The phylogenetic analyses of bacterial 16S rRNA gene sequences from clone libraries showed that the predominant phylotypes in horizon Ag were related to Alphaproteobacteria and Bacteroidetes. In contrast, the most abundant phylotypes in horizon Cr-2 were related to Deltaproteo-bacteria, Chloroflexi, Deferribacteres and Nitrospira. The high frequency of sequences with low levels of similarity to known bacterial species in horizons Ag and Cr-2 indicates that the bacterial communities in both horizons are dominated by novel bacterial species. (c) 2008 Elsevier Ltd. All rights reserved.

Increased electronegative LDL and decreased antibodies against electronegative LDL levels correlate with inflammatory markers and adhesion molecules in hemodialysed patients

Background: Chronic Kidney Disease (CKD) patients present high levels of electronegative LDL (LDL) that can modulate the expression of molecules involved in inflammation and it is closely linked to atherosclerosis. We investigated the association between LDL(-) and inflammatory markers in patients undergoing hemodialysis (HD). Methods: Forty-seven HD patients from a private clinic in Rio de Janeiro, Brazil were studied and compared with 20 age matched healthy individuals. Serum LDL(-) and anti-LDL(-) autoantibody levels were measured by ELISA; TNF-alpha, IL-6, VCAM-1 and ICAM-1 were determined by a multiplex assay kit. Results: HD patients presented higher IL-6 and TNF-alpha concentrations (4.1 +/- 1.6 and 5.5 +/- 2.1 pg/ml, respectively) than healthy subjects (2.6 +/- 0.2 and 2.4 +/- 1.1 pg/ml, respectively) (p = 0.0001). In addition, they presented higher VCAM-1 and ICAM-1 levels and, LDL(-) concentrations were also increased (0.18 +/- 0.12 U/I) when compared to healthy individuals (0.10 +/- 0.08 U/I) (p<0.02). In contrast, the anti-LDL(-) autoantibody levels were lower in HD patients (0.02 +/- 0.01 mg/l) than in healthy subjects (0.05 +/- 0.03 mg/l) (p<0.001). There was a positive correlation between LDL(-) and IL-6 (r = 0.25, p = 0.004) and ICAM-1 (r = 0.36; p = 0.003). There was also a negative correlation between anti-LDL(-) autoantibodies and TNF-alpha (r = -0.37; p = 0.003) and VCAM-1 (r = -0.50; p = 0.0001). Conclusions: The association between LDL(-) and inflammation and the lower levels of anti-LDL(-) autoantibodies are important risk factors related to atherosclerosis in CKD. (C) 2011 Published by Elsevier B.V.

Enhanced fibroblast adhesion and proliferation on electrospun fibers obtained from poly(isosorbide succinate-b-L-lactide) block copolymers

Block copolymers containing isosorbide succinate and L-lactic acid repeating units with different mass compositions were synthesized in two steps: bulk ring-opening copolymerization from L-lactide and poli(isosorbide succinate) (PIS) preoligomer, in the presence of tin(II) 2-ethylhexanoate as catalyst. followed by chain extension in solution by using hexamethylene diisocyanate. Poly(L-lactide) (PLLA) and a chain extension product from PIS were also obtained, for comparison. SEC, (1)H and (13)C NMR, MALDI-TOFMS, WAXD, DSC, TG, and contact angle measurements were used in their characterization. The incorporation of isosorbide succinate into PLLA main backbone had minor effect on the thermal stability and the T(g) of the products. However, it reduced the crystallinity and increased the surface energy in relation to PLLA. Nonwoven mats of the block copolymers and PLLA obtained by electrospinning technique were submitted to fibroblasts 3T3-L1 cell culture. The copolymers presented enhanced cell adhesion and proliferation rate as revealed by MTT assay and SEM images. (C) 2009 Elsevier Ltd. All rights reserved.

Cytotoxicity and Endothelial Cell Adhesion of Lyophilized and Irradiated Bovine Pericardium Modified With Silk Fibroin and Chitosan

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40-CD40L system

The vascular effects of nitrolinoleate (LNO(2)), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO(2) was capable to deliver free radical nitric oxide ((center dot)NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO(2) for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO(2) were observed in vivo by intravital microscopy assays. LNO(2) decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO(2) reduced mRNA and protein expression of 2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO(2) on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO(2) involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO(2) inhibited adhesion molecules expression and promoted (center dot)NO inactivation of the CD40-CD40L system, both important processes of the inflammatory response. (C) 2010 Elsevier Inc. All rights reserved.

Effect of process variables on fluiddynamics and adhesion efficiency during spouted bed coating of hard gelatine capsules

This article presents an evaluation of the effects of the spouted bed design and operating conditions on system fluiddynamics and process performance during enteric coating of hard gelatine capsules. The design parameters studied were the column diameter (150 mm and 200 mm), the included angle of the conical base, gamma (60 degrees or 40 degrees) and the presence or absence of a Venturi inserted before the inlet air orifice. The process variables studied were the ratio between the feed flow rate of the coating suspension to the spouting gas flow rate (W(s)/W(g)), the mass of capsules loaded to the equipment (M(0)), and the ratio between the Spouting gas flow rate to the gas flow rate at minimum spouting condition (Q/Q(ms)). The response variables were the rate of increase of the capsules mass (K(1)), and the adhesion efficiency (eta). The linear regression equation for the dependent variable K, in terms of the independent variables adequately described the process with an r(2) value of 0.872. Analysis of variance (ANOVA) revealed that increasing of W(s)/W(g), Q/Q(ms) and gamma significantly increased the adhesion efficiency. Adhesion efficiencies higher than 90% were achieved by selecting precise coating conditions, indicating the feasibility of the process for coating of hard gelatine capsules. (C) 2008 Elsevier B.V. All rights reserved.

Essential Oil of Thymus vulgaris: Preparation of Pharmaceutical Mouthwash Formulation and In Vitro Evaluation of the Bacterial Plaque-Inhibiting Properties.

Essential Oil of Thymus vulgaris: Preparation of Pharmaceutical Mouthwash Formulation and In Vitro Evaluation of the Bacterial Plaque-Inhibiting Properties. The aim of this study was to evaluate the in vitro effect of the essential oil of Thymus vulgaris (thyme) pure or incorporate in a alcohol-free pharmaceutical mouthwash formulation, against Streptococcus mutans (ATCC 25175), being determined the Minimal Inhibitory Concentration (MIC) and the effect in the bacterial plate formation. The MIC value obtained for the essential oil was 100 mu g/mL (1 %). The mouthwash pharmaceutical formulation containing commercial essential oil of T. vulgaris was preparated. Microbiological and macroscopic analysis as well as analyses for MEV confirmed the effectiveness of this new alcohol-free mouthwash formulation containing essential oil of T. vulgaris as agent with plaque-inhibiting properties and possible application in the preventive dentistry. The chemical characterization of the bioactive essential oil was accomplished by CG-MS, being verified the presence of carvacrol, p-cimene and alpha-pinene as major constituents.