PROGRESSO NA SELE����O DE PROG��NIES DE TOMATE RESISTENTES �� MURCHA BACTERIANA ATRAV��S DA AVALIA����O EPIDEMIOL��GICA DA DOEN��A

Dados obtidos em experimentos realizados durante a d��cada 83/92 pelo Programa de Melhoramento Gen��tico do Tomateiro foram utilizados para avaliar a efici��ncia da utiliza����o do par��metro epidemiol��gico Taxa de Infec����o para sele����o de prog��nies resistentes ao pat��geno Pseudomonas solanacearum. Foram obtidas evid��ncias que as prog��nies selecionadas de uma gera����o para a seguinte apresentaram n��veis crescentes de resist��ncia e maior capacidade produtiva sob condi����o de cultivo em solos infestados pelo pat��geno, usando-se como padr��es de susceptibilidade variedades do grupo Santa Cruz e de resist��ncia a variedade Caraiba. A Taxa de Infec����o consegue detectar elevado n��mero de contrastes significativos quando comparada com outros par��metros que n��o consideram a resist��ncia como resultante de um processo na qual a express��o �� modificada com o tempo.

Popula����o microbiana em solo cultivado com soja e tratado com diferentes herbicidas em ��rea de cerrado no estado de Roraima

Este trabalho objetivou avaliar o impacto de herbicidas �� base de glyphosate, imazaquin e trifluralin na biomassa microbiana do solo, na comunidade bacteriana associada ao rizoplano de soja e tamb��m na nodula����o das plantas de soja. As avalia����es foram realizadas por um per��odo de 60 dias, em dois sistemas de manejo do solo: semeadura direta na palha (SD) e semeadura convencional (SC), que receberam a aplica����o dos herbicidas glyphosate e, imazaquin e trifluralin, respectivamente. Ao longo do per��odo estudado o imazaquin, na ��rea de SD, ocasionou redu����o da biomassa microbiana e, tamb��m alterou o perfil bacteriano analisado por eletroforese em gel com gradiente desnaturante (DGGE) de forma mais intensa, que o glyphosate. Na ��rea de SC n��o houve efeito significativo dos herbicidas sobre a biomassa microbiana, tendo ocorrido grande variabilidade entre repeti����es de um mesmo tratamento nos perfis de DGGE, o que dificultou a observa����o do efeito dos herbicidas. O seq��enciamento de fragmentos do 16S rDNA retirados dos g��is de DGGE mostrou que o glyphosate restringiu o desenvolvimento de uma bact��ria com 90% de homologia com Herbaspirillum sp., enquanto, o imazaquin estimulou uma bact��ria com 96% de homologia com Ralstonia sp. e, outras bact��rias com pelo menos 92% de homologia com Burkholderia, Thiomonas e Pseudomonas n��o foram afetadas. Tamb��m n��o houve efeito dos herbicidas sobre o n��mero de n��dulos nas plantas de soja.

Deteriora����o bacteriol��gica do jaraqui Semaprochilodus spp. capturado no estado do Amazonas e conservado em gelo

Neste trabalho foram analisados 73 esp��cimes de jaraqui Semaprochilodus spp. conservados em caixas de poliestireno expandido entre camadas de gelo. Foram realizadas as seguintes an��lises: avalia����o sensorial pela tabela de Torry modificada e pelo ��ndice de qualidade por dem��ritos; determina����o do pH e das bases vol��teis totais (N-BVT); contagem total dos microrganismos aer��bios psicr��filos a 20 ��C por 4 dias, psicrotr��ficos a 7 ��C por 10 dias, dos mes��filos a 37 ��C por 2 dias; contagem, isolamento e identifica����o das bact��rias Aeromonas sp. Bacillus sp. e Pseudomonas sp. a 20 ��C por 24 horas e de Plesiomonas sp. a 37 ��C por 2 dias. O jaraqui se manteve em condi����es de consumo, pela avalia����o sensorial, por 18 e 21 dias. O pH e as bases vol��teis totais n��o foram bons indicadores de qualidade; as contagens totais de psicr��filos, psicrotr��ficos e mes��filos n��o apresentaram diferen��a significativa e as bact��rias n��o apresentaram comportamento deteriorador pela aus��ncia da produ����o de H2S.

Engineering modular viral scaffolds for targeted bacterial population editing

Bacteria are central to human health and disease, but��existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to precisely manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E.��coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision this approach accelerating phage biology studies and enabling new technologies for bacterial population editing.

Antimicrobial activity of bergenin from Endopleura uchi (Huber) Cuatrec

Endopleura uchi (Huber) Cuatrec. is an Amazon species traditionally used as treatment for inflammations and female disorders. Bergenin was isolated from ethyl acetate fraction of bark of E. uchi by using column chromatography over sephadex LH-20 and then silica gel 60 flash. Its structure was identified on the basis of its NMR spectra. The antimicrobial activity of bergenin and fractions of methanol extract of E. uchi were evaluated against ATCC microorganisms (Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Candida albicans, C. guilliermondii, Aspergillus flavus, A. nidulans). Clinically isolated strains of all of these microorganisms, along with C. tropicalis, A. niger, Shigella sonnei, Serratia marcenses and Klebsiella pneumoniae were also evaluated. The growth inhibition caused by bergenin, extracts and fractions of E. uchi against ATCC microorganisms were similar to the inhibition to microorganisms clinically isolated. The ethyl acetate fraction and the isolate bergenin inhibit the growth of the yeasts C. albicans, C. tropicalis, and C. guilliermondii, but present lower activity against filamentous fungi Aspergillus flavus, A. nidulans, A. niger, and did not inhibit the Gram positive and Gram negative bacteria. The activity of the ethyl acetate fraction and bergenin are in agreement wit its high concentration found in bark extract of E. uchi. Moreover, the selective activity against three Candida species helps to understand its traditional use against infections that affect women.

Data quality in biofilm high-throughput routine analysis: intralaboratory protocol adaptation and experiment reproducibility

Biofilm research is growing more diverse and dependent on high-throughput technologies and the large-scale production of results aggravates data substantiation. In particular, it is often the case that experimental protocols are adapted to meet the needs of a particular laboratory and no statistical validation of the modified method is provided. This paper discusses the impact of intra-laboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development, considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the XTT assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non-rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intra-laboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.

Comparison of oleate conversion under microaerophilic and anaerobic conditions

Excessive accumulation of Long Chain Fatty Acids (LCFA) in methanogenic bioreactors is the cause of process failure associated to a severe decrease in methane production. In particular, fast and persistent accumulation of palmitate is critical and still not elucidated. Aerobes or facultative anaerobes were detected in those reactors, raising new questions on LCFA biodegradation. To get insight into the influence of oxygen, two bioreactors were operated under microaerophilic and anaerobic conditions, with oleate at 1 and 4 gCOD/(L d). Palmitate accumulated up to 2 and 16 gCOD/L in the anaerobic and microaerophilic reactor, respectively, which shows the importance of oxygen in this conversion. A second experiment was designed to understand the dynamics of oleate to palmitate conversion. A CSTR and a PFR were assembled in series and fed with oleate under microaerophilic conditions. HRT from 6 to 24 h were applied in the CSTR, and 14 to 52 min in the PFR. In the PFR a biofilm was formed where palmitate accounted for 82% of total LCFA. Pseudomonas was the predominant genus (42 %) in this biofilm, highlighting the role of aerobic and facultative anaerobic bacteria in LCFA bioconversion.

Strategies to combat infections of Acinetobacter baumannii biofilms

Disserta����o de mestrado integrado em Engenharia Biom��dica (��rea de especializa����o em Engenharia Cl��nica)

Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region

ABSTRACT Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

PEGylated ofloxacin nanoparticles render strong antibacterial activity against many clinically important human pathogens.

The rise of bacterial resistance against important drugs threatens their clinical utility. Fluoroquinones, one of the most important classes of contemporary antibiotics has also reported to suffer bacterial resistance. Since the general mechanism of bacterial resistance against fluoroquinone antibiotics (e.g. ofloxacin) consists of target mutations resulting in reduced membrane permeability and increased efflux by the bacteria, strategies that could increase bacterial uptake and reduce efflux of the drug would provide effective treatment. In the present study, we have compared the efficiencies of ofloxacin delivered in the form of free drug (OFX) and as nanoparticles on bacterial uptake and antibacterial activity. Although both poly(lactic-co-glycolic acid) (OFX-PLGA) and methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (OFX-mPEG-PLGA) nanoformulations presented improved bacterial uptake and antibacterial activity against all the tested human bacterial pathogens, namely, Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, OFX-mPEG-PLGA showed significantly higher bacterial uptake and antibacterial activity compared to OFX-PLGA. We have also found that mPEG-PLGA nanoencapsulation could significantly inhibit Bacillus subtilis resistance development against OFX.

Hydrocarbonoclastic bacteria: from bioremediation to bioenergy feedstock

Tese de Doutoramento em Engenharia Qu��mica e Biol��gica.

Bacteriophage-encoded depolymerases: their diversity and biotechnological applications

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Valorization of agro-industrial wastes towards the production of rhamnolipids

In this work, oil mill wastewater (OMW), a residue generated during olive oil extraction, was evaluated as an inducer of rhamnolipid production. Using a medium containing as sole ingredients corn steep liquor (10%, v/v), sugarcane molasses (10%, w/v) and OMW (25%, v/v), Pseudomonas aeruginosa #112 produced 4.5 and 5.1 g of rhamnolipid per liter in flasks and reactor, respectively, with critical micelle concentrations as low as 13 mg/l. Furthermore, in the medium supplemented with OMW, a higher proportion of more hydrophobic rhamnolipid congeners was observed comparing with the same medium without OMW. OMW is a hazardous waste which disposal represents a serious environmental problem; therefore, its valorization as a substrate for the production of added-value compounds such as rhamnolipids is of great interest. This is the first report of rhamnolipid production using a mixture of these three agro-industrial by-products, which can be useful for the sustainable production of rhamnolipids.

Vermiculite bio-barriers for Cu and Zn remediation: an eco-friendly approach for freshwater and sediments protection

The increase in heavy metal contamination in freshwater systems causes serious environmental problems in most industrialized countries, and the effort to ���nd ecofriendly techniques for reducing water and sediment contamination is fundamental for environmental protection. Permeable barriers made of natural clays can be used as low-cost and eco-friendly materials for adsorbing heavy metals from water solution and thus reducing the sediment contamination. This study discusses the application of permeable barriers made of vermiculite clay for heavy metals remediation at the interface between water and sediments and investigates the possibility to increase their ef���ciency by loading the vermiculite surface with a microbial bio���lm of Pseudomonas putida, which is well known to be a heavy metal accumulator. Some batch assays were performed to verify the uptake capacity of two systems and their adsorption kinetics, and the results indicated that the vermiculite bio-barrier system had a higher removal capacity than the vermiculite barrier (?34.4 and 22.8 % for Cu and Zn, respectively). Moreover, the presence of P. putida bio���lm strongly contributed to fasten the kinetics of metals adsorption onto vermiculite sheets. In open-system conditions, the presence of a vermiculite barrier at the interface between water and sediment could reduce the sediment contamination up to 20 and 23 % for Cu and Zn, respectively, highlighting the ef���ciency of these eco-friendly materials for environmental applications. Nevertheless, the contribution of microbial bio���lm in open-system setup should be optimized, and some important considerations about bio���lm attachment in a continuous-���ow system have been discussed.