Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

ABSTRACT Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Three-dimensional characteristics of bacteriae in the filiform and fungiform papillae of rat: a TEM and HRSEM study / Características tridimensionales de bacterias en las papilas filiformesy fungiformes de rata: un estudio en MET y ARMEB

Tongue mucosae of one day postnatal rat was examined by transmission electron microscopic and HRSEMmethods.The specimens were fixed using Karnovsky solution and embedded in Spurr resin for transmission electron microscopy. For HRSEM methods, the samples were fixed in 2 percent osmiun tetroxide, dehydrated in alcohol, critical point dried and coated with gold-palladium. The results demonstrated that the surface of tongue present the filiform and fungiform papillae covered by numerous keratinized epithelial cells. The bacteriae are attached to the surfaces of microplicae at random, demonstrating in their three-dimensional HRSEM images. At high magnification, the transmission electron microscopic images revealed the adhesion of bacteriae to the cell membrane by glycocalyx. The fibrillar structures surrounding the bacteriae are clearly seen.

Un día después del nacimiento, la mucosa de la lengua una rata fue examinada por el microscopio electrónico de transmisión y método de ARMEB. Los especímenes fueron fijados mediante uan solución Karnovsky y embebido en resina Spurr para microscopía electrónica de transmisión. Para el método ARMEB, las muestras fueron fijadas en tetróxido de osmio 2 por ciento, deshidratados en alcohol, secados al punto crítico y recubierto con oro-paladio. Los resultados demostraron que la superficie de la lengua presentaba papilas filiformes y fungiformes cubiertas por numerosas células epiteliales queratinizadas. Las bacterias se unen a las superficies de las microplicas al azar, lo que se demuestra en sus tres dimensiones las imágenes en ARMEB. A gran aumento, las imágenes del microscopio electrónico de transmisión revelan la adhesión de bacterias a la membrana celular por el glicocalix. Las estructuras fibrilares que rodean a las bacterias son claramente visibles.

Ultrastructure of the adhesion of bacteria to the epithelial cell membrane of three-day postnatal rat tongue mucosa: a transmission and high-resolution scanning electron microscopic study

Togue mucosa surface of 3-day postnatal rats was examined under transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM). For HRSEM analysis, the specimens were fixed in the same solution for 24 h, postfixed in 2 percent osmiun tetroxide, critical-point dried and coated with platinum-palladium. For TEM analysis, the specimens were fixed using modified Karnovsky solution and embedded in Spurr resin. The results revealed the presence of numerous microplicae in the membrane surface of keratinized epithelial cells to which groups of bacteria were attached. These bacteria were staphylococcus and coccus organized either in rows or at random, which were visualized in three-dimensional HRSEM images. At high magnification, the TEM images revealed the adhesion of bacteria to the cell membrane through numerous filamentous structures comprising the glycocalyx. The fine fibrillar structures rising from each bacterium and from cell membrane were clearly seen. These characteristics on bacteria structure may be used for future control or prevention of bacterial diseases and for installation of the oral native flora.

A superfície lingual de ratos de três dias de idade foi examinada em microscópia eletrônica de transmissão (MET) e em microscópia eletrônica varredura de alta resolução (MEVAR). Para o método de MEVAR, os espécimes foram fixados na mesma solução por 24 h, pós fixados em solução de tetróxido de ósmio a 2 por cento, secos em ponto crítico e cobertos com platina- paládio. Para análise em MET, os espécimes foram fixados utilizando-se solução de Karnovsky modificada e emblocadas em resina Spurr. Os resultados mostraram a presença de numerosas micropregas na membrana superficial das células epiteliais queratinizadas, nas quais estavam aderidos grupos de bactérias. Estas bactérias eram estafilococos e cocos, organizados em fileiras ou a esmo, e puderam ser observadas em imagens tri-dimensionais em MEVAR. Em maiores aumentos, as imagens em MET revelaram a adesão de bactérias nas células por meio de numerosas estruturas filamentares compondo o glicocálice. As delicadas estruturas filamentares na periferia das bactérias e das células foram nitidamente identificadas. Estas características da estrutura bacteriana podem ser utilizadas, no futuro, para controle e prevenção de doenças bacteriana, bem como para a instalação da flora oral nativa.

Crystal habits and magnetic microstructures of magnetosomes in coccoid magnetotactic bacteria

Nós relatamos a aplicação de holografia não-axial e microscopia eletrônica de alta resolução para estudar os hábitos cristalinos de magnetossomos e a microestrutura magnética de dois morfotipos de cocos de bactérias magnetotáticas coletadas em uma lagoa salobra em Itaipu, Brasil. Itaipu-1, o organismo cocóide maior, contémduas cadeias separadas de magnetossomos atipicamente grandes; os cristais dos magnetossomos possuem projeções aproximadamente quadradas, comprimentos deaté 250 nm e são ligeiramente alongados na direção [111] (razão largura/comprimento de aproximadamente 0.9).Os cristais dos magnetossomos em Itaipu-3 possuemcomprimentos até 120 nm, maior alongamento na direção [111] (largura/comprimento ~ 0.6), e proeminentes facetas nas extremidades. Os resultados mostram que os hábitos cristalinos dos magnetossomos em Itaipu-1 eItaipu-3 são relacionados, diferindo apenas nos tamanhos relativos das suas faces cristalinas. Em ambos os casos, os cristais são alinhados com seus eixos de alongamento [111] paralelos à direção da cadeia. Em Itaipu-1, mas não em Itaipu-3, o posicionamento cristalográfico, perpendicular à direção [111], de cristais sucessivos na cadeia de magnetossomos parece estar sobre controlebiológico. Enquanto os magnetossomos grandes em Itaipu-1 são monodomínios magnéticos metaestáveis, em Itaipu-3 eles são monodomínios magnéticos permanentes como na maioria das bactérias.

Ultrastructure of bacteria adhesion in cell membrane of young mouse lingual mucosa

Lingual mucosa of young mouse was examined by transmission and high resolution scanning electron microscopic images (HRSEM). The specimens were fixed with modified Karnovsky solution and embedded in Spurr resin for transmission electron micrascopy. Thin sections of 80 nm were cut and examined in the Jeol 1010 transmission electron microscope. For HRSEM method, the specimens were fixed in the same solution, postfixed in osmiun tetroxide, critical point dried and coated with palladium. The samples were examined under Hitachi S-900, SEM microscope. The results revealed groups of bacteria attached to the surface of keratinized epithelial cells. These streptococcus and coccus attached on the cell membrane were noted in the three-dimensional SEM images- At high magnification, the transmission electron microscopic images demonstrated the adhesion of bacteria to the cell membrane through numerous fimbria comprising the glycocalyx. The fine fibrillar structure rising from bacteria were clearly seen.

A mucosa lingual de camundongos jovens foi examinada através de imagens de microscopia eletrônica de transmissão e de varredura de alta resolução. Os espécimes foram fixados em solução modificada de Karnovsky e emblocadas em resina Spurr para a microscopia eletrônica de transmissão. Cortes finos de 80 nm foram feitos e examinados em um microscópio eletrônico de transmissão Jeol 1010. Para a microscopia eletrônica de varredura de alta resolução os espécimes foram imersos na mesma solução, pós fixados em tetróxido de ósmio, secos e cobertos com paládio. As amostras foram examinadas em um microscópio eletrônico de varredura Hitachi S-900. Os resultados revelaram grupos de bactéria aderidos à superfície queratinizada das células epiteliais. Estes estreptococos e cocos aderidos à membrana celular foram notados em imagens tridimensionais. Em aumentos maiores, as imagens de microscopia eletrônica de transmissão demonstraram a adesão de bactéria à membrana celular através de numerosas fimbrias compondo o glicocalice. A estrutura fibrilar emergindo da bactéria foi claramente observada.

Isolation of Herpetosiphon giganteus and ultrastructure analysis by electron microscopy

Herpetosiphon giganteus is a filamentous gliding bacterium. Gliding motility is the movement of the cells over surfaces without the aid of flagella. The mechanism responsible for bacterial gliding motility has not been known and there are only a few data on Herpetosiphon giganteus. The aim of this study was to observe the ultrastructure and negative staining of isolated strains of Herpetosiphon giganteus to find any organelles of locomotion. First, 35 strains of gliding bacteria were isolated from soil, freshwater, mud and activated sewage sludge. Then, 8 strains very closely related to Herpetosiphon giganteus were used for further examination. For extracellular slime and fibril observation, photoelectron micrographs were taken from different patterns on the cell surface of strains that were negatively stained. Thin sections with and without lysozyme treatment were prepared and examined by transmission electron microscopy. When the filaments were negatively stained, fibrils were detected in young cultures. There were two different kinds of fibrils in this study. The extracellular slime of these organisms was clearly visible. Examination with the electron microscopy revealed neither flagella nor an axial filament of any kind. There was no evidence for external organelles of locomotion. The results indicated that ring like structure localized at the cell surface connected with fibrils is responsible for gliding movement. The secretion of slime is necessary for adhesion of the cell to a solid surface and for ease of movement

Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms

A simple apparatus for harvesting uncultured magnetotactic microorganisms is described. This apparatus consists of a glass container with two openings. A large opening on the topside is used to introduce the sediment and water. The sediment and water are previously stored in loosely capped bottles previously tested for the presence of magnetotactic bacteria. The apparatus is exposed to a properly aligned magnetic field of a homemade coil and the bacteria are removed through the capillary end of the second opening of the container. Harvested bacteria can then be used to ultrastrucutral studies using electron spectroscopic imaging. Large numbers of magnetotactic bacteria consisting of cocci and rod-shaped cells were efficiently collected from different environments. This apparatus is useful for microbiological studies on uncultured magnetotactic bacteria, especially in molecular approaches for phylogenetic investigations that give information on the natural diversity of microbial communities.

Ultra-estrutura de bactérias periodontopatogênicas / Ultrastructure of pathogenic bacteria in periodontal tissue

É objetivo deste estudo, analisar a ultra-estrutura da Porphyromonas gingivalis, Prevotella intermedia e do Actinobacillus actinomycetemcomitans pelas técnicas mais atuais de microscopia eletrônica, dando ênfase às estruturas de superfície e às diferenças entre os microorganismos. Os resultados mostraram que os três microorganismos estudados apresentaram estrutura típica de parede celular de bactérias Gram-negativas. Também foram encontradas vesículas de membrana externa no meio intercelular das três bactérias estudadas

Role of plasmids in the antibiotic resistance of bacteria in Asir Province

One hundred and five clinical isolated with different antibiotic susceptibility patterns were analyzed for the presented of plasmid DNA. The highest percentages of plasmid DNA were found in Escherichia coli isolated [55%], Klebsiella pneumoniae isolated [50%], and Psudomonas aeruginosa isolated [12.5%]. The number of plasmid species per cell did not always correlated with the antibiotic resistance pattern; however, the plasmids isolated were found correlated with the resistance phenotypes. The significance of and resulting concern stemming from the absence of plasmid DNA in the antibiotic resistance isolated are diseased. The potential use of this approach for tracing infections is also discussed

Importancia de la microscopía electrónica en enfermedades infecciosas / Importance of the electron microscopy on infectious diseases

Con el advenimiento del microscopio electrónico casi todas las disciplinas del área biológica se han visto favorecidas. La infectología tal vez constituye uno de los ejemplos más claros del importante apoyo que esta técnica ha tenido en su desarrollo. El conocimiento morfológico a nivel molecular de los microrganismos ha sido trascendente, no sólo en el desarrollo de la infectología misma, sino también por las perspectivas que ha abierto en otros campos; por ejemplo: es indiscutible que en la bioquímica y fisiología dichos conocimientos incidieron de alguna forma en la generación de bases sobre las cuales se apoyó el nacimiento de la biología molecular. De igual manera, estos conocimientos han contribuido al desarrollo de la biología celular, que a su vez ha beneficiado a la patología, y por último, algunos aspectos taxonómicos se han resuelto a medida que se amplían nuestros conocimientos sobre la estructura fina de organismos infectantes. No se exagera al afirmar que miles de ejemplos podrían citarse, ya que al hablar de infectología se implica a la virología, bacteriología, micología y parasitología