Ultraestrutura de vesículas de membrana externa em bactérias de um ecossistema aquático amazônico

Abstract

Outer membrane vesicles (OMVs) released by Gram negative bacteria play an important role in a variety of biological processes, such as bacterial survival, nutrient acquisition, biofilm establishment, cell communication and pathogenesis. Despite the importance of these vesicles in pathogenic bacteria, the formation and occurrence of OMVs in bacteria of aquatic ecosystems is still poorly understood. The present work aimed to investigate the occurrence and formation of OMVs in cultured bacteria from an Amazonian aquatic ecosystem. For bacterial cultures, samples were collected from Negro River (AM), serially diluted and spread-plate in two non-selective solid culture media: R2A and TSA. Colonies were characterized through macroscopic aspects and microscopic characteristics. Colonies morphotypes were transferred to non-selective liquid media (TSB) and the growth curve was analyzed by optical and cellular density, through spectrophotometry and fluorescence microscopy, respectively. Our results showed that R2A media enabled the growth of most colonies morphotypes (10). Microscopic analyses by light microscopy showed that Gram negative bacteria (58,33%), bacillus shape (51%) and isolate arrangement (58,33%) were predominant among bacterial morphotypes. The density curve showed a growth stabilization around the fourth hour of culture, indicating the end of the exponential phase and the begging of the stationary phase. Transmission electron microscopy revealed bacteria with different sizes (0,93 μm2 à 1.290 μm2) showing gram-positive and gram-negative envelopes. TEM quantitative analyses showed higher frequency of Gram negative bacteria (77,28%) exhibiting cell envelope composed by plasma membrane, periplasmic space and outer membrane. TEM enabled the visualization of intra and extracellular bacterial structures, such as capsule, flagellum, nucleiod, granules and cellular septa. TEM analyses revealed the occurrence of OMVs extruded from the outer membrane of Gram negative bacteria. The higher OMVs proportion was found in process of budding, appearing isolated or in clusters adhered to cellular envelope. Free OMVs were also detected in the extracellular medium, near or distant from bacterial clusters. Morphometric analyses showed vesicles with various sizes (15 µm-300 µm in diameter). However, vesicles in process of formation showed smaller size than free vesicles in the extracellular medium. Altogether, the present work demonstrated, for the first time, the ability of bacteria from Negro River to generate OMVs during growth in culture media. The ultrastructural characterization of OMVs emphasizes the fact that bacteria from aquatic ecosystems are structurally organized microorganisms denoting its functional and adaptive complexity.