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Aeromonas  spp. are Gram-negative rod-shaped bacteria ubiquitously distributed in diverse water sources. Several  Aeromonas  spp. are known as human and fish pathogens. Recently, attention has been focused on the relationship between bacterial biofilm formation and pathogenicity or drug resistance. However, there have been few reports on biofilm formation by  Aeromonas . This study is the first to examine the  in vitro  formation and components of the biofilm of several  Aeromonas  clinical and environmental strains. A biofilm formation assay using 1% crystal violet on a polystyrene plate revealed that most  Aeromonas  strains used in this study formed biofilms but one strain did not. Analysis of the basic components contained in the biofilms formed by  Aeromonas  strains confirmed that they contained polysaccharides containing GlcNAc, extracellular nucleic acids, and proteins, as previously reported for the biofilms of other bacterial species. Among these components, we focused on several proteins fractionated by SDS-PAGE and determined their amino acid sequences. The results showed that some proteins existing in the  Aeromonas  biofilms have amino acid sequences homologous to functional proteins present in the outer membrane of Gram-negative bacteria. This result suggests that outer membrane components may affect the biofilm formation of  Aeromonas  strains. It is known that Gram-negative bacteria often release extracellular membrane vesicles from the outer membrane, so we think that the outer membrane-derived proteins found in the  Aeromonas  biofilms may be derived from such membrane vesicles. To examine this idea, we next investigated the ability of  Aeromonas  strains to form outer membrane vesicles (OMVs). Electron microscopic analysis revealed that most  Aeromonas  strains released OMVs outside the cells. Finally, we purified OMVs from several  Aeromonas  strains and examined their effect on the biofilm formation. We found that the addition of OMVs dose-dependently promoted biofilm formation, except for one strain that did not form biofilms. These results suggest that the OMVs released from the bacterial cells are closely related to the biofilm formation of  Aeromonas  strains.

Outer Membrane Vesicles Released From Aeromonas Strains Are Involved in the Biofilm Formation