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

Soshi Seike,Hiroyasu Yamanaka,Mitsunobu Ueda,Eizo Takahashi,Hidetomo Kobayashi,Keinosuke Okamoto

doi:10.3389/fmicb.2020.613650

Abstract

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.

Highlights

Aeromonas spp. are Gram-negative, facultative anaerobic rodshaped bacteria and are ubiquitously distributed in diverse water sources and in foods (Janda and Brenden, 1987)
Among these components contained in the Aeromonas biofilms, we found that some proteins in extracellular matrix (ECM) might be closely related to the Aeromonas biofilm formation
It is likely that these components of the ECM function to sustain the biofilm formed in Aeromonas strains, the results showed that the amount of each of these ECM constituents varied among the bacterial strains

Summary

Introduction

Aeromonas spp. are Gram-negative, facultative anaerobic rodshaped bacteria and are ubiquitously distributed in diverse water sources and in foods (Janda and Brenden, 1987). Several Aeromonas species, such as A. hydrophila and A. sobria, are known as human pathogens that cause foodborne gastrointestinal diseases (Janda and Abbott, 2010). These pathogens cause severe extraintestinal diseases in an opportunistic manner, including sepsis, peritonitis, and meningitis, especially in immunocompromised patients or elderly persons (Michael Janda and Abbott, 1998). Various kinds of extracellular virulence factors, including cytolysins, enterotoxins and proteases, are thought to be deeply involved in the pathogenesis of these conditions (Pemberton et al, 1997; Fujii et al, 1998; Okamoto et al, 2000). Other unknown factors may be involved in the virulence of these pathogens

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Conclusion