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

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          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.

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          Most cited references64

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          NIH Image to ImageJ: 25 years of image analysis

          For the past twenty five years the NIH family of imaging software, NIH Image and ImageJ have been pioneers as open tools for scientific image analysis. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            Investigation of the freely available easy-to-use software ‘EZR' for medical statistics

            Y Kanda (2012)
            Although there are many commercially available statistical software packages, only a few implement a competing risk analysis or a proportional hazards regression model with time-dependent covariates, which are necessary in studies on hematopoietic SCT. In addition, most packages are not clinician friendly, as they require that commands be written based on statistical languages. This report describes the statistical software ‘EZR' (Easy R), which is based on R and R commander. EZR enables the application of statistical functions that are frequently used in clinical studies, such as survival analyses, including competing risk analyses and the use of time-dependent covariates, receiver operating characteristics analyses, meta-analyses, sample size calculation and so on, by point-and-click access. EZR is freely available on our website (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html) and runs on both Windows (Microsoft Corporation, USA) and Mac OS X (Apple, USA). This report provides instructions for the installation and operation of EZR.
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              The biofilm matrix.

              The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                07 January 2021
                2020
                : 11
                : 613650
                Affiliations
                [1] 1Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University , Hiroshima, Japan
                [2] 2Laboratory of Medical Microbiology, Department of Health Pharmacy, Yokohama University of Pharmacy , Yokohama, Japan
                [3] 3Collaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera and Enteric Diseases , Kolkata, India
                Author notes

                Edited by: Anne Mai-Prochnow, The University of Sydney, Australia

                Reviewed by: Dirk Linke, University of Oslo, Norway; Jack Christopher Leo, Nottingham Trent University, United Kingdom

                *Correspondence: Hiroyasu Yamanaka, h-yamana@ 123456hirokoku-u.ac.jp

                This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2020.613650
                7817658
                33488556
                ab97823e-27ab-4c95-ba66-59baca207043
                Copyright © 2021 Seike, Kobayashi, Ueda, Takahashi, Okamoto and Yamanaka.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 October 2020
                : 07 December 2020
                Page count
                Figures: 6, Tables: 2, Equations: 0, References: 64, Pages: 14, Words: 0
                Funding
                Funded by: Japan Society for the Promotion of Science 10.13039/501100001691
                Award ID: 20K16255
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                aeromonas,biofilm,outer membrane vesicles (omvs),extracellular polymeric substances (eps),extracellular matrix (ecm)

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