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      Gardnerella vaginalis Enhances Atopobium vaginae Viability in an in vitro Model

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          Abstract

          Bacterial vaginosis (BV) is the most common vaginal infection among women of reproductive age. A hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, presumably initiated by facultative anaerobes of the genus Gardnerella, which then becomes a scaffold for other species to adhere to. One of the species often found incorporated in Gardnerella mediated biofilms is Atopobium vaginae. Interestingly, A. vaginae is very rarely found without the presence of Gardnerella. However, not much is known regarding the interactions between A. vaginae and Gardnerella species. This study assessed biological interactions between Gardnerella vaginalis and A. vaginae. In our in vitro model, by using specific Gardnerella and A. vaginae Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH) probes, we confirmed that A. vaginae was able to incorporate a pre-formed G. vaginalis biofilm, accounting for up to 20% of the total number of biofilm cells. However, our findings showed that almost 92% of A. vaginae cells lost viability after 48 h of mono-species planktonic growth, but were able to maintain viability when co-cultured with Gardnerella or after pre-conditioning with cell-free supernatant of Gardnerella cultures. While the in vitro conditions are very different from the in vivo microenvironment, this study contributes to a better understanding of why A. vaginae vaginal colonization rarely occurs in the absence of Gardnerella. Overall, this highlights the importance of microbial interactions between BV-associated bacteria and demands more studies focused on the polymicrobial bacterial communities found in BV.

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          Most cited references36

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          Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci.

          The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.
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            Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.

            In the present study six assays for the quantification of biofilms formed in 96-well microtiter plates were optimised and evaluated: the crystal violet (CV) assay, the Syto9 assay, the fluorescein diacetate (FDA) assay, the resazurin assay, the XTT assay and the dimethyl methylene blue (DMMB) assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these assays showed a broad applicability and a high repeatability for most isolates. In addition, the estimated numbers of CFUs present in the biofilms show limited variations between the different assays. Nevertheless, our data show that some assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV assay for P. aeruginosa).
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              Critical review on biofilm methods.

              Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                04 March 2020
                2020
                : 10
                : 83
                Affiliations
                [1] 1Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), Centre of Biological Engineering (CEB) , Braga, Portugal
                [2] 2Laboratory Bacteriology Research, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium
                Author notes

                Edited by: Shai Bel, Bar-Ilan University, Israel

                Reviewed by: Janneke Van De Wijgert, University of Liverpool, United Kingdom; Antonio Machado, Universidad San Francisco de Quito, Ecuador; Werner Mendling, Deutsches Zentrum für Infektionen in Gynäkologie und Geburtshilfe, Germany

                *Correspondence: Nuno Cerca nunocerca@ 123456ceb.uminho.pt

                This article was submitted to Microbiome in Health and Disease, a section of the journal Frontiers in Cellular and Infection Microbiology

                †These authors have contributed equally to this work

                Article
                10.3389/fcimb.2020.00083
                7064616
                32195197
                5275b376-cb4f-4cf5-a619-18b4864bff4f
                Copyright © 2020 Castro, Rosca, Cools, Vaneechoutte and Cerca.

                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
                : 16 December 2019
                : 18 February 2020
                Page count
                Figures: 3, Tables: 1, Equations: 0, References: 39, Pages: 9, Words: 5786
                Funding
                Funded by: Fundação para a Ciência e a Tecnologia 10.13039/501100001871
                Award ID: PD/BD/128037/2016
                Award ID: PTDC/BIA-MIC/28271/2017
                Categories
                Cellular and Infection Microbiology
                Brief Research Report

                Infectious disease & Microbiology
                bacterial vaginosis,polymicrobial biofilms,gardnerella,atopobium vaginae,pna-fish

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