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      The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer

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          Abstract

          Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

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          Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species.

          Despite recent advances in commercially optimized identification systems, bacterial identification remains a challenging task in many routine microbiological laboratories, especially in situations where taxonomically novel isolates are involved. The 16S rRNA gene has been used extensively for this task when coupled with a well-curated database, such as EzTaxon, containing sequences of type strains of prokaryotic species with validly published names. Although the EzTaxon database has been widely used for routine identification of prokaryotic isolates, sequences from uncultured prokaryotes have not been considered. Here, the next generation database, named EzTaxon-e, is formally introduced. This new database covers not only species within the formal nomenclatural system but also phylotypes that may represent species in nature. In addition to an identification function based on Basic Local Alignment Search Tool (blast) searches and pairwise global sequence alignments, a new objective method of assessing the degree of completeness in sequencing is proposed. All sequences that are held in the EzTaxon-e database have been subjected to phylogenetic analysis and this has resulted in a complete hierarchical classification system. It is concluded that the EzTaxon-e database provides a useful taxonomic backbone for the identification of cultured and uncultured prokaryotes and offers a valuable means of communication among microbiologists who routinely encounter taxonomically novel isolates. The database and its analytical functions can be found at http://eztaxon-e.ezbiocloud.net/.
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            Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics.

            The evolutionary relationships of 46 Shigella strains representing each of the serotypes belonging to the four traditional Shigella species (subgroups), Dysenteriae, Flexneri, Boydii, and Sonnei, were determined by sequencing of eight housekeeping genes in four regions of the chromosome. Analysis revealed a very similar evolutionary pattern for each region. Three clusters of strains were identified, each including strains from different subgroups. Cluster 1 contains the majority of Boydii and Dysenteriae strains (B1-4, B6, B8, B10, B14, and B18; and D3-7, D9, and D11-13) plus Flexneri 6 and 6A. Cluster 2 contains seven Boydii strains (B5, B7, B9, B11, B15, B16, and B17) and Dysenteriae 2. Cluster 3 contains one Boydii strain (B12) and the Flexneri serotypes 1-5 strains. Sonnei and three Dysenteriae strains (D1, D8, and D10) are outside of the three main clusters but, nonetheless, are clearly within Escherichia coli. Boydii 13 was found to be distantly related to E. coli. Shigella strains, like the other pathogenic forms of E. coli, do not have a single evolutionary origin, indicating convergent evolution of Shigella phenotypic properties. We estimate the three main Shigella clusters to have evolved within the last 35,000 to 270,000 years, suggesting that shigellosis was one of the early infectious diseases of humans.
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              Polyphasic taxonomy, a consensus approach to bacterial systematics.

              Over the last 25 years, a much broader range of taxonomic studies of bacteria has gradually replaced the former reliance upon morphological, physiological, and biochemical characterization. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them in a consensus type of classification, framed in a general phylogeny derived from 16S rRNA sequence analysis. In some cases, the consensus classification is a compromise containing a minimum of contradictions. It is thought that the more parameters that will become available in the future, the more polyphasic classification will gain stability. In this review, the practice of polyphasic taxonomy is discussed for four groups of bacteria chosen for their relevance, complexity, or both: the genera Xanthomonas and Campylobacter, the lactic acid bacteria, and the family Comamonadaceae. An evaluation of our present insights, the conclusions derived from it, and the perspectives of polyphasic taxonomy are discussed, emphasizing the keystone role of the species. Taxonomists did not succeed in standardizing species delimitation by using percent DNA hybridization values. Together with the absence of another "gold standard" for species definition, this has an enormous repercussion on bacterial taxonomy. This problem is faced in polyphasic taxonomy, which does not depend on a theory, a hypothesis, or a set of rules, presenting a pragmatic approach to a consensus type of taxonomy, integrating all available data maximally. In the future, polyphasic taxonomy will have to cope with (i) enormous amounts of data, (ii) large numbers of strains, and (iii) data fusion (data aggregation), which will demand efficient and centralized data storage. In the future, taxonomic studies will require collaborative efforts by specialized laboratories even more than now is the case. Whether these future developments will guarantee a more stable consensus classification remains an open question.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                18 April 2014
                : 9
                : 4
                : e95384
                Affiliations
                [1 ]Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium
                [2 ]Laboratory of Biochemistry and Brewing, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
                [3 ]Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium
                [4 ]Mycothèque de l'Université catholique de Louvain (MUCL), Belgian Coordinated Collection of Microorganisms (BCCM), Earth and Life Institute, Applied Microbiology, Mycology, Université catholique de Louvain, Louvain-la-Neuve, Belgium
                Auburn University, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: FS MJ AVL LDV PV. Performed the experiments: FS. Analyzed the data: FS ADW HMD AVL LDV PV. Contributed reagents/materials/analysis tools: ADW MA HMD. Wrote the paper: FS HMD AVL LDV PV.

                Article
                PONE-D-14-05061
                10.1371/journal.pone.0095384
                3991685
                24748344
                e496e24d-c269-495b-9ac1-72e9a254b681
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 February 2014
                : 25 March 2014
                Page count
                Pages: 13
                Funding
                This research was funded by a Ph.D. grant (FS) of the Agency for Innovation by Science and Technology (IWT) and by the Research Foundation Flanders (FWO-Vlaanderen). The authors further acknowledge their finances from the research fund of the University College Ghent (AW) and the Vrije Universiteit Brussel (HOA, SRP, IRP, and IOF projects; MJ and LDV) and from the Belgian Federal Science Policy (BCCM C4/10/017) (HMD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Proteomics
                Biotechnology
                Applied Microbiology
                Industrial Microbiology
                Ecology
                Microbial Ecology
                Microbiology
                Bacteriology
                Bacterial Taxonomy
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Mycology
                Fungal Classification
                Organisms
                Fungi
                Yeast

                Uncategorized
                Uncategorized

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