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      New insights into the gut microbiome in loggerhead sea turtles Caretta caretta stranded on the Mediterranean coast

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          Abstract

          Caretta caretta is the most common sea turtle species in the Mediterranean Sea. The species is threatened by anthropomorphic activity that causes thousands of deaths and hundreds of strandings along the Mediterranean coast. Stranded turtles are often cared for in rehabilitation centres until they recover or die. The objective of this study was to characterize the gut microbiome of nine sea turtles stranded along the Sicilian coast of the Mediterranean Sea using high-throughput sequencing analysis targeting V3–V4 regions of the bacterial 16S rRNA gene. Stool samples were collected from eight specimens hosted in the recovery centre after a few days of hospitalization (under 7) and from one hosted for many weeks (78 days). To better explore the role of bacterial communities in loggerhead sea turtles, we compared our data with published fecal microbiomes from specimens stranded along the Tuscan and Ligurian coast. Our results highlight that, despite the different origin, size and health conditions of the animals, Firmicutes, Bacteroidetes and Proteobacteria constitute the main components of the microbiota. This study widens our knowledge on the gut microbiome of sea turtles and could be helpful for the set up of rehabilitation therapies of stranded animals after recovery in specialized centres.

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          Environmental and Gut Bacteroidetes: The Food Connection

          Members of the diverse bacterial phylum Bacteroidetes have colonized virtually all types of habitats on Earth. They are among the major members of the microbiota of animals, especially in the gastrointestinal tract, can act as pathogens and are frequently found in soils, oceans and freshwater. In these contrasting ecological niches, Bacteroidetes are increasingly regarded as specialists for the degradation of high molecular weight organic matter, i.e., proteins and carbohydrates. This review presents the current knowledge on the role and mechanisms of polysaccharide degradation by Bacteroidetes in their respective habitats. The recent sequencing of Bacteroidetes genomes confirms the presence of numerous carbohydrate-active enzymes covering a large spectrum of substrates from plant, algal, and animal origin. Comparative genomics reveal specific Polysaccharide Utilization Loci shared between distantly related members of the phylum, either in environmental or gut-associated species. Moreover, Bacteroidetes genomes appear to be highly plastic and frequently reorganized through genetic rearrangements, gene duplications and lateral gene transfers (LGT), a feature that could have driven their adaptation to distinct ecological niches. Evidence is accumulating that the nature of the diet shapes the composition of the intestinal microbiota. We address the potential links between gut and environmental bacteria through food consumption. LGT can provide gut bacteria with original sets of utensils to degrade otherwise refractory substrates found in the diet. A more complete understanding of the genetic gateways between food-associated environmental species and intestinal microbial communities sheds new light on the origin and evolution of Bacteroidetes as animals’ symbionts. It also raises the question as to how the consumption of increasingly hygienic and processed food deprives our microbiota from useful environmental genes and possibly affects our health.
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            Honor thy symbionts.

            Our intestine is the site of an extraordinarily complex and dynamic environmentally transmitted consortial symbiosis. The molecular foundations of beneficial symbiotic host-bacterial relationships in the gut are being revealed in part from studies of simplified models of this ecosystem, where germ-free mice are colonized with specified members of the microbial community, and in part from comparisons of the genomes of members of the intestinal microbiota. The results emphasize the contributions of symbionts to postnatal gut development and host physiology, as well as the remarkable strategies these microorganisms have evolved to sustain their alliances. These points are illustrated by the human-Bacteroides thetaiotaomicron symbiosis. Interdisciplinary studies of the effects of the intestinal environment on genome structure and function should provide important new insights about how microbes and humans have coevolved mutually beneficial relationships and new perspectives about the foundations of our health.
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              The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies

              Background Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. Results We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. Conclusions Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0351-6) contains supplementary material, which is available to authorized users.
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                Author and article information

                Contributors
                Role: Data curation
                Role: Data curationRole: Formal analysisRole: Writing – original draft
                Role: Investigation
                Role: InvestigationRole: Methodology
                Role: MethodologyRole: Writing – review & editing
                Role: Methodology
                Role: Data curation
                Role: ConceptualizationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                14 August 2019
                2019
                : 14
                : 8
                : e0220329
                Affiliations
                [1 ] Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Università degli Studi di Palermo, Palermo, Italy
                [2 ] Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy
                Universita degli Studi della Tuscia, ITALY
                Author notes

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

                [¤]

                Current address: Groningen Institute for Evolutionary Life Sciences, University of Groningen, Netherlands

                Author information
                http://orcid.org/0000-0003-4325-9728
                http://orcid.org/0000-0001-7794-2918
                http://orcid.org/0000-0002-0890-5192
                http://orcid.org/0000-0003-1054-6915
                Article
                PONE-D-19-06261
                10.1371/journal.pone.0220329
                6693768
                31412070
                245b1e4b-4984-43b6-941c-ec13a94abbdb
                © 2019 Arizza et al

                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
                : 4 March 2019
                : 12 July 2019
                Page count
                Figures: 4, Tables: 4, Pages: 14
                Funding
                This work was supported by Università degli Studi di Palermo (Palermo University) - FFR to RA, MA, VA. This work was supported as part of the Project “Biotechnologies for Human Health and Blue Growth – BYTHOS”, BYTHOS-C1-1.1-9, CUP – B76H18000180005, funded by 2014 - 2020 INTERREG V-A Italy - Malta EU Programme. 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
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Reptiles
                Testudines
                Turtles
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbiome
                Biology and Life Sciences
                Genetics
                Genomics
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Microbiology
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Organisms
                Bacteria
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Fusobacteria
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Fusobacteria
                Biology and Life Sciences
                Organisms
                Bacteria
                Gut Bacteria
                Fusobacteria
                Biology and Life Sciences
                Organisms
                Bacteria
                Gut Bacteria
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Databases
                Earth sciences
                Marine and aquatic sciences
                Bodies of water
                Oceans
                Mediterranean Sea
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Reptiles
                Custom metadata
                The sequence dataset was deposited in the GenBank database (Bioproject PRJNA481425, Submission ID: SUB4304187).

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