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      Variation in the microbiome of the urogenital tract of Chlamydia-free female koalas ( Phascolarctos cinereus) with and without ‘wet bottom’

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          Abstract

          Koalas ( Phascolarctos cinereus) are iconic Australian marsupials currently threatened by several processes, including infectious diseases and ecological disruption. Infection with Chlamydia pecorum, is considered a key driver of population decline. The clinical sign of ‘wet bottom’, a staining of the rump associated with urinary incontinence, is often caused by chlamydial urinary tract infections. However, wet bottom has been recorded in koalas free of C. pecorum, suggesting other causative agents in those individuals. We used 16S rRNA diversity profiling to investigate the microbiome of the urogenital tract of ten female koalas in order to identify potential causative agents of wet bottom, other than C. pecorum. Five urogenital samples were processed from koalas presenting with wet bottom and five were clinically normal. All koalas were negative for C. pecorum infection. We detected thirteen phyla across the ten samples, with Firmicutes occurring at the highest relative abundance (77.6%). The order Lactobacillales, within the Firmicutes, comprised 70.3% of the reads from all samples. After normalising reads using DESeq2 and testing for significant differences ( P < 0.05), there were 25 operational taxonomic units (OTUs) more commonly found in one group over the other. The families Aerococcaceae and Tissierellaceae both had four significantly differentially abundant OTUs. These four Tissierellaceae OTUs were all significantly more abundant in koalas with wet bottom. This study provides the foundation for future investigations of causes of koala wet bottom, other than C. pecorum infection. This is of clinical relevance as wet bottom is often assumed to be caused by C. pecorum and treated accordingly. Our research highlights that other organisms may be causing wet bottom, and these potential aetiological agents need to be further investigated to fully address the problems this species faces.

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          EMPeror: a tool for visualizing high-throughput microbial community data

          Background As microbial ecologists take advantage of high-throughput sequencing technologies to describe microbial communities across ever-increasing numbers of samples, new analysis tools are required to relate the distribution of microbes among larger numbers of communities, and to use increasingly rich and standards-compliant metadata to understand the biological factors driving these relationships. In particular, the Earth Microbiome Project drives these needs by profiling the genomic content of tens of thousands of samples across multiple environment types. Findings Features of EMPeror include: ability to visualize gradients and categorical data, visualize different principal coordinates axes, present the data in the form of parallel coordinates, show taxa as well as environmental samples, dynamically adjust the size and transparency of the spheres representing the communities on a per-category basis, dynamically scale the axes according to the fraction of variance each explains, show, hide or recolor points according to arbitrary metadata including that compliant with the MIxS family of standards developed by the Genomic Standards Consortium, display jackknifed-resampled data to assess statistical confidence in clustering, perform coordinate comparisons (useful for procrustes analysis plots), and greatly reduce loading times and overall memory footprint compared with existing approaches. Additionally, ease of sharing, given EMPeror’s small output file size, enables agile collaboration by allowing users to embed these visualizations via emails or web pages without the need for extra plugins. Conclusions Here we present EMPeror, an open source and web browser enabled tool with a versatile command line interface that allows researchers to perform rapid exploratory investigations of 3D visualizations of microbial community data, such as the widely used principal coordinates plots. EMPeror includes a rich set of controllers to modify features as a function of the metadata. By being specifically tailored to the requirements of microbial ecologists, EMPeror thus increases the speed with which insight can be gained from large microbiome datasets.
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            Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction.

            Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.
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              Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus.

              Members of genus Peptostreptococcus have previously been found to be distantly related to the type species, Peptostreptococcus anaerobius, on the basis of 16S rDNA sequence similarities. They were divided into three major phylogenetic groups, and their peptidoglycan structure and biochemical traits differed between groups. The reclassification of the species of these three groups into three new genera, Peptoniphilus gen. nov., Anaerococcus gen. nov. and Gallicola gen. nov., is proposed. The genus Peptoniphilus gen. nov. includes the following butyrate-producing, non-saccharolytic species that use peptone and amino acids as major energy sources: Peptoniphilus asaccharolyticus comb. nov. (type species), Peptoniphilus lacrimaris comb. nov., Peptoniphilus harei comb. nov., Peptoniphilus indolicus comb. nov. and Peptoniphilus ivorii comb. nov. The genus Anaerococcus gen. nov. contains the saccharolytic, butyrate-producing species Anaerococcus prevotii comb. nov. (type species), Anaerococcus tetradius comb. nov., Anaerococcus lactolyticus comb. nov., Anaerococcus hydrogenalis comb. nov., Anaerococcus vaginalis comb. nov. and Anaerococcus octavius sp. nov. The genus Gallicola gen. nov. contains a single species, Gallicola barnesae comb. nov.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: 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
                26 March 2018
                2018
                : 13
                : 3
                : e0194881
                Affiliations
                [1 ] Asia Pacific Centre for Animal Health, The University of Melbourne, Parkville, Victoria, Australia
                [2 ] Veterinary Department, Melbourne Zoo, Parkville, Victoria, Australia
                [3 ] Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Victoria, Australia
                [4 ] Centre for Equine Infectious Diseases, The University of Melbourne, Parkville, Victoria, Australia
                Midwestern University, UNITED STATES
                Author notes

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

                ‡ FMS and JMD are joint senior authors on this work.

                Author information
                http://orcid.org/0000-0003-2407-4496
                Article
                PONE-D-17-42771
                10.1371/journal.pone.0194881
                5868818
                29579080
                01662e8f-1480-460e-9ceb-461c1af75da8
                © 2018 Legione 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
                : 6 December 2017
                : 12 March 2018
                Page count
                Figures: 4, Tables: 4, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100008190, Holsworth Wildlife Research Endowment;
                Award Recipient :
                The research was supported by a grant from the Holsworth Wildlife Research Endowment (HOLSW2015-1-F052) ( https://equitytrustees.smartygrants.com.au/holsworth). Alistair Legione was supported by an Australian Postgraduate Award scholarship. 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
                Taxonomy
                Computer and Information Sciences
                Data Management
                Taxonomy
                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
                Medicine and Health Sciences
                Infectious Diseases
                Sexually Transmitted Diseases
                Chlamydia Infection
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Ribosomal RNA
                Biology and life sciences
                Biochemistry
                Ribosomes
                Ribosomal RNA
                Biology and life sciences
                Cell biology
                Cellular structures and organelles
                Ribosomes
                Ribosomal RNA
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Clostridium
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Clostridium
                Biology and Life Sciences
                Organisms
                Bacteria
                Gut Bacteria
                Clostridium
                Biology and Life Sciences
                Organisms
                Bacteria
                Chlamydia
                Research and Analysis Methods
                Database and Informatics Methods
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                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
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                Biology and Life Sciences
                Microbiology
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                Microbial Pathogens
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                Medicine and Health Sciences
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                Pathogens
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                Biology and Life Sciences
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                Gut Bacteria
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                Custom metadata
                All relevant data are within the paper and its Supporting Information files. Illumina reads for each sample used in this study are available to download from the NCBI Sequence Read archive (accession numbers: SRX2464137 – SRX246146).

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