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      Differences of lung microbiome in patients with clinically stable and exacerbated bronchiectasis

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          Abstract

          Background

          Molecular-based diagnostic techniques can compensate for the inherent limitations of culture-based microbiology and provide a more comprehensive description of an entire community of bacteria at a particular anatomical site. Using culture-independent DNA-based molecular techniques, the aim of the present study was to characterize, differentiate, and compare the composition of lower airway bacterial microbiome between clinically stable and acutely infected patients with bronchiectasis experiencing exacerbation.

          Methods

          Patients with clinically stable bronchiectasis and those experiencing acutely exacerbated bronchiectasis were recruited. All patients underwent bronchoscopy. Paired sputum and bronchoalveolar lavage (BAL) samples were collected for microbiological tests. Molecular analysis was performed for BAL samples using 16S ribosomal RNA (rRNA) gene sequencing.

          Results

          The mean age of the 14 recruited patients was 60 years (range 42 to 78 years), and nine (64%) were female. Using quantitative culture and 16S rRNA sequencing, the common organisms identified from 14 BAL samples were Haemophilus influenzae, Pseudomonas aeruginosa and Moraxella catarrhalis, and Prevotella. Molecular techniques revealed Prevotella and Veillonella as potentially pathogenic anaerobic species. 16S rRNA gene sequencing yielded similar relative abundances and distributions of taxa in the stable and exacerbated bronchiectasis groups. Alpha diversity with richness, Simpson’s and Shannon indices, and beta diversity using principal coordinate analysis revealed no significant differences in lung microbiome between patients with clinically stable and exacerbated bronchiectasis.

          Conclusion

          Culture-based microbiological and molecular-based techniques did not reveal significant differences in the lung microbiome of patients who were clinically stable and those experiencing exacerbated bronchiectasis. Patient-specific microbial communities were dominated by one or several genera, regardless of clinical status. DNA sequencing could identify potentially pathogenic organisms unable to be identified using microbiological methods.

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          Most cited references16

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          Advancing our understanding of the human microbiome using QIIME.

          High-throughput DNA sequencing technologies, coupled with advanced bioinformatics tools, have enabled rapid advances in microbial ecology and our understanding of the human microbiome. QIIME (Quantitative Insights Into Microbial Ecology) is an open-source bioinformatics software package designed for microbial community analysis based on DNA sequence data, which provides a single analysis framework for analysis of raw sequence data through publication-quality statistical analyses and interactive visualizations. In this chapter, we demonstrate the use of the QIIME pipeline to analyze microbial communities obtained from several sites on the bodies of transgenic and wild-type mice, as assessed using 16S rRNA gene sequences generated on the Illumina MiSeq platform. We present our recommended pipeline for performing microbial community analysis and provide guidelines for making critical choices in the process. We present examples of some of the types of analyses that are enabled by QIIME and discuss how other tools, such as phyloseq and R, can be applied to expand upon these analyses. © 2013 Elsevier Inc. All rights reserved.
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            The lung tissue microbiome in chronic obstructive pulmonary disease.

            Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others. To extend these findings to human lung tissue samples. DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing. Total bacterial populations within lung tissue were small (20-1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007). There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.
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              Performance comparison of Illumina and ion torrent next-generation sequencing platforms for 16S rRNA-based bacterial community profiling.

              High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common "benchtop" sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: ResourcesRole: SupervisionRole: Validation
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: ValidationRole: Visualization
                Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 August 2017
                2017
                : 12
                : 8
                : e0183553
                Affiliations
                [1 ] Division of Pulmonology, Department of Internal Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
                [2 ] Division of Pulmonology, Department of Internal Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
                [3 ] Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
                Lee Kong Chian School of Medicine, SINGAPORE
                Author notes

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

                Author information
                http://orcid.org/0000-0003-1525-1745
                Article
                PONE-D-17-14808
                10.1371/journal.pone.0183553
                5567645
                28829833
                d988846d-7725-48b2-8e35-add79b873feb
                © 2017 Byun 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
                : 16 April 2017
                : 7 August 2017
                Page count
                Figures: 5, Tables: 4, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100008005, Yonsei University College of Medicine;
                Award ID: 6-2014-0146
                Award Recipient :
                This study was supported by a faculty research grant of Yonsei University College of Medicine for 2014 (6-2014-0146). 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
                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
                Haemophilus Influenzae
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Haemophilus Influenzae
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                Haemophilus Influenzae
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                Anatomy
                Body Fluids
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                Custom metadata
                All data are available from a public repository (The Dryad Digital Repository), available at the following link: http://datadryad.org/review?doi=doi:10.5061/dryad.gj03f.

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