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      Longitudinal assessment of sputum microbiome by sequencing of the 16S rRNA gene in non-cystic fibrosis bronchiectasis patients

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          Abstract

          Background

          Bronchiectasis is accompanied by chronic bronchial infection that may drive disease progression. However, the evidence base for antibiotic therapy is limited. DNA based methods offer better identification and quantification of microbial constituents of sputum than standard clinical culture and may help inform patient management strategies. Our study objective was to determine the longitudinal variability of the non-cystic fibrosis (CF) bronchiectasis microbiome in sputum with respect to clinical variables. Eighty-five patients with non-CF bronchiectasis and daily sputum production were recruited from outpatient clinics and followed for six months. Monthly sputum samples and clinical measurements were taken, together with additional samples during exacerbations. 16S rRNA gene sequencing of the sputum microbiota was successful for 381 samples from 76 patients and analysed in conjunction with clinical data.

          Results

          Microbial communities were highly individual in composition and stability, usually with limited diversity and often containing multiple pathogens. When compared to DNA sequencing, microbial culture had restricted sensitivity in identifying common pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, Moraxella catarrhalis. With some exceptions, community characteristics showed poor correlations with clinical features including underlying disease, antibiotic use and exacerbations, with the subject showing the strongest association with community structure. When present, the pathogens mucoid Pseudomonas aeruginosa and Haemophilus influenzae may also shape the structure of the rest of the microbial community.

          Conclusions

          The use of microbial community analysis of sputum added to information from microbial culture. A simple model of exacerbations driven by bacterial overgrowth was not supported, suggesting a need for revision of principles for antibiotic therapy. In individual patients, the management of chronic bronchial infection may be improved by therapy specific to their microbiome, taking into account pathogen load, community stability, and acute and chronic community responses to antibiotics.

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          Most cited references5

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          New strains of bacteria and exacerbations of chronic obstructive pulmonary disease.

          The role of bacterial pathogens in acute exacerbations of chronic obstructive pulmonary disease is controversial. In older studies, the rates of isolation of bacterial pathogens from sputum were the same during acute exacerbations and during stable disease. However, these studies did not differentiate among strains within a bacterial species and therefore could not detect changes in strains over time. We hypothesized that the acquisition of a new strain of a pathogenic bacterial species is associated with exacerbation of chronic obstructive pulmonary disease. We conducted a prospective study in which clinical information and sputum samples for culture were collected monthly and during exacerbations from 81 outpatients with chronic obstructive pulmonary disease. Molecular typing of sputum isolates of nonencapsulated Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa was performed. Over a period of 56 months, the 81 patients made a total of 1975 clinic visits, 374 of which were made during exacerbations (mean, 2.1 per patient per year). On the basis of molecular typing, an exacerbation was diagnosed at 33.0 percent of the clinic visits that involved isolation of a new strain of a bacterial pathogen, as compared with 15.4 percent of visits at which no new strain was isolated (P<0.001; relative risk of an exacerbation, 2.15; 95 percent confidence interval, 1.83 to 2.53). Isolation of a new strain of H. influenzae, M. catarrhalis, or S. pneumoniae was associated with a significantly increased risk of an exacerbation. The association between an exacerbation and the isolation of a new strain of a bacterial pathogen supports the causative role of bacteria in exacerbations of chronic obstructive pulmonary disease. Copyright 2002 Massachusetts Medical Society
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            PCR primers that amplify fungal rRNA genes from environmental samples.

            Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.
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              Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease

              Chronic lung infections in cystic fibrosis (CF) patients are composed of complex microbial communities that incite persistent inflammation and airway damage. Despite the high density of bacteria that colonize the lower airways, nutrient sources that sustain bacterial growth in vivo, and how those nutrients are derived, are not well characterized. In this study, we examined the possibility that mucins serve as an important carbon reservoir for the CF lung microbiota. While Pseudomonas aeruginosa was unable to efficiently utilize mucins in isolation, we found that anaerobic, mucin-fermenting bacteria could stimulate the robust growth of CF pathogens when provided intact mucins as a sole carbon source. 16S rRNA sequencing and enrichment culturing of sputum also identified that mucin-degrading anaerobes are ubiquitous in the airways of CF patients. The collective fermentative metabolism of these mucin-degrading communities in vitro generated amino acids and short chain fatty acids (propionate and acetate) during growth on mucin, and the same metabolites were also found in abundance within expectorated sputum. The significance of these findings was supported by in vivo P. aeruginosa gene expression, which revealed a heightened expression of genes required for the catabolism of propionate. Given that propionate is exclusively derived from bacterial fermentation, these data provide evidence for an important role of mucin fermenting bacteria in the carbon flux of the lower airways. More specifically, microorganisms typically defined as commensals may contribute to airway disease by degrading mucins, in turn providing nutrients for pathogens otherwise unable to efficiently obtain carbon in the lung.
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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, CA USA )
                1932-6203
                7 February 2017
                2017
                : 12
                : 2
                : e0170622
                Affiliations
                [1 ]National Heart and Lung Institute, Imperial College London, London, United Kingdom
                [2 ]Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom
                Universite Paris-Sud, FRANCE
                Author notes

                Competing Interests: DB received a grant from Novartis UK for sequencing costs. The funder had no involvement in study design, collection or analysis of data, or in the decision to publish. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                • Conceptualization: MJC ML WOCC MFM.

                • Data curation: MJC CH MC ML.

                • Formal analysis: MJC PLJ MC AJ ML.

                • Funding acquisition: DB MFM WOCC ML.

                • Methodology: MJC EMT CH GJM PLJ ML.

                • Project administration: MJC ML MFM WOCC.

                • Resources: CH AJ ML RW DB MFM WOCC.

                • Supervision: ML MFM WOCC.

                • Visualization: MJC ML.

                • Writing – original draft: MJC.

                • Writing – review & editing: MJC EMT CH GKM PLJ MC AJ RW DB WOCC MFM ML.

                Author information
                http://orcid.org/0000-0002-4002-1506
                Article
                PONE-D-16-38695
                10.1371/journal.pone.0170622
                5295668
                28170403
                8c4c4409-31b9-481d-a48c-c492726030ab
                © 2017 Cox 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
                : 27 September 2016
                : 6 January 2017
                Page count
                Figures: 3, Tables: 2, Pages: 17
                Funding
                The study was funded by the Wellcome Trust under WT077959 and WT096964. This project was funded and supported by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. The views expressed in this publication are those of the authors and not necessarily those of the NHS, The National Institute for Health Research or the Department of Health. Funding for sequencing was provided by a grant to DB from Novartis UK. WOCC and MFM are supported by a Wellcome Trust Joint Senior Investigator’s Award, which also supports MJC and EMT and WOCC is an NIHR Senior Investigator. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Antimicrobials
                Antibiotics
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobials
                Antibiotics
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Mucus
                Sputum
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Mucus
                Sputum
                Biology and Life Sciences
                Physiology
                Body Fluids
                Mucus
                Sputum
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Mucus
                Sputum
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Ribosomal RNA
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                Biochemistry
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                Cell biology
                Cellular structures and organelles
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                Haemophilus Influenzae
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                Haemophilus Influenzae
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                Organisms
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                Haemophilus Influenzae
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                Genetics
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                Pseudomonas Aeruginosa
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                Staphylococcus
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                Custom metadata
                Sequence data has been submitted to the European Nucleotide Archive and is available under accession number PRJEB14304 and sample accessions ERS1201047 to ERS1201427. In addition, the denoised reads, OTU table and clinical data are available to download at [ http://lungen.bioinformatics.ic.ac.uk/data/microbiome_LAMB_bronchiectasis/] and at BioStudies accession S-BSST1 [ https://www.ebi.ac.uk/biostudies/studies/S-BSST1/]. The clinical data is also available as S1 Table.

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