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      The lung microbiome in children with HIV-bronchiectasis: a cross-sectional pilot study

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          Abstract

          Background

          Data on the lung microbiome in HIV-infected children is limited. The current study sought to determine the lung microbiome in HIV-associated bronchiectasis and to assess its association with pulmonary exacerbations.

          Methods

          A cross-sectional pilot study of 22 children (68% male; mean age 10.8 years) with HIV-associated bronchiectasis and a control group of 5 children with cystic fibrosis (CF). Thirty-one samples were collected, with 11 during exacerbations. Sputum samples were processed with 16S rRNA pyrosequencing.

          Results

          The average number of operational taxonomy units (OTUs) was 298 ± 67 vs. 434 ± 90, for HIV-bronchiectasis and CF, respectively. The relative abundance of Proteobacteria was higher in HIV-bronchiectasis (72.3%), with only 22.2% Firmicutes. There was no correlation between lung functions (FEV 1% and FEF 25/75%) and bacterial community ( r = 0.154; p = 0.470 and r = 0.178; p = 0.403), respectively. Bacterial assemblage of exacerbation and non-exacerbation samples in HIV-bronchiectasis was not significantly different (ANOSIM, R HIV-bronchiectasis = 0.08; p = 0.14 and R CF = 0.08, p = 0.50). Higher within-community heterogeneity and lower evenness was associated with CF (Shannon-Weiner (H′) = 5.39 ± 0.38 and Pielou’s evenness (J) 0.79 ± 0.10 vs. HIV-bronchiectasis (Shannon-Weiner (H′) = 4.45 ± 0.49 and Pielou’s (J) 0.89 ± 0.03.

          Conclusion

          The microbiome in children with HIV-associated bronchiectasis seems to be less rich, diverse and heterogeneous with predominance of Proteobacteria when compared to cystic fibrosis.

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          Most cited references18

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          Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.

          Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.
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            Analysis of the Upper Respiratory Tract Microbiotas as the Source of the Lung and Gastric Microbiotas in Healthy Individuals

            ABSTRACT No studies have examined the relationships between bacterial communities along sites of the upper aerodigestive tract of an individual subject. Our objective was to perform an intrasubject and intersite analysis to determine the contributions of two upper mucosal sites (mouth and nose) as source communities for the bacterial microbiome of lower sites (lungs and stomach). Oral wash, bronchoalveolar lavage (BAL) fluid, nasal swab, and gastric aspirate samples were collected from 28 healthy subjects. Extensive analysis of controls and serial intrasubject BAL fluid samples demonstrated that sampling of the lungs by bronchoscopy was not confounded by oral microbiome contamination. By quantitative PCR, the oral cavity and stomach contained the highest bacterial signal levels and the nasal cavity and lungs contained much lower levels. Pyrosequencing of 16S rRNA gene amplicon libraries generated from these samples showed that the oral and gastric compartments had the greatest species richness, which was significantly greater in both than the richness measured in the lungs and nasal cavity. The bacterial communities of the lungs were significantly different from those of the mouth, nose, and stomach, while the greatest similarity was between the oral and gastric communities. However, the bacterial communities of healthy lungs shared significant membership with the mouth, but not the nose, and marked subject-subject variation was noted. In summary, microbial immigration from the oral cavity appears to be the significant source of the lung microbiome during health, but unlike the stomach, the lungs exhibit evidence of selective elimination of Prevotella bacteria derived from the upper airways.
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              Optimizing methods and dodging pitfalls in microbiome research

              Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0267-5) contains supplementary material, which is available to authorized users.
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                Author and article information

                Contributors
                +27 31 260 4399 , masekelar@ukzn.ac.za
                solizev@gmail.com
                fanus.venter@fabi.up.ac.za
                wilhelm.debeer@fabi.up.ac.za
                robin.green@up.ac.za
                Journal
                BMC Pulm Med
                BMC Pulm Med
                BMC Pulmonary Medicine
                BioMed Central (London )
                1471-2466
                22 May 2018
                22 May 2018
                2018
                : 18
                : 87
                Affiliations
                [1 ]ISNI 0000 0001 2107 2298, GRID grid.49697.35, Department of Paediatrics and Child Health, Faculty of Health Sciences, , University of Pretoria, ; Pretoria, South Africa
                [2 ]ISNI 0000 0001 0723 4123, GRID grid.16463.36, Department of Maternal and Child Health, Nelson R Mandela School of Medicine, College of Health Sciences, , University of KwaZulu-Natal, ; 719 Umbilo Road, Congella, Durban, 4013 South Africa
                [3 ]ISNI 0000 0001 2107 2298, GRID grid.49697.35, Department of Microbiology and Plant Pathology, , University of Pretoria, ; Pretoria, South Africa
                Author information
                http://orcid.org/0000-0001-9665-2035
                Article
                632
                10.1186/s12890-018-0632-6
                5964725
                29788934
                70fd070d-85ff-4e0f-9c73-e531b05ed4b4
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 7 April 2017
                : 25 April 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100008422, Genomics Research Institute, University of Pretoria;
                Award ID: Theme Genomica
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2018

                Respiratory medicine
                paediatrics,microbiology,hiv-associated bronchiectasis,bacterial diversity,lung microbiome

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