title: Bacterial Topography of the Healthy Human Lower Respiratory Tract.
abstract: Although culture-independent techniques have refuted lung sterility in health, controversy about contamination during bronchoscope passage through the upper respiratory tract (URT) has impeded research progress. We sought to establish whether bronchoscopic sampling accurately reflects the lung microbiome in health and to distinguish between two proposed routes of authentic microbial immigration, (i) dispersion along contiguous respiratory mucosa and (ii) subclinical microaspiration. During bronchoscopy of eight adult volunteers without lung disease, we performed seven protected specimen brushings (PSB) and bilateral bronchoalveolar lavages (BALs) per subject. We amplified, sequenced, and analyzed the bacterial 16S rRNA gene V4 regions by using the Illumina MiSeq platform. Rigorous attention was paid to eliminate potential sources of error or contamination, including a randomized processing order and the inclusion and analysis of exhaustive procedural and sequencing control specimens. Indices of mouth-lung immigration (mouth-lung community similarity, bacterial burden, and community richness) were all significantly greater in airway and alveolar specimens than in bronchoscope contamination control specimens, indicating minimal evidence of pharyngeal contamination. Ecological indices of mouth-lung immigration peaked at or near the carina, as predicted for a primary immigration route of microaspiration. Bacterial burden, diversity, and mouth-lung similarity were greater in BAL than PSB samples, reflecting differences in the sampled surface areas. (This study has been registered at ClinicalTrials.gov under registration no. NCT02392182.)IMPORTANCEThis study defines the bacterial topography of the healthy human respiratory tract and provides ecological evidence that bacteria enter the lungs in health primarily by microaspiration, with potential contribution in some subjects by direct dispersal along contiguous mucosa. By demonstrating that contamination contributes negligibly to microbial communities in bronchoscopically acquired specimens, we validate the use of bronchoscopy to investigate the lung microbiome.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA.
authors.forename: Robert P
authors.identifier: 0000-0002-6875-4277
authors.initials: RP
authors.lastname: Dickson
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Erb-Downward
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA.|Research Service, VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Freeman
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: McCloskey
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA.
authors.forename: Nicole R
authors.initials: NR
authors.lastname: Falkowski
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA.|Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.|Graduate Program in Immunology, Rackham Graduate School, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Gary B
authors.identifier: 0000-0002-4505-9642
authors.initials: GB
authors.lastname: Huffnagle
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA jlcurtis@umich.edu.|Graduate Program in Immunology, Rackham Graduate School, University of Michigan, Ann Arbor, Michigan, USA.|Pulmonary and Critical Care Medicine Section, Medical Service, VA Ann Arbor Healthcare System, Ann Arbor, Michigan, USA.
authors.forename: Jeffrey L
authors.identifier: 0000-0001-5191-4847
authors.initials: JL
authors.lastname: Curtis
mesh_terms: D000328:Adult; D000368:Aged; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D018893:Bronchoalveolar Lavage / Q000295:instrumentation / Q000592:standards; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D001999:Bronchoscopy / Q000009:adverse effects / Q000592:standards; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008168:Lung / Q000382:microbiology*; D008297:Male; D054892:Metagenome; D064307:Microbiota / Q000235:genetics; D008875:Middle Aged; D009055:Mouth / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S