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.)

The lung microbiome in health and disease

“…are characteristic. 13,[16][17][18] In common with other mucosal surfaces, many of the consistently observed OTUs are facultative anaerobes. Anaerobic incubation is not a routine component of standard clinical microbiology for sputum, perhaps explaining the early presumption that normal healthy lungs were sterile.…”

“…However, the use of bronchoscopy samples for investigation of the lung microbiome has been validated by careful studies of the bacterial topography in the healthy human airways. 13,16,17 The existence of a healthy lung microbiome is now generally accepted, together with recognition that its disturbances influence many lung diseases. There is consensus from 16S rRNA studies that, at the family level, the healthy lung is typified by the presence of Firmicutes , Bacteriodetes , Proteobacteria , Fusobacteria and Actinobacteria , while, at the OTU level, Prevotella , Veillonella and Streptococcus spp.…”

“…In health, the oropharynx and intrathoracic airways might carry very similar microbiota 15,16 (although nasal swabs do not reflect the lower airway microbiome 17 ) and oropharyngeal swabs might be useful epidemiologically. However, in disease states, the oropharynx does not consistently match the lower airways 15 and regional differences might be present within the lung itself.…”

“…However, in disease states, the oropharynx does not consistently match the lower airways 15 and regional differences might be present within the lung itself. 17 Sputum is used clinically as an effective surrogate for airway samples, despite acknowledged contamination from upper airways and saliva. In the absence of sputum production, a reliable means of lower airway sampling remains an obstacle to large-scale investigations of lung disease or serial studies requiring frequent sampling.…”

Commensal Fungi in Health and Disease

“…In healthy hosts with no structural lung disease and intact immune defenses, the extent to which these fungal organisms represent transient inhaled environmental or microaspirated upper airway organisms versus a self-renewing microbial community is not known. A recent well-designed study of the respiratory tract bacterial microbiome suggests that microaspiration is the primary source of lung bacteria in healthy humans, but this study did not examine fungi which may predominantly arrive in the lung via a different source such as inhalation of environmental spores (Dickson et al, 2017). It should be noted that the profile of fungal organisms in the healthy lower respiratory tract is likely distinct from the upper airway, as Candida is usually the dominant fungal genus identified in oral wash specimens from healthy individuals (Charlson et al, 2012; Ghannoum et al, 2010).…”