Topographical Continuity of Bacterial Populations in the Healthy Human Respiratory Tract

  • Charlson E
  • Bittinger K
  • Haas A
 et al. 
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Abstract

Rationale: Defining the biogeography of bacterial populations in human body habitats is a high priority for understanding micro-bial–host relationships in health and disease. The healthy lung was traditionally considered sterile, but this notion has been challenged by emerging molecular approaches that enable comprehensive ex-amination of microbial communities. However, studies of the lung are challenging due to difficulties in working with low biomass samples. Objectives: Our goal was to use molecular methods to define the bacterial microbiota present in the lungs of healthy individuals and assess its relationship to upper airway populations. Methods: We sampled respiratory flora intensively at multiple sites in six healthy individuals. The upper tract was sampled by oral wash and oro-/nasopharyngeal swabs. Two bronchoscopes were used to collect samples up to the glottis, followed by serial bronchoalveolar lavage and lower airway protected brush. Bacterial abundance and compo-sition were analyzed by 16S rDNA Q-PCR and deep sequencing. Measurements and Main Results: Bacterial communities from the lung displayed composition indistinguishable from the upper airways, but were 2 to 4 logs lower in biomass. Lung-specific sequences were rare and not shared among individuals. There was no unique lung microbiome. Conclusions: In contrast to other organ systems, the respiratory tract harbors a homogenous microbiota that decreases in biomass from upper to lower tract. The healthy lung does not contain a consistent distinct microbiome, but instead contains low levels of bacterial sequences largely indistinguishable from upper respiratory flora. These findings establish baseline data for healthy subjects and sam-pling approaches for sequence-based analysis of diseases. Intensive efforts are being directed at understanding microbial populations living in association with the human body, the hu-man microbiome (1, 2). Microbial populations play important roles in health. In the gastrointestinal tract they instruct immune development, promote intestinal angiogenesis, and assist diges-tion (3–6). Disturbance of the healthy gut or genital tract micro-biome, as with antibiotic therapy, can allow colonization with Clostridium difficile or Candida spp. Whether there is a normal microbiome in the lower respiratory tract (LRT) that contributes to health, or to disease when disrupted, has been unclear. Traditional culture-based studies and classic teaching indicate that the healthy LRT is sterile (7–9), but recent culture-independent techniques have suggested otherwise (10, 11). Using molecular methods based on DNA sequencing or microarrays, it is possible to investigate microflora composition without requiring culture of individual microbes (12, 13). In such studies, DNA is prepared from the sampled microbiota, and composition analyzed by deep sequencing or DNA hybridization, allowing comprehen-sive profiling of full communities. Prior knowledge of organisms expected or the ability to culture and identify individual bacteria is not necessary. These approaches are being increasingly applied to the lung, and several reports have proposed that distinctive mi-crobial populations reside in the healthy LRT. However, studies of the lung microbiome face challenges not present in body sites that are easily accessible or contain high bac-terial biomass such as skin, gut, oral cavity, or genital tract. Sam-pling by bronchoscopy requires passage through the upper respiratory tract (URT), which harbors large microbial popula-tions and where contaminating organisms may be acquired. In ad-dition, samples from lung may be of low microbial biomass, meaning that low-level admixture of sequences from dust,

Author-supplied keywords

  • 16s rdna
  • healthy lung colonization
  • microbiome

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Authors

  • Emily S Charlson

  • Kyle Bittinger

  • Andrew R Haas

  • Ayannah S Fitzgerald

  • Ian Frank

  • Anjana Yadav

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