The Human Respiratory Microbiome: Implications and Impact

Alicia B. Mitchell, BMedSci (Hons); Allan R. Glanville, MBBS, MD, FRACP

Disclosures

Semin Respir Crit Care Med. 2018;39(2):199-212. 

In This Article

Human Respiratory Microbiome

The respiratory microbiome includes all airway and lung-tissue associated microbes, and is more specifically defined as the lower respiratory tract beneath the larynx. Above this, the oropharyngeal and nasal-associated microbiota are separate to the lower respiratory tract microbiome and have received more attention previously due to ease of access.

The majority of studies investigating the microbiome have focused on the gut due to the relatively large biomass present and the ease of sampling. Conversely, there are many challenges when attempting to characterize the microbiome of the respiratory tract. In particular, there are difficulties in obtaining samples from the distal airways due to the risk of upper airway contamination. The dominant methodologies employed in sampling the lower respiratory tract include bronchoalveolar lavage (BAL) obtained via bronchoscopy, and spontaneously expectorated or induced sputum. Sputum presents a less invasive method of sampling the lower respiratory tract but has an increased risk of oropharyngeal contamination due to passing directly through the upper respiratory tract. However, when used as a sampling technique in diseases such as cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD), the biological signals detected have been significantly and meaningfully associated with multiple measures including severity of illness, airway inflammation, antibiotic use, and risk of subsequent exacerbations.[20–25] A further study found that expectorated sputum samples accurately represented the dominant microbes in the relatively homogenous respiratory tract of individuals with end-stage CF who had undergone lung transplantation; however, sputum samples over-represented the diversity and representation of atypical bacterial species.[26] Bronchoscopy is a more invasive option, which may have a lower rate of upper respiratory tract contamination. The bronchoscope is passed through either the oral or nasal cavity to sample the lungs, but once inserted, lavage is collected directly from distal airways without direct passage through the upper respiratory tract. A recent study[27] using serial BAL analysis demonstrated that sampling the lungs via bronchoscopy was not significantly confounded by the oral microbiome, consistent with previous serial bronchoscopy studies.[28] The evidence from these studies supports the understanding that minimal contamination from the upper respiratory tract is present in lower respiratory tract samples when utilizing bronchoscopic techniques.

Sampling of the lower respiratory tract and lung tissues has indicated that there are ~10–100 bacterial cells per 1000 human cells within the lungs,[29] a greatly reduced biomass compared with other colonized body compartments. This may be due to lower levels of nutrient sources in the lungs supporting microbiota growth when compared with the gastrointestinal tract. Variable physiological conditions are present within the lungs which may also affect bacterial presence. These factors include pH, relative blood perfusion, relative alveolar ventilation, temperature, oxygen concentration, epithelial cell structure, deposition of inhaled particles, and number of inflammatory cells.[30–32] These local selective pressures appear to play less of a role in healthy subjects. The greatest impact is seen in severe cases of chronic respiratory conditions where the lung microenvironment becomes maladapted due to remodeling of the airways and extracellular matrix.[33,34]

Additionally, lung-associated biomass appears to be significantly lower than the upper respiratory tract biomass. Total bacterial signal level as measured by quantitative polymerase chain reaction (qPCR) for 16S rRNA-encoding genes in DNA showed that the signal level was 100- to 1000-fold lower in BAL samples when compared with an oral wash.[27] A similar pattern was also observed with species richness, with a higher bacterial species richness in the oral cavity compared with both the lungs and the nasal cavity. This low concentration of microbiota within the lungs may be associated with the early understanding of lung sterility, but even after clear demonstration of a resident lung microbiota using sequencing-based approaches, the low biomass still presents further challenges in characterizing the burden and diversity of microbial species contained within the lower respiratory tract.

Culture-based protocols characteristic of clinical microbiology laboratories are optimized to detect acute infections with respiratory pathogens in individuals and to distinguish their absence. They rely on sufficient microbial load and specific growth conditions to encourage growth of these bacterial species.[35] These conditions selectively disadvantage the growth of anaerobes and bacteria which do not optimally flourish at 37°C, encompassing the majority of bacterial species which have now been determined to compose the healthy lung microbiota. Many of the species present within the healthy lung determined by culture-independent techniques have now been grown in culture under modified conditions. The first study to utilize these culture-independent sequencing methodologies to characterize the microbiota in the lungs of healthy subjects showed that the lungs contain a distinct and diverse microbiome. Furthermore, the bacterial composition in the lungs of healthy controls was compared with asthmatic subjects, demonstrating early evidence of variations in the lung microbiota in patients with chronic respiratory diseases, specifically the relative enrichment of Proteobacteria.[36]

Using culture independent techniques, it has since been determined that the most common bacterial phyla in the lower respiratory tract include Bacteroidetes, Firmicutes, and Proteobacteria. The most prominent genera present in healthy controls are Prevotella, Veillonella, and Streptococcus. Studies comparing concurrently collected upper and lower respiratory tract samples show that the microbial communities are distinct from each other, but there are similarities that prevail. This suggests that the traditional understanding of the respiratory tract comprised of discrete, independent compartments is likely outdated. A more accurate representation of the respiratory tract is a single continuous, internally heterogeneous ecosystem from the anterior nares through to the alveoli.[27] Spatial heterogeneity of the microbial topography within this system occurs within different areas, but there is constant movement of species between these areas.[11]

This clear spatial heterogeneity within the different lobes of the lung[37] and at different levels of the respiratory tract has been observed in chronic respiratory conditions. The turbulent act of coughing may play a role in homogenizing the microbial species within the lumen of the airways, thus decreasing spatial heterogeneity in healthy subjects.[28,37] In disease states such as CF, where thick, sticky secretions prevent effective clearance of microbes through mucociliary and coughing mechanisms, spatial differences in microbes may be more commonly observed.[38] Lung disease alters the population dynamics and the lung terrain,[37] which translate into unique environmental conditions in each disease state which facilitate changes to the local microbial communities.

The microbiota of the lungs is determined by a balance between immigration of species, likely from microaspiration and direct movement into the lungs, elimination due to mucociliary mechanisms, cough and the innate and adaptive immune responses and selective pressures present within the lungs themselves. Previous studies have shown that microbiota from the oral cavity is constantly being introduced into the lower respiratory tract, often during sleep as a result of microaspiration[9,10] and likely due to the direct mucosal extension between the oral cavity and the lungs. It has also been suggested that microaerosols generated in the oral cavity may be inhaled into the lungs and further contribute to the composition of the lung microbiome. The bacterial communities within healthy lungs demonstrate significant overlap with the bacterial species found in the oral cavity but not the nasal cavity. However, the relative abundance of bacterial phyla in healthy lungs appears to be significantly different from the oral bacterial community. Selective elimination of Prevotella species was observed in the lung,[27] providing further evidence for the influence of selective pressures within the lower respiratory tract in shaping the microbiome composition.

Elimination mechanisms are involved in clearance of microbial species from the respiratory tract. Mucociliary clearance and cough allow organisms to be mechanically removed from the lungs. Microbes are trapped within the mucus secreted from goblet cells and through the constant beating of cilia or coughing are moved up and out of the lower respiratory tract into the pharynx where they are either expectorated or swallowed. Host inflammatory cells and cytokines are involved in the immune response in the lungs leading to the clearance of potential pathogens. The composition of effector cells present within the airway appears to be associated with features of the microbiota.[28] Therefore, a constant flux in the lung microbiota from introduction of oral microbes and elimination of species present within the lungs as well as selective pressures induced by the lung microenvironment create a "steady state" during health (Figure 1).[27]

Figure 1.

Dynamics of the human respiratory microbiome. The human respiratory microbiome comprises all organisms that live in or on the human lung and respiratory tract including bacteria, fungi, archaea, viruses, and bacteriophages. Numerous forces combine to create a dynamic situation such that the net result represents the balance between acquisition, elimination, and local defense measures designed to maintain an equilibrium between resident and transient species.

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