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Curr Opin Microbiol. Author manuscript; available in PMC 2021 August 01.
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Published in final edited form as:

Curr Opin Microbiol. 2020 August ; 56: 24–29. doi:10.1016/j.mib.2020.06.001.

Perspectives in lung microbiome research

Imran Sulaiman1, Sheeja Schuster1, Leopoldo N. Segal1
1Division of Pulmonary, Critical Care, & Sleep Medicine, Department of Medicine, New York

University School of Medicine, NY

Abstract
Our understanding of the existence and role of the lung microbiome has grown at a slower pace
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than other microbiome research areas. This is likely a consequence of the original dogma that the
lung was a sterile environment although there are other barriers that are worth discussing. Here we
will not be conducting an exhaustive review of the current literature on the lung microbiome but
rather we will focus on what we see as some important challenges that the field needs to face in
order to improve our mechanistic understanding of the lung microbiome and its role on human
health.

Keywords
microbiome

Lung microbiome research is at its infancy. While the gut microbiota is resilient and varies
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broadly between different individuals [1], we are just now starting to appreciate that the
lungs are not sterile and in fact harbor a complex microbial landscape. Additionally, we now
know that the gut microbiome modulates the systemic immune tone contributing to the
differentiation of IL17 producing T cells and regulatory T cells [2], whereas the lung
microbiome field has so far only show associations between lung microbiota signatures and
host immune phenotype [3-5]. It is understood that gut dysbiosis affects the metabolic local
and systemic microenvironment [6,7] and contributes to the susceptibility to pathogens such
as Clostridium difficile [8], however we do not know whether lung microbiota dysbiosis
contributes to the acquisition of respiratory pathogens. Finally, while we are now uncovering
how the gut microbiota modulates the effectiveness of drugs such as immunomodulators for
cancer [9,10], we currently have not studied the effects of the lung microbiota on drugs such
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Corresponding Author/ Address for Reprints: Leopoldo N. Segal, MD1 Leopoldo.Segal@nyumc.org, NYU School of Medicine 462
First Ave 7W54 New York, NY 10016, Tel: (212) 562-3752, Fax: (212) 263-7445.
Financial Disclosure: None
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.
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 Sulaiman et al. Page 2

as inhaled therapy for COPD, asthma or even on immunomodulators that have become the
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front line therapy for non-small cell lung cancer.

In order to improve our mechanistic understanding of the lung microbiome we first need to
understand the reasons for its delay in development. Here we identify some of the key
obstacles in lung microbiome research that are important to consider.

Difficulty in sample collection

Analysis of the gut microbiome has concentrated on the use of stool samples [2,6-8,10,11], a
somewhat easily acquired specimen that is a direct product of transit through the entire
gastrointestinal tract. Although some have noted regional differences in the lower
gastrointestinal microbiota [12], the use of stool samples as a surrogate of the microbiome of
the gastrointestinal tract is widely accepted. In lung microbiome research, a large number of
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investigations have concentrated on the use of sputum samples [9,13,14] given the
accessibility of these samples, whether this be spontaneous (easier among subjects with
respiratory conditions with high mucus production such as cystic fibrosis) or induced.
However, the heterogeneity of the microbial topography throughout the respiratory tract
complicates this approach. For example, the upper airway microbiota (including the dental
microbiome, oral microbiome and supraglottic microbiota) is invariantly colonized with
anaerobes that coexist at high concentration (similar to the ones measured in the lower
gastrointestinal tract) [15-17]. In contrast, the lower airway microbiota has significantly
reduced bacterial concentration and with clear differences in diversity when compared to the
upper airway microbiota (Figure 1A) [18-21]. Since expectorated sputum is the result of the
sum of secretions throughout the respiratory tract, multiple topographical sources can
contribute to microbes found in the microbial assessment of these samples. Thus, sputum
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can be: 1) a perfect representation of the upper airways (including oral and dental); 2) a
mixture of upper and lower airway microbes; or 3) a perfect representation of the lower
airways. Given the significantly higher bacterial burden in the upper airways than in the
lower airways most evidence supports that the first two scenarios are the most likely. This is
further confirmed by two investigations that collected paired upper and lower airway
samples (one in a cohort of subjects with asthma and a second on patients with
bronchiectasis) and demonstrated that the microbial similarity between sputum and upper
airway samples was greater than the similarity between sputum and lower airway samples
[22,23]. If the microbes present in sputum are a variable mixture of microbes from the lower
and upper airways, it is unclear how to determine the relative contribution of those two
different topographical sources to the final collection of microbes observed in sputum.
Further, recent investigations have suggested that the method for sputum collection
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(spontaneous vs. induced) will affect the results [24]. Some have proposed the exhaled
breath condensate (EBC) as a less invasive alternative for lung microbiome sampling
[25-28]. This involves condensing exhaled vapor into a liquid which is then sent for
sequencing, a much less invasive procedure when compared to bronchoscopy. However, in
an animal model the microbial composition of EBC samples was not representative of the
microbiota in the lower airways [29]. A comparison of bronchoscopy samples to EBC
samples in humans has not yet been performed. Thus, we currently do not have any accurate
surrogates for lung microbiome evaluation. This does not necessarily mean that sputum

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samples are without use in the evaluation of the lung microbiome, but it is our opinion that
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investigators should be mindful about these limitations when planning their research. While
sputum has been used in culture-based approaches for decades. The focus of those studies
was primarily the detection of microbes with potential pathogenic role rather than a
comprehensive description of the microbial communities. Additionally, with current sputum
culture approaches we have accepted that the microbial yield from the lower airways is
variable as exemplified by the clinical need to have repeated sputum cultures in
Tuberculosis.

Low biomass samples

Most microbes in the human body are contained in the gastrointestinal tract. Many other
mucosae in topographical contact with the exterior world are also exposed to microbes but
the microbial biomass contained in those are significantly lower [30,31]. This imposes
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several challenges for sample processing and data analysis. One such challenge is the
presence of bacterial DNA contamination from exogenous sources acquired through sample
collection and sample processing (Figure 1A). In a lower airway sample, these
“contaminant” bacterial DNA may be in higher concentration than the bacterial DNA
originating form “true” lung bacteria. Thus, it is expected that the amplification process and
subsequent sequencing will be biased towards these contaminants resulting in a large
proportion of the sequenced microbiota being dominated by them [32-34]. Importantly, the
nature of these contaminant bacterial DNA will depend on the method used. In addition,
further noise, in the sequencing output, could be expected from PCR error, index switching,
and chimera formation [35,36]. The downstream effects on the data will then be variable,
affecting diversity metrics and the relative abundance of the true bacteria present in the
lower airways. Different methods have been developed to identify a bacterial signal as
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contaminant which are dependent on the quality and quantity of data on background samples
(e.g., DNA isolation negative controls, bronchoscopy or instrument controls). These
bioinformatic methods include abundance filtering, where a threshold is set to remove low
abundance bacteria (probably not useful in low biomass samples); removing all sequences
present at high prevalence in negative control samples (again a threshold can be set);
removing sequences based on an inverse correlation with bacterial DNA concentration; and
Bayesian methods. With these different methods, one could then remove sequences
identified as contaminants from the rest of the data set. However, given the compositional
nature (and thus not quantitative) of the sequencing data the effects on what’s left after
removal can be quite variable and difficult to predict or reproduce. In fact, a recent
consensus of lung microbiome experts have not recommend routine bioinformatic removal
of contaminants [37].
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What do we know and why is it important to foster lung microbiome

research:
Even though we know now, with culture independent techniques, that the lungs are not
sterile, there are significant microbial ecological differences with other mucosae where the
presence of microbes has never been questioned. For example, in health, while the gut

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microbiota harbors a large microbial biomass that occupies the mucosal lumen, shows high
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dynamics but also high resilience [38], the microbiota of the healthy lung behaves like a near
desert island. This is not surprising, since the main function of the lung is to allow for gas
exchange between our blood and the ambient air. Thus, the mucosal lumen of the airways
should be occupied by mostly air. It is now well accepted that the presence of microbes in
the lower airways of healthy lungs behave similar to an island with a tight balance of
microbial immigration (mostly from microaspiration of oral secretions) and elimination [39].
Further, the lung is not a single continuous longitudinal topographical system but rather a
branching series of parallel topographical systems that allows large amount of regional
microbial heterogeneity. Additionally, several conditions for bacterial growth vary within the
lung, such as blood flow, local pH, temperature and oxygen tension [40]. This, together with
individual patient factors such as inadequate airway clearance/subclinical aspiration or the
presence of chronic lung disease (such as COPD or bronchiectasis), can explain significant
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heterogeneity within each subject and between subjects with similar diagnosis.

Despite these limitations, it is now clear that the lung microbiome affects the host immune
tone in the lower airways [41-43]. For example, several studies have shown that aspiration is
a common phenomenon [44,45], and the presence of oral commensals in the lower airways
(even in healthy lungs) is associated with a distinct metabolic profile and with an increased
immune tone characterized by IL17 producing T cells [46]. Multiple lines of investigations
support the effect of the lower airway microbiota on the host in multiple disease states such
as asthma, COPD, bronchiectasis, interstitial lung disease, lung cancer, etc [23,47-50].
However, most of these studies are based on cross-sectional human studies where
directionality of the associations is difficult to establish. To aid in understanding causality
better, there is a need for more longitudinal studies. Further, while we can support that the
lung microbiome affects the host immune tone, the molecular mechanisms are not clearly
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established. Research into the metabolic profiling of the lower airway environment has
shown that the lower airways in fact contain multiple metabolites that could be end products
of microbial metabolism or else affected by microbial metabolic activity [51,52]. One
example of this is the identification of short chain fatty acids (SCFAs) in the lower airways,
which are end product of microbial fermentation done by anaerobic bacteria and have
significant immunomodulatory effects [53].

Next steps in lung microbiome research:

The lung microbiome field has been dominated by descriptive cross-sectional studies
focused on the “1,000 feet view” of the microbiome. It is becoming increasingly clear that it
is important to not only identify the presence of organisms in different environments but to
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also understand the function of these organisms and how they may impact the host. How can
we accomplish these goals? Shotgun sequencing methods have been developed over the last
two decades [54-56]. The use of this untargeted sequencing approach of all microbial genes
provides information on both taxonomic composition (with increased resolution when
compared to 16S rRNA gene sequencing) as well as functional potential of different
microbes. While this technology is useful in better understanding how microbes function
there has been very few publications using this technology in analyzing the airway
microbiome, and in most cases focused on its use for taxonomy [57-61], primarily due to

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issues mentioned previous (low biomass and contamination). It is also important to note that
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by studying genomic DNA we are not able to identify active microbial function nor whether
these sequences are from viable or non-viable microbes [62]. Another possible approach to
study microbial communities is through the use of metatranscriptomic RNA sequencing
approaches, which allow for characterization of microbial gene expression [62]. Using such
methods, we are able to ascertain which microbial genes are actively being transcribed,
therefore proving information on ongoing microbial function, and microbial viability (given
the short live nature of RNA). Even fewer investigations exists using metatranscriptomic
approaches in the lung and it is likely that the dynamics of these microbial signatures will
follow a different dynamic than DNA signatures (Figure 1B) [63,64]. Ultimately, microbial
functional molecules will need to be studied measuring proteins (metaproteomics) and
metabolites (metabolomics) both of which have rarely been used in lung microbiome
research [46,53].
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While these novel methods will likely have significant impact on the lung microbiome field,
classical mechanistic investigations will be required to help progress the understanding of
the lower airway microbiome. These include the use of preclinical models [50,65,66],
longitudinal studies [67,68], and, ultimately, clinical trials modulating the microbial
environment.

Prior studies have provided a full state of the art review of lung microbiome [37,69]. In this
publication we provide a critical view of where the research field is, the major limitations
present and the strong belief that the search for mechanistic insights will depend on moving
on from descriptive, hypothesis free, cross sectional studies and focusing more on
hypothesis-driven, mechanistic studies.
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Acknowledgments
Research support funding: R37 CA244775 (LNS, NIH/NCI); R01 HL125816 (SBK, LNS, NIH/NHLBI); PACT
grant (LNS, FNIH); Chest Foundation Grant (IS).

Financial support for the PACT project are made possible through funding support provided to the FNIH by:
AbbVie Inc., Amgen Inc., Boehringer-Ingelheim Pharma GmbH & Co. KG, Bristol-Myers Squibb, Celgene
Corporation, Genentech Inc., Gilead, GlaxoSmithKline plc, Janssen Pharmaceutical Companies of Johnson &
Johnson, Novartis Institutes for Biomedical Research, Pfizer Inc., and Sanofi.

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Figure 1: Technical challenges for lung microbiome research using culture independent
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techniques.
A. Variable biomass through the respiratory tree affect the composition and signal to noise
ratio of each type of sample. B. While bacterial clearance in the lower airways is a well-
established physiological event the dynamics of the microbial molecular signatures need to
be studied.
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Curr Opin Microbiol. Author manuscript; available in PMC 2021 August 01.