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The role of the local microbial ecosystem
in respiratory health and disease
rstb.royalsocietypublishing.org
Wouter A. A. de Steenhuijsen Piters1, Elisabeth A. M. Sanders1,2
and Debby Bogaert1
1
Review
Cite this article: de Steenhuijsen Piters WAA,
Sanders EAM, Bogaert D. 2015 The role of the
local microbial ecosystem in respiratory health
and disease. Phil. Trans. R. Soc. B 370:
20140294.
http://dx.doi.org/10.1098/rstb.2014.0294
Accepted: 8 April 2015
One contribution of 17 to a theme issue
‘Within-host dynamics of infection: from
ecological insights to evolutionary predictions’.
Subject Areas:
microbiology, ecology, health and disease
and epidemiology
Keywords:
microbiome, upper respiratory tract,
bacterial interaction, colonization resistance,
pathogenesis, respiratory infections
Author for correspondence:
Debby Bogaert
e-mail: [email protected]
Department of Paediatric Immunology and Infectious Diseases, The Wilhelmina Children’s Hospital/University
Medical Centre Utrecht, Utrecht, The Netherlands
2
Centre for Infectious Disease Control, National Institute of Public Health and the Environment (RIVM),
Bilthoven, The Netherlands
Respiratory tract infections are a major global health concern, accounting
for high morbidity and mortality, especially in young children and elderly individuals. Traditionally, highly common bacterial respiratory tract infections,
including otitis media and pneumonia, were thought to be caused by a limited
number of pathogens including Streptococcus pneumoniae and Haemophilus influenzae. However, these pathogens are also frequently observed commensal
residents of the upper respiratory tract (URT) and form—together with harmless commensal bacteria, viruses and fungi—intricate ecological networks,
collectively known as the ‘microbiome’. Analogous to the gut microbiome,
the respiratory microbiome at equilibrium is thought to be beneficial to the
host by priming the immune system and providing colonization resistance,
while an imbalanced ecosystem might predispose to bacterial overgrowth
and development of respiratory infections. We postulate that specific ecological
perturbations of the bacterial communities in the URT can occur in response to
various lifestyle or environmental effectors, leading to diminished colonization
resistance, loss of containment of newly acquired or resident pathogens, preluding bacterial overgrowth, ultimately resulting in local or systemic bacterial
infections. Here, we review the current body of literature regarding nichespecific upper respiratory microbiota profiles within human hosts and the
changes occurring within these profiles that are associated with respiratory
infections.
1. Introduction
The human body harbours a large variety of microbial communities: intricate
interacting networks consisting of a myriad of bacteria, fungi, viruses, bacteriophages, archaea and eukaryotes, that colonize different body surfaces,
including the skin [1], vagina, oral cavity, gut [2], upper respiratory tract
(URT) [3] and lung [4]. The recent advent of high-throughput sequencing
methods has made it possible to study these communities and their relationship
with health and (chronic) disease in detail. Particular interest has been paid to
the bacterial communities or ‘microbiome’ of the gut [5], and its role in metabolism [6], immune maturation, mucosal barrier functions [7] and colonization
resistance [8,9]. These studies have led to a paradigm shift where the old
‘one pathogen/one disease’ theory as postulated by Dr Robert Koch in the nineteenth century, has been gradually replaced by the theory that human health is
the outcome of a complex, interconnected network of interactions between
microbes and their host [10].
Although much less extensively studied, we postulate that the URT microbiome is, analogously to the gut microbiome, a strong determinant of
respiratory health. When the microbiome is perturbed, for example by antibiotic
treatment or disease state, potential pathogens (hereafter termed ‘pathobionts’)
such as Streptococcus pneumoniae, can overgrow, spread and ultimately result in
acute local or disseminated respiratory infections, such as acute otitis media
(AOM), pneumonia, septicaemia or meningitis.
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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N
pH
M
S Spn
H2O2
D
CB
+
BF
Hi
(a)
M
CB
S
Sa
PB pH
N
+
+
sebum O2 + moisture
Sa
+
FF
nasal cavity
F
Sv
Hi
(c)
nasopharynx
V
Spn
P
L
B
pH ? Mc
–
elderly +
Spy
anterior nares
oral cavity
oropharynx
Sag
Sd
hypoharynx
LB
trachea
oesophagus
Figure 1. Niche-specific bacterial community structure of the anterior nares, the nasopharynx and the oropharynx (which resembles the oral cavity). Microbial
interaction networks associated with these anatomical locations are depicted, with potential driving environmental forces (acidity, oxygen availability and presence
of antimicrobial compounds). Coloured circles represent individual bacterial community members, which are interconnected by dotted blue and red lines, respectively, representing positive and negative associations. Microbial community members are colour-coded based on phylum level; Firmicutes: red/brown, Actinobacteria:
yellow, Bacteroidetes: green, Proteobacteria: blue and Fusobacteria: purple. S: Streptococcus spp.; Spn: S. pneumoniae; Sv: S. salivarius; Spy: S. pyogenes; Sd:
S. dysgalactiae; Sag: S. agalactiae; Sa: S. aureus; V: Veillonella spp.; D: Dolosigranulum spp.; LB: Lactobacillus spp.; CB: Corynebacterium spp.; PB: Propionibacterium
spp.; B: Bacteroidetes; P: Prevotella spp.; M: Moraxella spp.; Mc: M. catarrhalis; Hi: H. influenzae; N: Neisseria spp.; L: Leptotrichia spp. and F: Fusobacterium spp.
The URT exhibits many important physiological functions
such as filtering, humidifying and warming of inhaled air
[11] and consists of a communicating system comprising
the anterior nares, nasal cavity, nasopharynx, sinuses, Eustachian tube, middle ear cavity, oral cavity, oropharynx and
larynx, forming the interface between the external environment and the lower respiratory and gastrointestinal tract
(figure 1) [12,13]. The mucosal surfaces of these niches are
colonized with a wide array of bacteria, mostly members of
the phyla Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria and Fusobacteria [3,14,15]. However, large differences in
microbial profiles can be observed between niches at lower
taxonomic levels. We hypothesize that these differences arise
from specific niche characteristics, resulting from variations
in humidity, acidity and epithelial cell type. Interestingly, the
interplay between these abiotic components and microbiota
seems to adhere to known principles in general ecology, such
as environmental selection, dispersal, speciation and drift
[16], which enables us to describe and potentially even predict
ecosystem dynamics in health and disease.
In this review, we will focus on the ecological interactions
within the bacterial URT microbiome in health and in acute
respiratory diseases. First, we describe the relevant ecological
processes that direct bacterial community assembly in the
human URT (§2). Then, in §§3 –6 we discuss niche-specific
variations in microbial composition and elaborate on possible
mechanistic explanations for the observed co-occurrence and
co-exclusion patterns. Finally, we will describe changes in
bacterial community composition in relation to local and
disseminated infectious diseases (§§7,8).
2. The human upper respiratory tract niches
from an ecological perspective
The human URT is a unified system of skin and mucosal
surfaces that stands in direct and continuous contact with
the outer world. The nostrils or anterior nares are closest
to the outside environment and skin surface, and are lined
with skin-like keratinized squamous epithelium, containing
sebaceous and serous glands [17]. This niche acts as the
vestibule to the nasal cavity, which is characterized by
pseudostratified ciliated columnar epithelium containing
mucin-secreting goblet cells (respiratory epithelium) [18].
The nasal cavity serves as the collection site for secretions of
the frontal, maxillary, ethmoidal and sphenoidal sinuses and
constitutes a continuum with the more posteriorly located
nasopharynx, which reaches from the choanae anteriorly to
the posterior wall of the nasopharynx and is covered with adenoid gland tissue. The lateral wall of the nasopharynx contains
the pharyngeal ostium of the Eustachian tube, connecting
the nasopharynx to the middle ear. Like the nasal cavity, the
lateral nasopharyngeal cavity is carpeted with respiratory
epithelium, but patches of stratified squamous epithelium
appear with increasing age [18]. The nasopharynx is separated from the oropharynx by the soft palate, which enables
Phil. Trans. R. Soc. B 370: 20140294
middle ear
frontal sinus
sphenoidal sinus
2
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adults –
(b)
Mc
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3. The human upper respiratory tract niches: the
anterior nares
The microbial ecosystem of the anterior nares is typically
enriched for members of the phyla Actinobacteria (i.e. Corynebacterium and Propionibacterium spp.) and Firmicutes (i.e.
Streptococcus in children and Staphylococcus spp. in adults)
with a low abundance of anaerobes belonging to the Bacteroidetes phylum [28,31,32]. The number of Proteobacteria
strongly varies, with studies reporting high abundance of
Moraxellaceae in children [31,33], enrichment for Proteobacteria in adult ICU-patients (i.e. members of the orders
Enterobacteriales and Pseudomonadales) [34] and low abundance of members of the class Gammaproteobacteria in
healthy adults [32].
Corynebacterium, Propionibacterium and Staphylococcus are
the most frequently carried genera in the anterior nares, which
is independent of ethnical backgrounds and geographical
location [31,35]. The microbiome of the adult anterior nares
can generally be sub-classified in one of four distinct microbial
profiles or ‘types’, which are characterized by predominance of either Corynebacterium, Propionibacterium, Moraxella or
Staphylococcus spp., suggesting that complex synergistic and
antagonistic relationships between these bacteria are driving
within-niche variation [35]. Interestingly, Corynebacterium- and
Propionibacterium-enriched profiles seem to be more ‘tolerant’
to members of other genera, like staphylococci, in contrast to
Moraxella-dominated bacterial communities.
The keratinized squamous epithelium of the anterior nares
includes sebum-producing glands that seem responsible for the
selective enrichment of lipophilic bacteria, like Propionibacterium
spp. [14,36]. Propionibacterium spp. are capable of hydrolysing
lipid-rich sebum, hence releasing short-chain free fatty acids
[37]. As a consequence, the pH is lowered, inducing outgrowth
of especially corynebacteria and coagulase-negative staphylococci, whereas the relatively moist and oxygen-rich conditions
in the anterior nares will more specifically select for outgrowth
of Staphylococcus aureus and corynebacteria [38]. Thus, the simultaneous presence of Propionibacterium and Staphylococcus spp.
might be at least partly driven by different features of the local
environment, although true synergism between both genera
might also occur by the production of coproporphyrin III by
Propionibacterium spp., promoting S. aureus biofilm formation
[39]. The observation that co-presence between Corynebacterium
and S. aureus is species-specific (it holds for Corynebacterium
accolens but not for Corynebacterium pseudodiphteriticum) [14],
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Phil. Trans. R. Soc. B 370: 20140294
and ecological niche-specialization, though little evidence is
available to support this hypothesis.
In concert, these four ecological processes structure interaction networks of bacterial species, resulting in ecosystems
that can be characterized by their biodiversity. In ecosystem
theory, biodiversity is a term used to quantify both species
richness (i.e. number of species) and evenness (i.e. distribution of species) and is thought to give rise to ecosystem
stability [27,28] and enhance ecosystem functioning [29,30].
In the URT, bacterial richness and evenness vary dramatically
between niches, with the highest richness and evenness
observed in the oropharynx and oral cavity [3,28]. Contrastingly, the anterior nares harbour a bacterial microbiome
exhibiting low biodiversity and are thereby more comparable
to other areas covered by skin-like epithelium [1,28].
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us to breathe through our nose and prevents nasal regurgitation of food [12]. Both the oropharynx and the oral
cavity are lined with non-keratinized stratified squamous
epithelium [18] and are part of both the respiratory and
gastrointestinal tracts.
The URT is an interesting area from an ecological perspective: the constant exposure to the external environment and its
accompanying conditions, and influences of respiration and
gastrointestinal processes shape different habitats for a wide
palette of microorganisms. Community ecology provides a
large number of theoretical frameworks to explain bacterial
community assemblage, composition, diversity and its underlying processes. Historically, it was believed that microbes
were cosmopolitan and arranged in ecosystems that were
shaped purely by the force of environmental selection [19].
However, more recently, the idea that microbiological ecosystems are structured by a combination of both selective and
chance-driven processes—selection, ecological drift, speciation
and dispersal—has been increasingly embraced [20].
Of the processes involved, dispersal (i.e. movement of
organisms across space) seems responsible for initial bacterial
colonization of the URT. Directly after birth, the skin, gut,
nasopharynx and mouth microbial communities are undifferentiated and colonized by bacterial species reflective of
delivery mode: in vaginally delivered children Lactobacillus,
Prevotella, Atopobium and Sneathia are predominant genera,
whereas in infants born by caesarean section typical skin
inhabitants such as staphylococci are initially observed [21].
Immaturity of the host’s immune system enables tolerance
of these initial colonists in the absence of a manifest inflammatory response; instead, early microbial exposure is believed to
be vital to consecutive development of the immune system
[22], which in turn contributes to local bacterial containment
in a later stage through selection. Selection results from
between-species differences in functionality or microbial fitness and drives variation in growth or replication rates,
further shaping the bacterial community structure, both with
regard to composition and relative abundance of species.
Environmental pressure formed by constant exposure of the
URT to the external environment and its accompanying conditions, as well as differentials in oxygen, pH, humidity,
immunological factors, nutrients and epithelial cell type, ultimately shape different (micro)habitats within the URT [18,23].
Within these habitats, additional selection is mediated by
interspecies interactions that can vary in both strength and
directionality, i.e. can either be mutual, commensal or antagonistic [24]. Existing microbial assemblages are constantly
challenged by new colonizers originating from both exogenous (i.e. inhalation of airborne microorganisms and contact
with food-associated microbes) [25,26] and endogenous (i.e.
draining middle ears and sinuses, regurgitation from stomach
content and sputum from the lungs) sources. Apart from
dispersal and selection, a third ecological process, speciation,
might be involved in the development of a given microbial
community. Speciation is the result of genetic isolation and
local diversification, the latter of which is caused by adaptation
of a bacterial species to its new environment by mutation and
recombination (i.e. horizontal gene transfer). The fourth ecological process affecting bacterial community assembly is
ecological drift, a chance-driven process that occurs in a community of functionally equivalent species. Speciation and
ecological drift might play a role in development of respiratory
bacterial communities after initial community assemblage
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In line with the gut and anterior nares, variations in microbial
profiles can be observed in the nasopharynx of individuals
from early in life onwards. These initial nasopharyngeal bacterial assemblages are typified by predominance of
species belonging to the genera Moraxella, Corynebacterium,
Dolosigranulum, Streptococcus or Staphylococcus spp. [46] and
are likely related to dispersal of bacterial species derived from
the maternal skin (Staphylococcus and Corynebacterium spp.)
or vaginal microbiome (Staphylococcus, Streptococcus and
Dolosigranulum spp.) during the perinatal period. Additionally,
the type of infant feeding seems to have an important role in
both community establishment and subsequent development:
exclusive breastfeeding at six weeks of life was associated
with increased Corynebacterium/Dolosigranulum profiles, whereas
S. aureus predominance was observed in infants who were exclusively formula fed [26]. Although several of the early profiles
disappeared within the first months of life, initial profile signatures were indicative of successive nasopharyngeal microbiome
composition and stability. More stable patterns were marked by
early high abundance of Moraxella and Corynebacterium/Dolosigranulum spp., whereas the less stable profiles, characterized by
high abundance of Streptococcus spp. and acquisition of Haemophilus influenzae over time, were associated with higher rates of
parent-reported respiratory infections and wheezing in the first
years of life [46]. In adults, apart from a clear absence of Moraxella
spp., similar bacterial inhabitants of nasopharyngeal microbiota
were identified, although the power is lacking to clearly identify
distinct microbiome profiles in this age group [3,23,47–49].
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4. The human upper respiratory tract niches: the
nasopharynx
Species observed in the nasopharynx show high overlap
with the inhabitants of topographically proximate niches like
the anterior nares (mainly Gram-positive aerobes, including Staphylococcus, Dolosigranulum, Corynebacterium and
Propionibacterium spp.) and the oropharynx (Streptococcus spp.).
Gram-negative anaerobes that are encountered primarily in the
oropharynx and oral cavity, such as Prevotella and Veillonella
spp., were also observed in low abundance in the nasopharynx
of young children [23,26], which might be explained by frequent
nasal regurgitation of oral content in this age group.
Mechanistic insights into the observed interactions between
putative commensals and pathobionts are sparse. Cultivationbased colonization studies typically date from before the era of
next-generation sequencing techniques and exclusively focused
on a selection of well-known pathobionts, including S. pneumoniae, Moraxella catarrhalis, H. influenzae, S. aureus and haemolytic
streptococci. Well recognized is the co-exclusion of S. pneumoniae
and S. aureus in the URT of immunocompetent individuals [50],
which may partially be mediated by hydrogen peroxide
production of S. pneumoniae [51], although CD4þ T-cell involvement is suggested by the apparent lack of this interaction in HIVinfected patients [52]. Exclusion patterns are also observed
between S. pneumoniae and H. influenzae and could be driven
by competition for adherence to the human platelet-activating
factor receptor (PAF-r) [53,54], but also by modulation of the
host innate immune system; for example, in animal models,
H. influenzae colonization has been demonstrated to induce
neutrophil recruitment and potentiate opsonophagocytic killing
of co-colonizing pneumococci [55,56]. In return, resistance to
H. influenzae-induced opsonophagocytosis is conveyed by natural selection for thicker pneumococcal capsules, consequently
enhancing virulence and metabolic costs [57]. Xu & Pichichero
[58] have recently shown the additional involvement of the
adaptive immune system in bacterial interactions in healthy
children, with pneumococcal-specific IgG and IgA antibody
responses being enhanced by co-colonization of S. pneumoniae
together with H. influenzae or M. catarrhalis.
On the other hand, synergistic co-occurrence patterns
between species have also been observed, for example between
M. catarrhalis and H. influenzae. This interaction is conferred
indirectly by secretion of ubiquitous surface proteins carried
by outer membrane vesicles (OMVs) of M. catarrhalis, that
bind to the third component of complement (C3), hampering
complement-mediated killing of H. influenzae [59]. Moreover,
Schaar et al. [60] described that these OMVs carry b-lactamase,
conveying resistance to amoxicillin-induced killing to
susceptible M. catarrhalis, S. pneumoniae and H. influenzae.
Intriguingly, although co-exclusion patterns were observed
between S. pneumoniae, H. influenzae and M. catarrhalis in in
vitro studies, symbiotic relationships between these bacterial
community members have been observed in vivo, suggesting
a major influence of selection by host epithelium and
immune system on these microbe–microbe interactions [61].
The commensal genus Dolosigranulum, which is merely
observed in the anterior nares and nasopharynx in both children and adults [3,14,62], has only recently been identified as
a regular commensal of the URT. It is a fermenting lactic-acidproducing bacterium that is commonly co-occurring with
corynebacteria, which are lipophilic and non-fermenting.
We speculate that as the fermentation process of Dolosigranulum spp. generates lactic acid, which reduces the local pH, it
might select for Corynebacterium spp. outgrowth. The presence of corynebacteria is strongly related to breastfeeding,
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might explain previous ambiguous reports on either the synergistic or antagonistic interaction between these species in both
mechanistic and epidemiological studies [34,35,40,41].
Although the niche characteristics seem similar between adults and children, a relatively higher abundance of
Streptococcaceae, Moraxellaceae and Neisseriaceae was
reported in the anterior nares of healthy children compared
with healthy adults [31]. As these families are even more
abundant in the adjacent nasopharynx, it remains uncertain
whether their predominance in the anterior nares of children
is merely a reflection of dispersal from their primary niche
rather than a true difference in microbial equilibrium in the
anterior nares of children compared with adults (figure 1a).
Alternatively, age-related niche-specific local immunity
may play a role in keeping resident pathobionts at bay. In the
anterior nares, both epithelial cells and local immune cells,
such as neutrophils and natural killer (NK)-cells, produce
antimicrobial peptides (AMPs) and proteins, which form the
first-line defence against invading microbes and coordinate
downstream immune signalling [42]. Moreover, various fatty
acids produced by the epidermis exhibit antimicrobial activity
against for example S. aureus [43,44]. Microbial homeostasis in
the anterior nares is additionally governed by cellular elements
of immune system as illustrated by a study on nasal decolonization of S. aureus in mice, which was shown to be T-cell
dependent and reliant on upregulation of proinflammatory
cytokines such as IL-17A [45]. Especially the latter process
will mature with age, potentially directing resident microbial
community structure in an age-dependent manner.
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The oropharynx links the mouth, the nasopharynx, the larynx,
the lower airways and the gastrointestinal tract, and is thereby
exposed to a wide variety of microorganisms, of both exo- and
endogenous origins. The species pool from which the oropharyngeal niche can sample is therefore generally large, plausibly
contributing to the highly diverse bacterial communities
observed in both adults and the elderly. The oropharynx is
also the ecological niche for potential pathogenic bacteria that
may cause local ( pharyngitis) or disseminated ( pulmonary)
disease [66]. Dispersal of oropharyngeal bacterial communities
to the lower respiratory tract by (micro-)aspiration or inhalation is presumably a frequently occurring event both in
health and disease, owing to the significant overlap between
the oropharyngeal microbiome and bacterial communities
observed in the healthy lung [67–69].
Metagenomic sequencing has demonstrated that the
healthy adult oropharynx is colonized with pathobionts such
as Streptococcus, Haemophilus and Neisseria spp. and the Gramnegative, anaerobic putative commensal genera Veillonella,
Prevotella, Leptotrichia and Fusobacterium [3,70–74]. A recent
study in children showed a similar oropharyngeal microbiome
composition to that observed in adults, with enrichment of,
most notably, Neisseria, Granulicatella, Prevotella, Porphyromonas,
Fusobacteriaceae and certain Prevotella spp. [23].
The oropharynx is especially known to harbour several
potentially pathogenic members of the genus Streptococcus,
including S. pneumoniae [75], S. pyogenes (group A b-haemolytic Streptococcus) [76], S. agalactiae (group B b-haemolytic
Streptococcus) [77] and Streptococcus dysgalactiae subsp.
equisimilis (group C and G b-haemolytic streptococci) [78]. Particularly, S. pyogenes is known to cause a wide spectrum of
diseases, varying greatly in severity from self-limiting pharyngitis to life-threatening sepsis and streptococcal toxic shock
syndrome [79]. To this day, no definitive explanation has
6. The human upper respiratory tract niches:
the oral cavity
Apart from the oropharynx, various sites in the oral cavity have
been extensively characterized, particularly by the Human
Microbiome Project, including the tongue dorsum, hard palate,
sub- and supragingival plaque, buccal mucosa, palatine tonsils,
keratinized gingiva and saliva. Similar to the oropharynx, the
oral microbiome is subject to major, potentially perturbing factors, including hygiene measures (i.e. tooth brushing and
mouthwash rinsing), mechanical forces through mastication,
enzymatic activity in saliva, and temperature, pH and dietary
alterations [88]. Moreover, like the oropharynx, the mouth is in
close contact with the surrounding environment and is thus
exposed to large numbers of food- and airborne bacteria, as
well as indigenous pathogens from other URT niches. Despite
these factors, the oral microbiome has been demonstrated to be
highly stable over time [28], which might result from the large
core microbiome shared among unrelated individuals [89].
The initial oral microbiome composition in children is, like
the other niches, greatly influenced by mode of delivery [21],
feeding type, with higher abundance of lactobacilli observed
in breastfed children [25], and horizontal transfer of microbes
[90]. Initiated by the eruption of teeth, local microbial compositions show an increase in Bacteroidetes (i.e. Veillonellaceae
and Prevotella spp.) and a decrease of Proteobacteria (i.e. Moraxella spp.) with age, gradually transitioning towards an adult-like
microbiome configuration [91,92] distinguished by Streptococcus
(presumably oralis/mitis/peroris), Veillonella, Selenomonas,
Gemella, Fusobacterium, Prevotella, Lactobacillus and Neisseria
spp. [15,88]. Interestingly, saliva is characterized by relatively
high amounts of Prevotella, Neisseria and Haemophilus spp.,
whereas sub- and supragingival plaques and keratinized gingiva are enriched for corynebacteria; this implies that there is
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Phil. Trans. R. Soc. B 370: 20140294
5. The human upper respiratory tract niches: the
oropharynx
been found for this great spread in disease severity, although
it has been postulated that interactions between different streptococcal species may play a role in pathogenesis [80]. These
interactions are mediated by various quorum-sensing systems
(i.e. systems regulating communication between bacterial cells
by signalling molecules), which were shown to be involved in
induction of biofilm formation in vitro [81] and virulence
modulation in vitro and in vivo [82,83].
Contrariwise, antagonistic interactions involving streptococcal species have also been demonstrated. For example,
S. viridans, a Group A a-haemolytic Streptococcus, reduces
growth of known otitis media pathogens, S. pneumoniae,
M. catarrhalis and H. influenzae in vitro [84], suggesting a
possible role in containment of these pathobionts (figure 1c).
Apart from synergistic and antagonistic interactions
between different streptococcal species, interactions between
members of the Streptococcus genus and other genera have
also been described; for example, S. pyogenes has been shown
to co-occur with M. catarrhalis and non-typeable H. influenzae
[85]. This epidemiological phenomenon could be mediated
by enhanced adherence to and invasion of human epithelial
cells, as was observed for S. pyogenes and M. catarrhalis aggregates in vitro [86]. Furthermore, M. catarrhalis and H. influenzae
have been shown to reduce the susceptibility of S. pyogenes to
penicillin by their production of b-lactamase-containing
OMVs in vitro, potentially contributing to treatment failure in
streptococcal tonsillitis [87].
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which might be explained by the fact that this genus is a
core member of the human breast milk microbiome [63].
Interestingly, microbial communities characterized by high
abundance of Corynebacterium and Dolosigranulum spp.
lacked the presence of H. influenzae and S. pneumoniae,
suggesting colonization resistance against these potential
pathogens (figure 1b).
Additional host factors plausibly involved in shaping the
local ecosystem occur in the mucus layer that forms a physical barrier and segregates resident bacterial flora in the
nasopharyngeal lumen from the epithelial cell surface. The
central purpose of this barrier is to limit contact between
microbiota and host tissue to prevent low-grade local inflammation and thus translocation of pathobionts [22]. Within the
mucus layer AMPs and secretory IgA accumulate, further
restricting exposure of epithelial cells to resident microbiota
[64]. Except for direct action of innate immune components
on microbial community composition, it has recently been
postulated that mucus drives selection of bacterial communities adapted to metabolizing complex sugars contained
in mucus [65]. The mucosal barrier is supported by
mucosa-associated lymphoid tissue, which harbours antigen-presenting cells, B- and T-cells, and forms the interface
between local innate and adaptive immunity.
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Traditionally, microbiome alterations have been related to diseases with a chronic inflammatory character, such as obesity
[98], COPD [99] and asthma [71]. However, the microbiome’s
influence on (susceptibility to) acute upper and lower respiratory
infections is increasingly gaining recognition.
Uncomplicated URT infections are highly prevalent and
cause substantial morbidity worldwide; approximately 75%
of children have suffered from AOM by the age of 3 years
[100]. By contrast, lower respiratory tract infections are less
common, but are accompanied by a high case-fatality rate
[101]. In both types of infection, pathobionts generally residing asymptomatically in the URT are commonly involved
[3,102,103], underscoring the need to better understand the
pathogenesis of these infections.
Historically, the URT niche is regarded as the ecological
niche of most potential respiratory pathogens, such as
S. pneumoniae, H. influenzae and S. aureus. Up to 93% of children
under 2 years of age are colonized by at least one potential
pathogen when measured by conventional culture [61]; therefore, these pathobionts can without doubt be considered part
of the residential flora. Colonization with these bacterial species
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Phil. Trans. R. Soc. B 370: 20140294
7. Pathogenesis of respiratory infections: the
pathogen perspective
might actually have an ambiguous role in the development of
respiratory diseases: on the one hand, ‘pathogen’ carriage is a
prerequisite for development of disease; on the other hand,
pathobiont colonization does not necessarily lead to infection
and has even been suggested to provide resistance against
acquisition of new pathogenic strains [104]. As the risk of developing respiratory infections seems specifically related to recent
acquisition of a new strain [104,105], a state of bacterial symbiosis, even including potential pathogens, may be protective
against short-term infection and inflammation. Nevertheless,
carriage can develop towards respiratory disease in a fraction
of cases, a process that can be triggered by various exo- or
endogenous stimuli [106–108]. Especially, this fact supports
the hypothesis that overgrowth of a potential pathogen is not
a random event, but instead a co-occurrence of multiple factors
leading to lack of containment of the potential pathogen by the
resident commensals and local immunity. Both seem more
likely to fail following a recent acquisition of a new bacterium
or viral co-infection, inducing a combined state of temporary
dysbiosis in the absence of adaptive immunity.
Next-generation sequencing of the nasopharyngeal microbiome of healthy children and children suffering from mild
URT infections, revealed that colonization with and density of
a potential pathogen (S. pneumoniae, M. catarrhalis and
H. influenzae) is related to a less evenly distributed microbiome,
suggesting reduced containment by the commensal flora
[33,109]. This is supported by the observations that increased
abundance of the three AOM pathogens is accompanied by
decreased presence of the commensals Lactococcus, Anoxybacillus,
Corynebacterium and Dolosigranulum spp. Also, in contrast to the
increased abundance of Haemophilus and Streptococcus spp. and
members of the Pasteurellaceae during AOM, an increased abundance of Dolosigranulum and Corynebacteria spp. was associated
with absence of AOM [33]. In addition, both biodiversity and
bacterial density of the nasopharyngeal microbiome are lower
in children diagnosed with AOM compared with healthy children, altogether suggesting ecological dysbiosis to be key to
development of these infections [110]. This is further underlined
by a study on the tonsillar crypt microbiome of children and
adults suffering from recurrent tonsillitis, Proteobacteria
(mainly Haemophilus spp.) were associated with disease, whereas
Bacteroidetes (especially Prevotella spp.) abundance was associated with absence of disease [111]. Particularly, this latter
finding was in conjunction with a study on the oropharyngeal
microbiome in (elderly) pneumonia patients, which showed
that lack of members of the Bacteroidetes phylum including
Prevotella spp. as well as other Gram-negative anaerobic
species like Leptotrichia and Veillonella spp. is associated with
pneumonia [112].
The hypothesis that the abundance of pathogenic species
can be modulated by the resident bacterial flora has been comprehensively addressed in a study on the association between
sinus microbiome changes and chronic rhinosinusitis (CRS).
In patients suffering from CRS, the investigators observed
increased abundance of Corynebacterium tuberculostearicum and
reduced abundance of bacterial members belonging to the
families Lactobacillaceae, Enterococcaceae, Aerococcaceae and
Streptococcaceae. Strikingly, mice depleted from their resident
sinus microbiome developed a more severe CRS phenotype
upon administration of C. tuberculostearicum compared with
mice with an intact sinus microbiome [113], suggesting resident
bacterial community members are not mere bystanders, but
actually have an important role in containing pathogens.
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significant overlap between the oral bacterial community composition and the oropharyngeal microbiome, likely due to
local dispersion of bacterial assemblages and similar environmental pressure. The antagonistic and synergistic interactions
between bacteria observed in the oropharynx will therefore presumably similarly affect the spatial structure of microbial
communities in the mouth.
Although not extensively researched for the URT, variations in microbial community structure have also been
demonstrated to arise from differences in local availability of
resources; for example, the oral cavity is enriched in bacterial
carbohydrate-active enzymes responsible for degradation of
simple sugars, such as dextran, whereas intestinal flora is
capable of metabolising more complex carbohydrates [93].
Acute infection in the oral cavity is a rare event, presumably owing to the tolerogenic local circumstances mediated
by dendritic cells (DCs) and various T-cell subtypes. Similar
to the nasopharynx, the oropharynx and oral cavity are sustained by specialized mucosa-associated lymphoid tissue,
which is imperative for antigen recognition and initiation of
an adequate immune response [94]. The equilibrium between
oral mucosal tolerance and inflammation is strongly guarded
by DCs. DC-induced upregulation of Toll-like receptor (TLR)4
by resident microbes induces the production of immunosuppressive interleukins including IL-10, subsequently activating
regulatory T-cells, thereby dampening inflammation and
contributing to the tolerable local conditions [95]. Mucosal
inflammation is further reduced by saliva-derived secretory
IgA that covers the mucosal surfaces of the oral cavity, hindering contact between microbiota and host mucosa, thus
preventing microbial attachment and immune activation, a
mechanism referred to as ‘immune exclusion’ [96].
However, crosstalk between resident microflora and the
host can also have a potentially unfavourable outcome as
was illustrated in a murine model, showing that normal
oral commensals can activate inflammasome inducing local
release of the highly proinflammatory cytokine IL-1b [97].
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Colonization resistance against foreign pathogens can be provided by occupation of otherwise vacant URT niches by
indigenous flora. As a result, an invading pathogen has to
compete with the commensal flora for adhesion receptors on
the mucosal surface as well as nutrients, limiting the chances
that the intruding species is indeed able to adhere, replicate,
disseminate and cause disease. Direct evidence that this mechanism also takes place in the URT is sparse, although it has
been demonstrated that following antibiotic treatment, which
leads to a reduction of bacterial community density and diversity, the risk of (a new) AOM is temporarily increased [33,122].
Apart from environmental differences in the URT linked to
human physiology, bacteria are also able to modify their environment themselves. Short-chain fatty acid (SCFA) produced by
fermenters such as Bacteroidetes and Propionibacterium spp.,
has been shown to directly inhibit pathogenic growth and
expression of virulence genes of pathogens in the gut and is
able to indirectly modulate bacterial growth conditions by acidification of the environment [123]. Direct inhibition of competing
microbes by production of antimicrobial compounds is exemplified by the production of hydrogen peroxide by S. pneumoniae,
7
Phil. Trans. R. Soc. B 370: 20140294
8. Pathogenesis of respiratory infections: the
ecosystem perspective
which is bacteridical for S. aureus [51]. Moreover, some bacterial
species produce peptides with antimicrobial efficacy, which are
generally referred to as bacteriocins [124]. This mechanism is
illustrated by the commensal streptococcal species S. salivarius,
which presumably inhibits pathogenic S. pyogenes growth by
production of bacteriocins. Although the URT stands in direct
contact with the outside world, we postulate the large surface
of the nasal cavity contains niches covering a range of
oxygen levels and carbon dioxide levels additionally shaping
the ecosystem’s habitat (figure 2b).
Biodiversity has been linked to higher resilience against
disturbances, such as antibiotic treatment and invasion of
new pathogens [125]. Diverse microbial communities might
contribute to colonization resistance through various mechanisms, for example by heightening the chance that all
resources are used which would otherwise be readily available for pathogens [126]. The maintenance of biodiversity
in ecological communities has been related to the presence
of so-called keystone species, loss of which would result in
extinction of many other (micro-)organisms, hence decreasing
the community’s biodiversity (figure 2a) [127,128]. A clear
example is the family Christensenellaceae which was recently
identified as a potential keystone taxon of the gut microbiome
because its presence or absence directly modulated the host’s
phenotype [129]. Although keystone species involved in
maintenance of the community structure of the URT microbiome have not been identified yet, stability of bacterial
communities over time has been linked to the presence of certain putative commensals, such as Moraxella, Dolosigranulum
and Corynebacterium spp., resulting in a more healthy host
phenotype [46]. Contrarily, early colonization with pathogens
such as S. pneumoniae, H. influenzae and M. catarrhalis
has been shown to result in a long-term increased risk of
pneumonia and bronchiolitis in healthy neonates [130].
Colonization resistance might be diminished by bacterial
community structures that induce a low-grade, asymptomatic inflammation, which facilitates invasion by pathogenic
bacteria. Studies have shown that colonization with Proteobacteria such as non-typeable H. influenzae is accompanied by
mucosal inflammation, characterized by increased epithelial
thickness of the large airways and production of early proinflammatory cytokines [131]. Contrariwise, colonization
with commensal Prevotella spp. was associated with low cytokine production, reduced recruitment of neutrophils and
leucocytes, and no detectable lung tissue pathology. The
more severe inflammation caused by non-typeable H. influenzae
colonization is presumably mediated in a TLR4-dependent
manner [132]. The ligand for TLR4 is lipopolysaccharide
(LPS), a major proinflammatory constituent of the outer cell
membrane of Gram-negative bacteria [133]. The immunostimulatory capacity of LPS has been related to its structure: hepta- or
hexa-acylated LPS observed in Gammaproteobacteria such as
H. influenzae induces a strong TLR4-mediated inflammatory
response in contrast to tetra- and penta-acylated LPS, which is
typically found in Bacteroidetes members such as Prevotella
spp. and has very little TLR4-stimulatory activity [132].
Bacteroidetes-associated LPS even antagonises hexa-acylated
LPS-mediated TLR4-signalling [134], dampening downstream
signalling, thereby contributing to mucosal homeostasis [135]
and colonization resistance versus pathogens (figure 2c).
Apart from variations in the proinflammatory capacity
related to the bacterial composition of the human microbiome,
mucosal inflammation can also be induced by viral infection,
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Unfortunately, there are no studies available yet using
next-generation sequencing methods to determine a possible
association between URT microbiota and acute rhinosinusitis,
laryngitis or pharyngitis. Conventional studies, however,
suggest that acute rhinosinusitis is caused most frequently
by viral agents, although a secondary bacterial infection by,
among others, S. pneumoniae and H. influenzae has been
observed in a limited number of cases [114]. Similarly, the
majority of infectious laryngitis cases have a viral origin,
potentially predisposing to bacterial laryngitis, caused
by pathogens such as H. influenzae type b, S. pneumoniae,
S. aureus, b-haemolytic streptococci, M. catarrhalis and
Klebsiella pneumoniae [115]. Cultivation-based approaches in
pharyngitis patients demonstrated that b-haemolytic streptococci other than group A or C, S. pyogenes (group A
Streptococcus), S. equisimilis and Streptococcus anginosus were
isolated more frequently compared with healthy controls
[116]. However, the possible ( protective) role of commensals
in these pathogenic processes has so far been largely ignored.
Imbalance of the oral microbiome has been investigated,
especially in relation to the development of periodontal disease, caries and cardiovascular health [117], showing a
central role for keystone pathogens Porphyromonas gingivalis,
Treponema denticola and Tannerella forsythia [118,119]. We postulate that an imbalanced oral microbiome might also have
an effect on respiratory health through physiological episodes
of micro-aspiration [66]. Compelling evidence for the importance of oral health and micro-aspiration is generated by a
study in residents of nursing homes, showing that neglect
of oral health measures leads to a dramatic increase in
incidence of and mortality by pneumonia [120].
We therefore speculate that the healthy URT microbiome
in general provides us with colonization resistance, which is
defined as (i) resistance against acquisition and establishment
of a new pathogen or (ii) containment of potentially pathogenic bacteria residing amidst harmless commensals in a
balanced microbiome [121].
Downloaded from http://rstb.royalsocietypublishing.org/ on June 15, 2017
(a)
(b)
(c)
8
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biodiversity
(d)
S. pneumoniae/salivarius
+
keystone species
H2O2/bacteriocins
DO2
–
Prevotella spp.
H. influenzae
Propionibacterium
–
pH
S. aureus
Proteobacteria
(influenza) virus
SCFA
Bacteroidetes
LPS
+
–
translocation
-
mucins
NK-cell
proinflammatory cytokines;
TNFa, IL-8, IL-6, IL-1
neutrophil
Dcytokine profile
colonization resistance
high
ICAM-1
CEACAM-1
PAF-r
modulation
low
Figure 2. Hypothetical model on the relationship between the URT microbiome and pathogenesis of infectious respiratory diseases. This figure shows the potential
mechanistic explanations for the influence of the URT microbiome on colonization resistance, reduction of which is presumed to be at the core of the pathogenesis of
acute respiratory infections, such as AOM, pharyngitis and tonsillitis. In (a), we show the optimal situation in which keystones species, for example Bacteroidetes
members for the oropharynx, are at the base of a diverse ecological system of bacterial interactions. (b) Depicts a situation in which synergistic or antagonistic
interspecies interactions modulate microbial composition and biodiversity, consequently regulating colonization resistance. For example, the release of metabolic byproducts, such as short chain fatty acids (SCFAs) from fermenting bacteria, results in acidification of the environment and inhibition of growth of acidophobic
bacteria, such as S. aureus. Furthermore, production of bactericidal products such as hydrogen peroxide or bacteriocins results in exclusion of certain bacterial species.
In (c), we demonstrate that a Proteobacteria-rich microbiome is associated with a low-grade inflammation through activation of Toll-like receptor 4 (TLR-4) by heptaor hexa-acylated lipopolysaccharide (LPS), which might forego invasion of pathogenic species. This immunostimulatory effect is reduced and even counteracted when
the TLR-4 receptor is activated by tetra- and penta-acylated LPS, of members of the phylum Bacteroidetes, such as Prevotella spp. (d ) Demonstrates the situation in
which viral infection results in exposure of fibrinogen (not shown) through virus-induced epithelial cell damage. Additionally, viral infection, but also the presence of
potential pathogenic bacterial species such as H. influenzae results in upregulation of adhesion receptors. Moreover, viral infection reduces epithelial integrity, thus
contributing to bacterial translocation. Finally, the presence of influenza virus increases the availability of host-derived sialic acid-rich mucins, resulting in increased
pneumococcal replication. For colour-codes of various bacterial community members, see the legend of figure 1. ICAM-1, intercellular adhesion molecule 1; CEACAM1, carcinoembryonic antigen-related cell adhesion molecule 1; PAF-r, platelet-activating factor receptor; IL, interleukin; TNF, tumour necrosis factor. D: change in/of.
thereby facilitating adhesion and invasion by pathogenic
bacterial species. Viral infections may induce direct damage
to ciliated cells, diminishing mucociliary clearance [136,137],
resulting in denudation of the epithelium [136], exposing fibronectin, to which pathogens such as S. pneumoniae, and to a
lesser extent S. aureus and S. pyogenes, can adhere [138].
Except for fibronectin, other surface molecules or cell receptors,
including intercellular adhesion molecule 1 (ICAM-1), carcinoembryonic antigen-related cell adhesion molecule 1
(CEACAM-1) and PAF-r are upregulated in response to viral
infection, also enhancing bacterial adhesion [139]. Eventually,
viral infection can diminish epithelial integrity and promote
bacterial translocation [140]. In addition to facilitating adherence and translocation of bacterial cells, viruses can directly
affect a plethora of components of the innate immune system
in particular, such as impairment of neutrophil function
[141], NK cell activation and recruitment [142] and alteration
of cytokine profiles [143,144], which might all contribute to
reduced colonization resistance against pathobionts, thereby
predisposing to bacterial superinfection. Third, influenza
virus-induced inflammation leads to increased expression of
sialic acid-rich mucins such as Muc5AC, which enhances pneumococcal proliferation [145]. Besides evidence from in vitro
studies, there is also evidence from in vivo studies showing
that a broad panel of respiratory viruses enhance pathobiont
colonization in both adults and children [61,146].
The reverse relationship, i.e. pathobiont presence enhancing viral acquisition and replication, might also occur: for
example, H. influenzae has been shown to induce expression
of ICAM-1 and TLR3-receptors, which enhanced rhinovirus
binding and stimulated rhinovirus-induced chemokine
production [147]. Furthermore, a recent in vitro study has
shown that H. influenzae similarly increases viral replication
of respiratory syncytial virus, which was related to the release
of proinflammatory cytokines IL-6 and IL-8, suggesting
specific bacterial community members can aggravate (inflammatory response to) viral infection [148]. Furthermore,
pre-incubation of human bronchial epithelial cells with
S. pneumoniae leads to increased susceptibility to infection
with human metapneumovirus (figure 2d) [149].
9. Conclusion and future directions
In this review, we have made an attempt to summarize the
current body of evidence regarding the role of the healthy
respiratory microbiome in URT health. We postulate that its
composition is orchestrated by biotic components such as
Phil. Trans. R. Soc. B 370: 20140294
TLR4
+
Downloaded from http://rstb.royalsocietypublishing.org/ on June 15, 2017
Competing interests. E.A.M.S. declares to have received unrestricted
research support from Pfizer, grant support for vaccine studies
from Pfizer and GlaxoSmithKline and fees paid to the institution
for advisory boards or participation in independent data monitoring
committees for Pfizer and GSK. No other authors reported financial
disclosures.
Funding. This research has received funding from the Wilhelmina
Children’s Hospital Fund, ZonMW (grant no. 91209010).
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