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Curr Opin Microbiol. Author manuscript; available in PMC 2021 February 12.
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Published in final edited form as:

Curr Opin Microbiol. 2020 February ; 53: 1–8. doi:10.1016/j.mib.2020.01.014.

Pseudomonas aeruginosa polymicrobial interactions during

lung infection
Karishma Bisht1,#, Jiwasmika Baishya1,#, Catherine A. Wakeman1,*
1Texas Tech University, Department of Biological Sciences, Lubbock TX USA

Abstract
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Chronic infections often contain complex polymicrobial communities that are recalcitrant to
antibiotic treatment. The pathogens associated with these infectious communities are often studied
in pure culture for their ability to cause disease. However, recent studies have begun to focus on
the role of polymicrobial interactions in disease outcomes. Pseudomonas aeruginosa can colonize
patients with chronic lung diseases for years and sometimes even decades. During these prolonged
infections, P. aeruginosa encounters a plethora of other microbes including bacteria, fungi, and
viruses. The interactions between these microbes can vary greatly, ranging from antagonistic to
synergistic depending on specific host and microbe-associated contexts. These additional layers of
complexity associated with chronic P. aeruginosa infections must be considered in future studies in
order to fully understand the physiology of infection. Such studies focusing on the entire
infectious community rather than individual species may ultimately lead to more effective
therapeutic design for persistent polymicrobial infections.
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Introduction
The host environment encountered by invading pathogens is often heterogeneous and
contains physiologically distinct regions [1]. Even within a single organ such as the lung,
there are variations and gradients of environmental factors including but not limited to
fluctuations in nutrient and oxygen levels [2] and the endogenous microbiome structure [3].
Interestingly, these differences in the microenvironments present within the host can lead to
population diversity both within a single species resulting in sub-lineages [4] as well as
within the microbial species of infectious polymicrobial communities [5, 6]. The
microorganisms within these diverse communities are not passively coexisting but rather
actively responding to each other with interactions ranging from cooperation to competition
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[7]. These interactions are critical to disease outcomes [3] and are therefore the focus of
intense study. Pseudomonas aeruginosa, an opportunistic human pathogen associated with
chronic respiratory infections, has been particularly well-studied for its ability to shift

*
correspondence should be addressed to catherine.wakeman@ttu.edu.
#these authors contributed equally to this work.
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Declarations of interest: None
 Bisht et al. Page 2

between cooperative and competitive interactions [8]. In this review, we will discuss the
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extensive microbial interactions experienced by P. aeruginosa during chronic lung infection

including intraspecies, interspecies, interkingdom, and viral interactions (Figure 1).

Intraspecies interactions of P. aeruginosa

The relatively large genome of P. aeruginosa enables this microorganism to colonize and
adapt to multiple types of environments [9, 10]. Due to this flexibility, P. aeruginosa cells are
capable of extensive levels of physiological differentiation within distinct
microenvironments such as those experienced within biofilms [11], within different
infectious sites such as wounds versus lung [12], or even within distinct regions of the same
organ such as the different lobes of the lung [1, 13]. This type of physiological
differentiation can give rise to the evolution of sub-lineages that can efficiently colonize
different niches in the host. A study analyzing clonal bacterial isolates sampled from a
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chronically infected individual over a 32-year period showed the presence of two distinct
and dominant P. aeruginosa lineages occupying separate niches, the sinuses and the lower
airway [14]. The researchers demonstrated rapid diversification of the original infecting
strain into three distinct sub-lineages which were functionally distinct and diverse in their
genomic composition.

The interactions between sub-lineages and distinct strains of P. aeruginosa can be extremely
important over the course of chronic infection. A study conducted to characterize P.
aeruginosa populations during chronic lung infections of bronchiectasis patients showed that
P. aeruginosa sub-lineages adapt by accumulating several loss-of-function mutations leading
to changes in phenotypes [15]. Some loss-of-function mutations may promote intraspecies
interactions with other sub-lineages. For example, a study has shown that auxotrophic strains
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of P. aeruginosa isolated from a chronically infected cystic fibrosis (CF) patient exhibited
growth complementation where isolates deficient for arginine could only grow in presence
of isolates deficient for methionine [16] (Figure 2A). In other cases, loss-of-function
mutations can select for intraspecies parasitism exemplified by pyoverdine cheater
populations. Studies on this phenomenon have revealed the presence of sub-populations of P.
aeruginosa that have lost the capacity to produce iron-scavenging siderophores but retain the
ability to take up siderophores produced by the remainder population [17]. This loss allows
pyoverdine cheater cells to acquire an essential metal from the host cells without expending
energy to obtain it. Thus, the nature of mutation or adaptation within a species can not only
dictate the emergence of distinct sub-populations but also the interactions between them.

Even outside of the context of loss-of-function mutations and/or auxotrophies, intraspecies

competition and cooperation are important for shaping P. aeruginosa lung infections. For
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example, R-pyocins produced by a strain of P. aeruginosa can negatively affect established

biofilm communities of a sensitive P. aeruginosa strain and displace the latter to achieve
colonization dominance [18] (Figure 2B). Additionally the type six secretion system (T6SS),
an apparatus used by some microbes to kill competitors through the injection of toxins, can
lead to the fitness of some P. aeruginosa strains over others [19]. Conversely, P. aeruginosa
populations with unique adaptations can cooperate within the context of the lung
environment. For example, enhanced resistance to the host immune peptide LL-37 and H2O2

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has been seen in communities of P. aeruginosa consisting of both mucoid and non-mucoid
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strains [20]. In this interaction, the alginate produced as a part of the extracellular polymeric
substance (EPS) of mucoidy cells protects this multi-strain P. aeruginosa community from
LL-37 mediated killing and the catalase produced by non-mucoidy cells protects the
community from the effects of H2O2.

The physiological differentiation in P. aeruginosa populations does not always need to be

driven by mutation. For example, heterogeneity in cellular levels of cyclic diguanylate (c-di-
GMP), a secondary messenger signaling molecule commonly found in bacteria and
generated by the Wsp system in P. aeruginosa, can drive differentiation into two distinct sub-
populations [21]. The high c-di-GMP producers catabolize polysaccharides for the biofilm
EPS matrix, thus protecting the cells within the matrix, and the low c-di-GMP producers
explore and check the colonizing surface [21]. Elimination of this c-di-GMP heterogeneity
leads to defects in biofilm formation by the cells suggesting that both sub-populations have
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indispensable contributions in the formation of the biofilm [21]. The intraspecies

interactions that lead to the formation of robust P. aeruginosa biofilms are crucial to the
persistence of this microorganism in chronic infection as biofilm formation has been
associated with microbial recalcitrance to both the immune response and antibiotic treatment
[11, 22]. Thus, the interactions between distinct P. aeruginosa populations generated by
either mutation or by physiological adaptation can be important drivers of P. aeruginosa
disease progression.

Interspecies bacterial interaction with P. aeruginosa

While the interactions between differentiated P. aeruginosa cells can be quite diverse, the
range of cooperation to competition between different bacterial species is perhaps even more
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striking. The spatial structure of the lung can impact the interactions between P. aeruginosa
and other microbial species existing within the lung environment. The microbe-microbe
interaction within this environment can result in P. aeruginosa eliciting distinct physiological
responses and exhibiting adaptive mutations. These interactions can sometimes be beneficial
for surrounding microorganisms as P. aeruginosa is capable of mediating cooperative/
synergistic interactions which could lead to the survival of both the species against the host
immune system. However, these interactions are often competitive in nature as P. aeruginosa
strives to survive in the harsh environment of the host-pathogen interface. This section
focuses on several examples of this balance between conflict and cooperation existing in
communities containing both P. aeruginosa and S. aureus, another prevalent pathogen
coinfecting the lungs of immunocompromised patients.

To invade and survive in the host environment P. aeruginosa has defensive strategies to
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overcome the stresses imposed by the nearby microbes and in turn possesses its own
offensive strategies to compete for nutrients and space [7]. In the context of infection, iron is
a crucial but limited resource sequestered by the host’s nutritional immunity [23]. Under
iron-depleted conditions in the CF lungs P. aeruginosa produces antimicrobials such as 4-
hydroxy-2-heptylquinoline-N-oxide (HQNO) to suppress the growth of S. aureus [24] and
can utilize the stored iron from the lysed S. aureus cells [25]. The induction of HQNO and
siderophores mediated by iron limitation in P. aeruginosa can ultimately shift the respiratory

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profile of neighboring S. aureus cells from aerobic to fermentative and eventually lead to the
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death of S. aureus [26]. HQNO exposure in S. aureus also makes this microbe more
susceptible to antimicrobial agents known to permeabilize its membrane leading to its
complete eradication [27] (Figure 2C). The context of the host environment is an important
driver in the P. aeruginosa-S. aureus interaction. For example, the host-derived antimicrobial
peptide sPLA2-IIA can play a major role in shifting the balance of infection in favor of P.
aeruginosa. A study found that in mouse lung infections the secreted toxin ExoS produced
by P. aeruginosa could induce sPLA2-IIA to eradicate S. aureus and promote dominance of
P. aeruginosa [28].

It is intriguing how context can determine the balance between competition versus
cooperation for virtually any microbial pair and for P. aeruginosa and S. aureus in particular.
For example, the competitive HQNO molecule produced by P. aeruginosa can also promote
S. aureus tolerance to certain antibiotics including vancomycin and tobramycin [29–31]. In
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addition to S. aureus gaining benefit from the presence of P. aeruginosa during antibiotic
exposure, P. aeruginosa can also benefit from the presence of S. aureus. For example, the
alpha toxin of S. aureus can mediate infection and dissemination of opportunistic Gram-
negative infections such as P. aeruginosa and K. pneumophila by disrupting host immunity
and epithelial barriers [32]. It is possibly due to this benefit of coinfection that P. aeruginosa
can suppress its anti-staphylococcal factors in response to the presence of host proteins such
as the innate immune protein calprotectin and the abundant serum protein albumin [33, 34]
(Figure 2D). Furthermore, P. aeruginosa can acquire mutations suppressing the production of
these molecules during chronic infection. One study has highlighted the role of alginate in
diminishing the production of anti-staphylococcal molecules in P. aeruginosa. P. aeruginosa
is known to overproduce alginate in the lung environment which can protect the microbial
cells against reactive oxygen species (ROS) produced by the host. However, this
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overproduction can be beneficial to S. aureus as it leads to the reduced production of anti-

staphylococcal molecules like siderophores enabling S. aureus to co-infect with P.
aeruginosa as observed in a CF infection model [35]. Another interesting factor that can
impact the interspecific coexistence between P. aeruginosa and S. aureus in the CF lungs is
the mutation in the quorum sensing (QS) regulation system over the period. It is known that
P. aeruginosa utilizes QS to activate expression of many virulence genes and biofilm
formation in a cell density-dependent manner. However, many host-adapted P. aeruginosa
isolates, such as the lasR mutants commonly isolated from CF lungs, are known to exhibit a
deficient QS system which decreases its ability to outcompete S. aureus from a CF lung
environment [36]. Further details about the QS network and its role in both competition and
cooperation between these two pathogens is described in this issue in a review article by
Ibberson and Whiteley [37].
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Ultimately, synergistic interactions have the potential to aid in the survival of both S. aureus
and P. aeruginosa against the host immune system via the exploitation of both
microorganisms’ physiological adaptations (Figure 2D). Recent studies have used Tn-seq as
a unique method to identify the essential genes promoting survival in context of mono-
versus co-infections. In the case of P. aeruginosa-S. aureus co-infection, many genes that had
been previously identified as essential in mono-infection were found to be dispensable in co-
infection as observed in a murine wound model. These context-dependent gene sets were

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termed community dependent essential genes [38]. An interesting area of future study would
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be to recapitulate these studies in other host niches such as the lung as well as in other
microbial pairs in addition to P. aeruginosa and S. aureus.

Interkingdom fungal interactions with P. aeruginosa

Interactions of P. aeruginosa in the host environment are not just limited to bacterial species.
A range of synergistic and antagonistic interactions have been studied between P. aeruginosa
and the fungal opportunistic pathogens Candida albicans and Aspergillus fumigatus. The
outcome of these interactions depends on various host- and pathogen-specific factors, which
influence gene expression and/or metabolic processes of the involved microorganisms.

A proteomic study on P. aeruginosa and C. albicans in mono-species biofilms versus mixed

biofilms explored the differences in genes expression of both pathogens in presence of each
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other [39]. This study found differential expression of critical virulence factors such as the
mixed culture-induced upregulation of the siderophores pyoverdine and pyochelin as well as
heme-binding proteins in P. aeruginosa, indicating that the presence of the fungus leads to
competition for resources such as iron. In contrast with these results, a downregulation of
these molecules was shown in another study that analyzed the transcriptomic profile of P.
aeruginosa co-infecting the gastrointestinal tract of neutropenic mice with C. albicans,
demonstrating that microbial interactions are highly dependent on the context of the host
niche [40]. Another study using a CF lung infection model has demonstrated potential
synergism between P aeruginosa and C. albicans [41]. This study showed that ethanol
produced by C. albicans promotes production of the Pel exopolysaccharide in P. aeruginosa
leading to biofilm maturation and colonization of the lung epithelial cells (Figure 3A).
Interestingly, phenazines produced by P. aeruginosa increased ethanol production by the
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fungus in turn and gave rise to a positive feedback loop that worsens host health.

Interaction of P. aeruginosa with the fungus A. fumigatus during lung infections have also
been extensively studied. Many studies have shown that patients with A. fumigatus lung
infections exhibit reduced lung function and increased risk of pulmonary exacerbations in
presence of P. aeruginosa [42]. Interestingly, P. aeruginosa isolates from CF patients are
often more inhibitory towards biofilms formed by A. fumigatus than non-CF isolates,
indicating that the selective pressures at the host-pathogen interface are promoting
competition between these microbes [43]. The study showed that the non-mucoid CF
isolates were most inhibitory of all and that similar inhibition against the fungus was
achieved by the culture supernatant of P. aeruginosa isolates only. Several other studies have
discovered the mechanisms by which P. aeruginosa outcompetes A. fumigatus in host
environments. For example, a study has shown that in a polymicrobial setting such as in the
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CF lung, P. aeruginosa can antagonistically interact with A. fumigatus by impeding the

fungi’s growth and modulating its filamentation [44]. Another study demonstrated that P.
aeruginosa inhibits growth of A. fumigatus by inducing oxidative and nitrogenic stress in
conidia and hyphal structures of the fungus [45] (Figure 3B). P. aeruginosa phenazines,
namely pyocyanin, phenazine-1-carboxamide, 1-hydroxyphenazine and phenazine-1-
carboxylic acid, produce reactive oxygen and nitrogen species which can penetrate through
A. fumigatus cells leading to altered mitochondrial ultrastructure and fungal death. However,

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at low concentrations, the same phenazine molecules were shown to enhance the growth of
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A. fumigatus via reduction of iron in iron-starved environment. This last finding again
highlights the fact that the balance between cooperation and competition is highly context
dependent.

One study of particular interest has also shown that P. aeruginosa exhibits phage-mediated
inhibition of metabolic activity in pre-formed biofilms of A. fumigatus [46]. The
bacteriophage Pf4, produced by P. aeruginosa, can inhibit metabolic activity in a dose
dependent manner at the biofilm surface where Pf4 binds to iron. This activity deprives the
fungus of this essential molecule and deficiency of iron acts as a global signal in alteration
of the fungi’s metabolism. Thus, this finding emphasizes the importance of assessing viral
effects on the complex microbial interactions at the host-pathogen interface.

Viral interactions with P. aeruginosa

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Recent studies have begun to demonstrate the importance of viruses in polymicrobial

infections. For example, both pediatric and adult CF patients experience frequent viral
infections with respiratory syncytial virus (RSV), adenovirus, influenza, parainfluenza, and
rhinovirus. These CF-associated viral infections can impact the disease progression and
eventually lead to a decline in lung function [47, 48]. The complex interaction of P.
aeruginosa with viruses has not been fully elucidated to date. However, research groups have
recently begun testing these types of polymicrobial interactions to further understand the
role of viruses in the progression of lung diseases associated with P. aeruginosa infection.
One such key study has revealed the role of RSV isolated from CF patients in helping P.
aeruginosa adapt and colonize in the lungs. The virus infection was able to augment the
growth of P. aeruginosa biofilm in a CF lung disease model by inducing antiviral interferon
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(IFN) signaling pathways. Additionally, as a result of RSV infection, host nutritional

immunity was impaired and iron bioavailability for the bacteria was increased due to a
release of transferrin, a host iron binding protein. [49] This ultimately enabled P. aeruginosa
to transition to a biofilm phenotype (Figure 3C). This finding emphasizes how the individual
modes of action of different infectious agents can synergize to impact disease progression
and severity.

Another category of viruses that has been gaining interest from researchers is bacteriophages
(phages), which are viruses that infect bacteria. Phages can reach high abundance at sites of
chronic infections and have been known to exceed their bacterial host density [50]. During
chronic lung infections both the bacteria and phages coevolve and undergo rapid
evolutionary adaptation, resulting in extensive genetic diversification. The bacteria-phage
coevolution impacts the structure of the microbial community [51, 52]. This can result in
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gaining ecologically relevant traits in bacterial species, which might help them survive
extreme conditions like environmental stressors or antibiotic treatment [53]. Microbial
communities in the CF lungs are both spatially and temporally heterogeneous and
competition in these regional niches can lead to diversification [50]. However, phages can
also drive microbial diversification when temperate phages integrate themselves into the host
genome, forming a prophage [54] (Figure 3D). The switch between the lytic and lysogenic
cycles of temperate phages may have an impact on their bacterial hosts as well [55]. For

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example, phages can assist in the transfer of bacterial DNA that can potentially impart new
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traits like antibiotic resistance and introduce novel functions such as virulence factor
production to the bacterial host [56].

Recent studies have shown that an infection with phage in P. aeruginosa can result in an
increased mutation rate which can be beneficial during the course of evolution and
adaptation within the lung environment [57]. This process has been demonstrated in vitro
using experimental evolution and population genomics to study the role of phages on driving
species diversity and evolution in P. aeruginosa biofilms [58, 59]. In one of these studies, P.
aeruginosa population dynamics was assessed after growth in conditions mimicking the CF
lung for approximately 240 bacterial generations in the presence of three Liverpool
Epidemic Strain (LES) phages (LESϕ2, ϕ3 and ϕ4). P. aeruginosa LES is a common
transmissible strain infecting CF patients and therefore the phages derived from this strain
are highly relevant to studies of CF disease progression. These results highlighted the
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importance of phages in selecting for adaptive mutations which mainly disrupted the type-IV
pilus dependent motility and quorum sensing regulators in P. aeruginosa. Overall, the study
suggested a role for phages in adaptive evolution by selecting for mutations that help the
pathogen adapt and survive in the CF lung environment [54]. It thus becomes important to
pursue the understudied viral interactions with P. aeruginosa for a more definitive
understanding of their impact on the CF airways and the lungs.

Conclusions
In the context of polymicrobial infections, the scarcity of resources creates an inherently
competitive environment, yet the presence of greater genetic diversity can enable resource
sharing and drive synergistic coexistence. The balance between these types of interactions
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can be easily shifted by subtle environmental influences present within different regions of
the lung [60]. In turn, these interactions can influence disease progression, severity, and
persistence by mediating tissue destruction and altering community susceptibility to the host
immune response and antibiotic treatment. Therefore, more effective therapeutics must be
developed to treat polymicrobial chronic infections. Studies have already demonstrated that
microbial physiological requirements can be dramatically altered in mono- versus co-
infections [38]. These findings emphasize that therapeutic targets identified via less
physiologically relevant mono-infections may falsely indicate the importance of
physiological processes that become dispensable in polymicrobial contexts. This
phenomenon highlights that further study of polymicrobial interactions at the host-pathogen
interface is essential in the development of novel antimicrobial therapeutics. Of note, most
studies delving into polymicrobial interactions during infection utilize in vitro studies or
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animal models of infection. Therefore, the extent of relevance of polymicrobial interactions

during human infection requires further exploration. This topic is discussed in greater detail
in the review by Ibberson and Whiteley published in this issue [37].

Acknowledgements
We would like to thank the members of the Wakeman lab for critical reading of this manuscript. Work in the
Wakeman lab is supported by NIH/NIGMS (R15GM128072).

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**This article highlights how regional niches in the host can shape interactions in polymicrobial
communities and vice versa.

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Highlights
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• Spatial heterogeneity in the lung environment can influence microbial

interactions.

• Context dictates shifts between cooperative and competitive microbial

interactions.

• Pseudomonas aeruginosa interacts with bacteria, fungi, and viruses in

infection.

• The outcome of these interactions can shape disease progression within the
host.
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Figure 1. Microbial interactions experienced by P. aeruginosa in the lungs.

The lungs are colonized by diverse bacterial, fungal, and viral taxa. The interactions of P.
aeruginosa with the other microbes inhabiting the lungs can be both cooperative and
competitive in nature. In particular, this review focuses on (1A) bacterial interactions such as
intraspecies interactions between sublineages of P. aeruginosa as well as in interspecies
interactions with S. aureus, (1B) interkingdom interactions with fungi such as C. albicans
and A. fumigatus, and (1C) interactions with viruses such as RSV and bacteriophages.
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Figure 2. Intra- and interspecies bacterial interactions exhibited by P. aeruginosa in the lungs.
In the host tissues, P. aeruginosa cells interact with neighboring bacterial cells, competitively
or cooperatively, to establish persistent infections. Intraspecies interactions between P.
aeruginosa sublineages and strains can be cooperative (2A) such as when different P.
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aeruginosa isolates like the methionine auxotroph depicted as green cells and the arginine
auxotroph depicted as brown cells share the production of biosynthetic molecules or
competitive (2B) as certain strains can dominate over others via the production of pyocins.
Similarly interspecies interactions with the common co-infecting pathogen S. aureus can be
competitive (2C) as P. aeruginosa can produce antimicrobials that not only intoxicate S.
aureus but also increase susceptibility to certain antimicrobial agents or synergistic (2D) via
P. aeruginosa’s exploitation of S. aureus’s anti-host factors, a phenomenon that may in turn
select for P. aeruginosa’s repression of anti-staphylococcal molecules in the presence of host
proteins like calprotectin and serum protein albumin.
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Figure 3. Interactions between P. aeruginosa and other microbial inhabitants of the cystic fibrosis
lungs.
In addition to the bacterial interactions of P. aeruginosa, recent studies have also shown its
interaction with fungus and viruses. This figure highlights some of these interactions in the
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context of synergism or antagonism. Phenazines secreted by P. aeruginosa can help in

ethanol production by C. albicans which in turn increases production of Pel polysaccharide
in the former (3A) resulting in a synergistic interaction. On the contrary, P. aeruginosa can
interact competitively with other fungal species such as A. fumigatus via production of
reactive oxygen and nitrogen species (3B). Figures 3C and 3D represents P. aeruginosa’s
interaction with viruses which are critical but understudied. Viruses such as Respiratory
syncytial viruses (RSV) can impair host nutritional immunity making iron bioavailable for P.
aeruginosa and helping it to switch from planktonic to biofilm lifestyle (3C). Other viruses
such as phages can also help P. aeruginosa survive the hostile lung environment by
introducing mutations that can lead to emergence of divergent strains well adapted to the CF
lungs (3D).
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