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Drug Discovery Today: Disease Models Vol. 4, No. 3 2007

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Andrew McCulloch – University of California, SanDiego, USA

Infectious diseases

Animal models of chronic Pseudomonasaeruginosa lung infection in cysticfibrosisNadine HoffmannDepartment of Clinical Microbiology, Rigshospitalet, and Department of Bacteriology, Institute for medical Microbiology and Immunology, Panum Institute,

University of Copenhagen, Copenhagen, Denmark

Patients with cystic fibrosis (CF), chronic Pseudomonas

aeruginosa lung infections represent a major morbid

complication. The current state-of the-art research

suggests that alginate biofilm formation is crucial in

the pathogenesis of chronic P. aeruginosa lung infection

and that quorum sensing (QS)-regulated virulence fac-

tors that might be important for initiation of acute

infection are often selected against during chronic

infection. Hence, fine tuned balances of regulatory

virulence systems are adaptive traits of P. aeruginosa

during persistent infection.

E-mail address: N. Hoffmann (nadinehof@hotmail.com)

1740-6757/$ � 2007 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2007.11.008

Section Editor:Trevor Trust – AstraZeneca R&D Boston, 35 GatehouseDrive, Waltham, MA 02451, USA

Introduction

Pseudomonas aeruginosa is a Gram-negative bacterial species

causing several opportunistic human infections and is fre-

quently isolated in ventilator-associated pneumonias and in

chronic bronchopneumonias of CF patients. A hallmark of P.

aeruginosa infecting CF patients is the transition from a non-

mucoid to a less virulent mucoid, alginate-overproducing

phenotype (Fig. 1) initiating a fatal, chronic stage of infection

[20]. Before development of a chronic stage, an intermittent

colonization of typically non-mucoid, cytotoxic (type III

secretion system), fast growing antibiotic-susceptible P. aer-

uginosa occurs [23,26]. The ability of P. aeruginosa to adapt

and grow as a mucoid alginate biofilm is a survival strategy

and an important aspect of the pathogenesis in the CF lung

disease because this mucoid character protects the bacteria

against antibiotics and immune responses. The lung tissue

damage is due to immune complex mediated chronic inflam-

mation dominated by release of proteases and oxygen radi-

cals from polymorphonuclear leukocytes [20] where oxygen

radicals are able to induce mutations in mucA gene leading to

mucoid phenotype [27]. The lifelong chronic lung infection

is associated with accumulation of loss-of-function muta-

tions in specific P. aeruginosa genes [25,33] and extensive

genetic adaptation and microevolution [5,21].

P. aeruginosa produces a wide range of virulence factors,

which are expressed differently, depending on environmen-

tal and metabolic aspects of its current habitat. Many of these

virulence factors are regulated by cell-to-cell signaling,

termed QS that is facilitated by a high density of cells, such

as in bacterial biofilms [8]. The QS systems are responsive to

different signal molecules; N-acylhomoserine lactone (AHL)

and 4-quinolones (4 Qs). The AHL-based circuits are encoded

by the Las and Rhl systems. The two systems operate with

specific signal molecules: N-3-oxododecanoyl-L-homoserine

lactone (3-oxo-C12-HSL) for the lasR-encoded receptor and

N-butanoyl-L-homoserine lactone (C4-HSL) for the rhlR

encode receptor. These two systems (las and rhl) operate

hierarchially with the las system on top where the quinolone

system is placed in between the las and the rhl system [10].

99

Drug Discovery Today: Disease Models | Infectious diseases Vol. 4, No. 3 2007

Figure 1. Mucoid P. aeruginosa microcolonies from broncho-alveolar

lavage from a CF patient with chronic P. aeruginosa lung infection.

This review is discussing the impact of QS, alginate and the

genomic diversity of P. aeruginosa and their implications on

the CF pathogenesis that is important when animal model

that matches etiologically to CF human chronic lung infec-

tion are being developed. Furthermore, bacterial lung appli-

cation methods in the animal models will be discussed. The

outline of cftr knockout mice is not discussed in the present

but is addressed in a review by Scholte et al. [31].

PAO1 isolates versus CF isolates

The aim of all animal models is to mimic human infection

as close as possible and to provide useful data; the animal

model infections must show a close etiological relationship

to the human disease. For instance, a typical virulent non-

mucoid P. aeruginosa causing an acute overwhelmingly

toxaemic pneumonia that causes death in two to three days

in mice clearly has very little to do with non-toxaemic

chronic P. aeruginosa lung infection that persists for

20–30 years in CF patients – even though both are

P. aeruginosa pneumonias.

The non-mucoid standard laboratory-reference strain P.

aeruginosa PAO1 was originally a burn-wound isolate and

has a genomic profile that is not similar to the majority of

clinical CF lung isolates [42]. PAO1 is generally used in vitro

and in vivo evaluating the interaction between the bacteria,

the host and antibacterial therapy. For example, in experi-

mental lung infection models, PAO1 is artificially embedded

in, for example, agar beads to resemble a situation similar to a

chronic biofilm lung infection. A problem, in particular, that

interferes with PAO1is that the strain is non-mucoid, viru-

lent, motile and replicates fast and thus opposite the char-

acteristics of a typically chronic mucoid CF isolate. Here, the

100 www.drugdiscoverytoday.com

bacteria adopt the biofilm mode of growth using high-energy

resources to encapsulating themselves in alginate but loss

motility and slow down replication and virulence factors

production. Secondly, basic metabolic activities, such as

regulation of carbon catabolism are altered in chronically

CF isolates compared with PAO1 [32]. Finally, the recent

report by Smith et al. concluded that the genotypes of P.

aeruginosa strains present in chronic stage of CF patients differ

systematically from those of PAO1 [33]. Clearly, a PAO1 lung

infection will not relate to a chronic P. aeruginosa lung

infection; however PAO1 might be a model for early acute

infection stages. Because chronically CF isolates of P. aerugi-

nosa appear to utilize a different set of virulence determinants

and pathogenic mechanisms to cause persistent infection

than non-CF isolates and because pathogenic processes are

altered in CF lung isolates, these isolates might respond

differently than non-CF isolates. Despite the diversity

among isolates from CF patients, they do provide data that

are likely to be more clinically relevant than studies based

solely on PAO1. Therefore, to evaluate the pathogenesis and

develop more effective treatments for Pseudomonas lung

infections in CF patients or other patients chronically

infected with P. aeruginosa (e.g. chronic obstructive pulmon-

ary disease), it is crucial to establish experimental chronic P.

aeruginosa lung infection models using chronically bacterial

lung isolates.

Animal models of chronic P. aeruginosa lung infection

The impact of alginate

Mucoid alginate-biofilms of P. aeruginosa in CF sputum were

first described by Høiby [19] followed by Lam et al. [24]

observing P. aeruginosa alveolar microcolonies encapsulated

in alginate where the alginate occupies more space than do

the bacterial cells themselves. The protection against

immune systems provided by the alginate probably resulting

in niche expansion, might be causing infections of the alveoli

in the aerobic zone of the lung [16,18]. The highly stressful

and complex environmental conditions associated with bio-

film life in lungs of CF patients require that P. aeruginosa

exploit their entire repertoire of regulatory systems when

adapting to the challenge imposed by the host immune

system and huge levels of antibiotics.

Since the establishment of the first animal model of

chronic P. aeruginosa lung infection in rats by Cash et al.

[6], several animal models of acute and chronic P. aeruginosa

lung infection have been described [22,37], references herein.

These models, however, required that P. aeruginosa was

embedded in an artificial biofilm (agar, agarose, seaweed

alginate) to prevent mechanical clearing. These artificial bead

models might lead to bronchopulmonary infection with

histopathological features that resemble CF. However, mak-

ing beads is a crucial step [37] and a risk for infection only in

the conducting airways, not the alveoli, owing to the size

Vol. 4, No. 3 2007 Drug Discovery Today: Disease Models | Infectious diseases

Figure 2. Photographs showing histopathologic features of a mouse model of chronic P. aeruginosa lung infection mimicking CF (a and b) similar to those of

chronic P. aeruginosa lung infections in humans (c and d). Sections of mouse lung tissue seven days post-infection stained with HE and Alcian-blue for

detection of alginate. Mucoid P. aeruginosa NH57388A infected CF mouse lung showing large P. aeruginosa biofilms (black circles) encapsulated in alginate

(blue color) in the alveolar space surrounded by PMNs (white arrow) at �40 (a) and �100 (b) magnifications. Autopsy of a CF patient who died from

chronic P. aeruginosa lung infection showing alveoli sacs filled with P. aeruginosa biofilms (black circles) at magnifications of�100 (c) and�1000 (d) with HE

and Gram stain.

of the beads mechanically blocking the bronchi in mice

[40]. Another concern with the bead-model is that the

Pseudomonas-laden beads probably undergo some kind of

crippling after instillation in the lung, and thus are suscep-

tible to the immune system. Apparently, in, for example,

mice challenge with PAO1-laden non-acetylated seaweed-

alginate beads, bacterial load decreases faster than mouse

challenged with a free stable, mucoid isolate constitutive

secreting O-acetylated alginate. Acetylation of alginate is

crucial to resistance to complement-mediated phagocytosis

[30] and confers a selective survival advantage of mucoid P.

aeruginosa [14,16]. In addition, because the alveolar space is

associated with volume and shape changes during breathing,

the adaptation of a viscoelastic biofilm structure secreted by

P. aeruginosa native alginate might be advantageous than, for

example, a more rigid seaweed alginate gel [16]. Animal

models addressing the role of P. aeruginosa alginate in pul-

monary lung infection without the uses of artificial embed-

ding have not been used widely, and the mice were already

sacrificed within 48 h [3,4,34,38,41]. Coleman et al. [7] pro-

duced a chronic infection with P. aeruginosa added to the

drinking water, although infection was established only in

few CF mice and with a low bacterial load. Recently, we

established a mouse model of chronic P. aeruginosa lung

infection without artificial embedding based on a mucoid

CF isolate constitutive secreting O-acetylated alginate and

thus encapsulating itself in a biofilm during long-lasting

infection [16]. Hence, a more reliable model infection that

matches etiologically to CF human chronic lung infection

(Fig. 2).

The impact of QS

Animal models of Pseudomonas lung infection can be used for

identifying virulence genes important for pathogenesis and

results are highly dependent on the details of the bacterial

strain, animal strain and challenge techniques used in the

model. Several animal studies addressing the role of QS for

pathogenicity of P. aeruginosa in acute [29,35] or chronic

pulmonary lung infection have been performed [1,39]. Data

from these studies showed that P. aeruginosa mutants defec-

tive in QS were less pathogenic and promoted bacterial

clearance in contrast to their parental strain. Additionally,

blocking of QS by antipathogenic drugs as furanones [12] and

garlic [2] had a beneficial effect on clearance of P. aeruginosa

from the mouse lung. In all studies, isolate PAO1 and not a CF

isolate was used and cautious interpretation of these studies is

therefore warranted. Apparently, for both non-mucoid and

mucoid CF isolates, bacterial clearance from the lung was not

different in mice infected with QS-negative mutants com-

pared with isolates with functional QS; however, mortality

was lower in mice infected with QS-negative mutants.

Indeed, mice challenged with the CF strains defective in

QS had slightly higher bacterial load in lung than mice

challenged with P. aeruginosa with functional QS [15–17].

These data indicate that loss of QS activity in CF isolates

might provide the bacteria with selective advantages for

survival and living in lung environment and is in accordance

with clinical histories of CF patients [9,25,33]. In fact,

D’Argenio et al. [9] showed that loss of QS due to lasR

mutations conferred a growth advantage in a CF isolate

recovered from chronic infection stage. Partly, this was due

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Drug Discovery Today: Disease Models | Infectious diseases Vol. 4, No. 3 2007

to increased expression of the catabolic regulator CbrB of P.

aeruginosa thus supporting a key role for metabolic adapta-

tion during chronic infection. Finally, a therapeutic animal

study, using mucoid CF isolate with functional QS encapsu-

lating itself in an alginate biofilm, demonstrated that garlic

treatment in balb/c-mice displayed no difference in mortality

and bacterial clearance compared with placebo (Hoffmann N,

to be published), contradicts the results by Bjarnsholt et al.

using non-mucoid PAO1 embedded in seaweed-alginate as

biofilm model [2]. A finding that might reflect differences in

the P. aeruginosa strains and/or the biofilm models used.

Table 1. Comparison of the advantages and disadvantages of m

Route Advantages Dis

Aerosol challenge Possibility of infecting a large number of

animals simultaneously

De

bac

Non-invasive Hig

con

Req

Hig

the

for

neb

Hig

ma

Intranasal challenge Easy to perform and non-invasive De

bac

Hig

ma

Blind intratracheal

challenge

Less invasive than intratracheal challenge

with incision; allows the exclusion of the

surgery-related inflammation

On

rea

pos

diffi

can

sho

app

Dif

ow

Intratracheal

challenge

with incision

Allows deposition of a standard inoculum

to the lung in 100% of the animals

Th

inv

resAnesthesia inhibits coughing and thus

swallowing of the bacteria

Induces colonization of the lung resembling

the pathophysiology of human disease

Low variability and therefore relative few

animals needed

Microscope-controlled

intratracheal

challenge

Less invasive than intratracheal challenge

with incision; delivery of sufficient number

of bacteria to the lung is good; induces

colonization of the lung resembling the

pathophysiology of human disease; allows

the exclusion of the surgery-related

inflammation

Mic

Low variability and therefore relative few

animals needed

Dif

ow

102 www.drugdiscoverytoday.com

The question whether the QS machinery can have a nega-

tive impact on the fitness of the organism is raised by the fact

that a significant percentage of clinical and environmental

isolates of P. aeruginosa are defective for QS [9,13]. CF isolates

of P. aeruginosa display a great variation of QS activity, and the

environment of the CF lung not only induce mutations in P.

aeruginosa but also select those mutants best able to survive

and persist. Much attention should be paid to the recent

reports by D’Argenio et al. [9] Smith et al. [33] and Lee et al.

[14,25] showing a selection for QS negative mutants, for

example, lasR mutations during chronic infection. Thus,

ethods for application of bacteria in lung

advantages Repr

oducibility

Technical

skill

Cost

livery of sufficient number of

teria to the lung is very difficult

Low High High

h risk of extra pulmonary tissue

tamination

uires appropriate equipment

h antigen load because many of

bacteria are lysed by shear

ces when pressed through the

ulizer

h variability and therefore relative

ny animals needed

livery of sufficient number of

teria to the lung is difficult

Low Low Low

h variability and therefore relative

ny animals needed

ly a small fraction of the inoculum

ches the lung because correct

ition of the catheter in mice is

cult; mostly the esophagus is

nulated, and thus this technique

uld be called a pharyngeal

lication

Low Medium Low

ficult to adapt for mouse model

ing to small trachea

e surgery-related inflammation

olved in tracheotomy might

ult in moribund animals

High High Low

roscope by challenge is required High High High

ficult to adapt for mouse model

ing to small trachea

Vol. 4, No. 3 2007 Drug Discovery Today: Disease Models | Infectious diseases

inactivation of QS might play an important role in the

adaptive processes. lasR mutation, however, still leaves the

rhl QS system intact. Interestingly, results from our laboratory

showed that for non-mucoid CF sputum isolates recovered

from chronic infection phase, the rhl-regulated signal mole-

cule C4-HSL was produced in excess of 3OC12-HSL [25]. An

observation we also found in a chronically, mucoid CF spu-

tum isolate (NH57388A) where large amount of C4-HSL

(1053 nM) compared with 3OC12-HSL (3 nM) (T B Rasmus-

sen, personal communication) was determined. These obser-

vations might indicate that selective pressure favor loss of the

las signaling system during chronic stage of CF infections

while maintaining expression of rhl-encoded factors.

Although the rhl system requires a functional las system,

the rhl system can be switched on independently [36] of

the las system.

Concurrent with and apparently in support of above find-

ings, the metabolic cost of the maintenance of the fully QS

machinery together with the energy consuming alginate

production during chronic stage could have a negative

impact on the fitness of the bacterium. Moreover, loss of

lasR function might represent a marker of an early stage in

chronic infection of the CF lung as suggested by D’Argenio

et al. [9]. Hence, fine tuned balances of regulatory virulence

systems are adaptive traits of P. aeruginosa during persistent

infections.

Comparison of different bacterial application methods

A rather high challenge dose is needed to establish a lung

infection in rodents because they are inherently more resis-

tant to bacterial infections than human. Additionally, differ-

ent species of rodent exhibit differences in course of

susceptibility to infection; thus, careful inoculum titration

is important for the outcome of the infection. Mice provide a

versatile and flexible model of infections because they are

cheap to purchase and maintain and a vast number of anti-

bodies are available. In particular, the availability of geneti-

cally modified animals, for example, cftr knockout mice, has

made the mice the preferred model system. Robust experi-

mental chronic pulmonary infection models require a repro-

ducible method of application with defined number of

bacteria to the respiratory tract without contaminating extra-

pulmonary tissue. In the following, different application

methods are summarized in Table 1 and discussed.

The least invasive application methods, namely aerosol

and intranasal have several disadvantages. Delivery to the

lungs of a high enough number of bacteria to ensure fatal

pneumonia is very difficult, and the risk of extrapulmonary

tissue contamination is high [28]. Hence, these models of

infection might primarily be used to study the acute infection

response.

The highest reproducibility is achieved after applying bac-

teria in the trachea under microscope [11] or by tracheotomy

disease [16,22], allowing the deposition of an accurate inocu-

lum in 100% of the animals. These techniques induce colo-

nization of the lung and bronchopulmonary chronic

infection resembling the pathophysiology of human. How-

ever, the trauma caused by surgery involved in the tracheot-

omy might result in moribund animals; and thus, obviously

the microscope-controlled technique is less invasive owing to

the exclusion of the surgery-related inflammation [11,28].

Conclusion

If animal models are carefully matched to a human infection

disease and if several points of correspondence between the

animal and human infections can be established, we can

begin to place some confidence in the extrapolation of data

between them. The extensive genetic adaptation of P. aeru-

ginosa in CF lung environment still presents a major chal-

lenge to definition of a gold standard model.

Acknowledgements

The author would like to thank Søren Molin, Infection Micro-

biology Group, BioCentrum-DTU, Technical University of

Denmark and Niels Høiby, Department of Clinical Microbiol-

ogy, Rigshospitalet, Copenhagen, Denmark, for critical read-

ing of the manuscript.

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