REVIEW ARTICLE
Di¡erential adaptation of microbial pathogens to airways
of patients with cystic ¢brosis and chronic obstructive
pulmonary disease
Gerd Döring1, Iyer G. Parameswaran2 & Timothy F. Murphy2
1
Institute of Medical Microbiology and Hygiene, Universitätsklinikum Tübingen, Tübingen, Germany; and 2Department of Medicine and New York
State Center of Excellence in Bioinformatics and Health Sciences, Division of Infectious Diseases, University at Buffalo, State University of New York,
Buffalo, NY, USA
Correspondence: Gerd Döring, Institute of
Medical Microbiology and Hygiene,
Universitätsklinikum Tübingen,
Wilhelmstrasse 31, D-72074 Tübingen,
Germany. Tel.: 149 7071 298 2069;
fax: 149 7071 293 011; e-mail:
gerd.doering@med.uni-tuebingen.de
Received 11 February 2010; revised 21 May
2010; accepted 25 May 2010.
Final version published online 24 June 2010.
DOI:10.1111/j.1574-6976.2010.00237.x
Editor: Dieter Haas
MICROBIOLOGY REVIEWS
Keywords
Pseudomonas aeruginosa; Haemophilus
influenzae; Staphylococcus aureus ;
Burkholderia ssp.; mutator; adaptive radiation.
Introduction
Cystic fibrosis (CF) is the most common autosomal
recessive disorder in Caucasians, affecting 1 : 2500 chil-
dren, with a carrier frequency of 1 : 25. In the CF lung,
disease develops as a consequence of mutations in the CF
transmembrane conductance regulator (CFTR) gene,
which encodes a membrane-bound cAMP-regulated chlor-
ide channel (Kerem et al., 1989; Riordan et al., 1989;
Rommens et al., 1989). The primary structure of CFTR
indicates that it belongs to a family of transmembrane
proteins called ATP-binding cassette transporters, which
form a large family of proteins responsible for the translo-
cation of a variety of compounds across membranes of
both prokaryotes and eukaryotes. CFTR is composed of
five domains: two membrane-spanning domains, two
nucleotide-binding domains (NBDs) and a regulatory

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Abstract
Cystic fibrosis (CF), the most common autosomal recessive disorder in Cauca-
sians, and chronic obstructive pulmonary disease (COPD), a disease of adults, are
characterized by chronic lung inflammation, airflow obstruction and extensive
tissue remodelling, which have a major impact on patients’ morbidity and
mortality. Airway inflammation is stimulated in CF by chronic bacterial infections
and in COPD by environmental stimuli, particularly from smoking. Pseudomonas
aeruginosa is the major bacterial pathogen in CF, while in COPD, Haemophilus
influenzae is most frequently observed. Molecular studies indicate that during
chronic pulmonary infection, P. aeruginosa clones genotypically and phenotypi-
cally adapt to the CF niche, resulting in a highly diverse bacterial community that
is difficult to eradicate therapeutically. Pseudomonas aeruginosa clones from COPD
patients remain within the airways only for limited time periods, do not adapt and
are easily eradicated. However, in a subgroup of severely ill COPD patients,
P. aeruginosa clones similar to those in CF persist. In this review, we will discuss the
pathophysiology of lung disease in CF and COPD, the complex genotypic and
phenotypic adaptation processes of the opportunistic bacterial pathogens and
novel treatment options.
domain. Over 1500 mutations and sequence variants have
been described to date and reported to the Cystic Fibrosis
Genetic Analysis Consortium. Most of these mutations are
rare and only four mutations occur at a frequency of
4 1%. The most frequent mutation, F508del, occurs in
the DNA sequence that codes for the NBD1 (Döring &
Ratjen, 2005). Abnormal transport of chloride and sodium
ions caused by CFTR mutations affects water movement
across epithelia, leading to pathophysiological conse-
quences in various organs including the respiratory, gas-
trointestinal and reproductive tract, the pancreas and the
liver. The CF phenotype is rather heterogeneous due to
many different mutations in CFTR and the influence of
modifier genes (Davies, 2007). Chronic respiratory infec-
tions caused by several opportunistic bacterial pathogens
are recognized to have the largest impact on morbidity and
mortality in CF patients (Høiby & Frederiksen, 2000).
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD
Bacterial infections stimulate inflammatory defence me-
chanisms, leading to tissue destruction and extensive tissue
remodelling (Döring & Ratjen, 2007). The resulting
emphysema and fibrosis mainly determine the reduced life
expectancy in individuals with CF. Once established, these
infections are difficult to treat with antibiotics and the
pathogens are rarely eradicated (Döring et al., 2004).
Unlike CF, chronic obstructive pulmonary disease
(COPD), a chronic, progressive disorder of lung parenchy-
ma and airways, is exclusively a disease of adults, especially
of individuals over the age of 60 years. COPD is the fourth
leading cause of death globally, accounting for approxi-
mately 2.7 million deaths in 2003 (Lopez et al., 2006).
COPD is defined by airflow obstruction that is not fully
reversible, in contrast to asthma, which is associated with
reversible airflow obstruction (Celli & MacNee, 2004). The
degree of reduction in forced expiratory volume in 1 s
(FEV1) is used to classify COPD into four stages (mild,
moderate, severe and very severe). Although genetic factors
influence COPD (Wood & Stockley, 2006; Silverman et al.,
2009; Smolonska et al., 2009), these influences are complex
and mostly unknown. COPD results from responses to
environmental stimuli, particularly from smoking, in ge-
netically susceptible individuals (Celli & MacNee, 2004;
Wood & Stockley, 2006). Bacterial pathogens also play an
important role in the course and pathogenesis of COPD
(Sethi & Murphy, 2008).
Many similarities exist between CF and COPD. In both
diseases, exuberant inflammation causes airflow obstruc-
tion, tissue destruction and extensive tissue remodelling,
leading to emphysema and fibrosis. Thus, inflammation
plays an important role in the morbidity and mortality of
patients. However, CF and COPD differ with regard to
infection. While the role of chronic bacterial infections,
particularly caused by Pseudomonas aeruginosa, is well
established in CF (Høiby & Frederiksen, 2000), the impact
of these infections in patients with COPD is less clear
(Murphy et al., 2008). Furthermore, pathogens such as
Moraxella catarrhalis and Streptococcus pneumoniae are
frequently observed in patients with COPD (Sethi & Mur-
phy, 2001), and yet are seldom present in the airways of CF
patients. Because of improved symptomatic treatment
strategies and antibiotic therapy, the prognosis of CF
individuals has improved considerably and many children
now reach adulthood. While antibiotic therapy is beneficial
in treating acute exacerbations of COPD (Anthonisen, 2002;
Ram et al., 2006), this therapy has not been demonstrated to
affect the course of the disease. In this review, we will discuss
the pathophysiology of lung disease in CF and COPD,
bacterial colonization of airways and the complex genotypic
and phenotypic adaptation processes of the opportunistic
bacterial pathogens in these diseases and novel treatment
options.
FEMS Microbiol Rev 35 (2011) 124–146
125
Pathophysiology of CF lung disease
A variety of innate immune functions have been reported to
be dysregulated in CF, and a number of mechanisms have
been put forward to explain the fact that practically every CF
patient suffers from chronic bacterial lung infections. The
inability of mutated CFTR to effectively secrete chloride
from respiratory epithelial cells into the airway surface
liquid causes excessive water absorption from the airway
surface liquid, leading to impaired mucociliary clearance in
individuals with CF (Matsui et al., 1998; Boucher, 2007)
(Fig. 1). This in turn facilitates the colonization of the
viscous mucus layer on the respiratory epithelium with
bacteria, rather than the colonization of the epithelial sur-
face per se (Ulrich et al., 1998; Worlitzsch et al., 2002).
Furthermore, an abnormal accumulation of ceramide in
the lungs of CF mice and in epithelial cells from CF patients
has been shown to result in an increased death rate of
respiratory epithelial cells and DNA deposits on the respira-
tory epithelium, which facilitates bacterial adherence
(Teichgräber et al., 2008). In addition to these mechanisms,
which explain the high incidence of chronic bacterial lung
infections in CF, other investigators have demonstrated
that alveolar macrophages from CF mice exhibit defective
killing of internalized P. aeruginosa (Di et al., 2006). After
phagocytosis by alveolar macrophages, bacteria enter the
phagosome and lysosomes, exhibiting a low pH due to
CFTR and vacuolar ATPase, and fuse with the phagosome,
forming the phagolysosome. The mature organelle exhibits
an acid pH that ensures the final destruction of the ingested
bacteria by a variety of proteolytic enzymes. Defective
CFTR-dependent acidification of lysosomal pH (Barasch
et al., 1991) has been linked to significant survival of
P. aeruginosa (Di et al., 2006). The contribution of CFTR to
the regulation of pH in intracellular organelles has been
discussed controversially (Haggie & Verkman, 2007, 2009)
due to experimental difficulties to ensure targeting of
identical vesicle populations by pH-sensitive fluorescent
dyes. Pseudomonas aeruginosa infection may also be facili-
tated in CF airways directly by defective CFTR, which, in its
functional state, binds the pathogen within lipid rafts and
removes it from the epithelial surface via internalization
(Pier, 2000).
The viscosity of the secretions has several further negative
consequences for the pathophysiology of CF lung disease.
These secretions are present in submucosal gland ducts of
CF patients as a result of mutated CFTR (Engelhardt et al.,
1992), and may impair the transport of antimicrobial
oligopeptides (Lehrer & Ganz, 2002; Selsted & Quellette,
2003) onto the epithelium (Verkman et al., 2003; Joo et al.,
2004) (Fig. 1). They may also negatively affect the migration
of neutrophils towards the pathogens within the mucus
overlaying the respiratory epithelium (Matsui et al., 2005)

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126
(Fig. 1). This may allow bacterial multiplication and pro-
found changes in the bacterial phenotypes that facilitate
chronic infection. Furthermore, within the highly viscous
mucus, a microaerobic/anaerobic milieu prevails due to
oxygen consumption by bacterial pathogens such as P.
aeruginosa (Worlitzsch et al., 2002; Hassett et al., 2009) or
invading neutrophils (Kolpen et al., 2010). Consequently,
the generation of reactive oxygen species (ROS) by neutro-
phils and other cells is abolished and bacterial killing is
impaired (Fig. 2a and b), particularly with regard to patho-
gens, which are intrinsically resistant to nonoxidative kill-
ing, or capable of changing their phenotype towards
resistance to nonoxidative killing.
Lung inflammation is a key finding in patients with CF
(Konstan et al., 1994). While ceramide accumulation has
been shown to induce a proinflammatory status in the
respiratory tissue of CF mice, which precedes bacterial
infection, including an increased production of cytokines
and recruitment of macrophages (Teichgräber et al., 2008),
inflammation clearly increases considerably in response to
persistent bacterial infections. The exaggerated inflamma-

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
Fig. 1. Bacterial killing mechanism of innate
immunity in the respiratory tract of CF patients
and healthy individuals. (a) In healthy individuals,
bacteria entering the mucus layer overlaying the
respiratory epithelium are effectively removed
from the airways by a functional mucociliary
clearance system and killed by CAMPs derived
from submucosal glands or epithelial cells, by
functional neutrophils (PMN) or macrophages
(MF) or within epithelial cells after uptake via
functional CFTR. (b) In individuals with CF,
defective CFTR leads to a highly viscous mucus
layer that impairs mucociliary clearance, the
migration of CAMPs, neutrophils and
macrophages towards the bacterial targets, the
uptake of bacteria by CFTR itself, and induces a
pH shift to alkaline values in epithelial lysosomes.
Because of ceramide accumulation, this results in
DNA deposits, which in turn serve as adhesion
matrices for bacteria. A similar pH shift in the
phagolysosomes of macrophages impairs
bacterial killing. Ceramide accumulation also
triggers cytokine release, which induces further
neutrophil influx (adapted from Döring &
Gulbins, 2009).
tory response dominated by neutrophils not only facilitates
tissue destruction but also induces tissue remodelling
(Döring & Gulbins, 2009; Ulrich et al., 2010). An essential
role in these pathogenic reactions is related to the imbalance
of serine and metalloproteases to their respective inhibitors
(Döring, 1999; Ratjen et al., 2002). Particularly, neutrophil
elastase has been implicated in the pathophysiology of CF
lung disease. The serine protease has been shown to cleave
the endogenous serine protease inhibitor a1-antitrypsin,
immunoglobulins, surfactant proteins, complement com-
ponents and a variety of cell receptors (Döring & Gulbins,
2009). Cleavage of the complement receptor 1 on neutro-
phils and the activated complement component C3bi on
opsonized bacteria creates an ‘opsonin-receptor mismatch’
(Berger et al., 1989; Tosi et al, 1990), which significantly
impairs P. aeruginosa killing.
The vicious cycle of persisting bacterial pathogens, en-
cased in biofilms, and decaying neutrophils leads to increas-
ing endobronchial DNA deposition and large mucus plugs,
which further obstruct the airways of the patients (Whit-
church et al., 2002; Worlitzsch et al., 2002).
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD
Fig. 2. Bacterial sensitivity and resistance to neutrophil-mediated killing
in aerobic and anaerobic environments. (a) On the respiratory epithelium
of healthy individuals, neutrophils effectively kill the CF-related bacterial
pathogens Pseudomonas aeruginosa (red), Staphylococcus aureus (blue)
and Burkholderia cepacia (green) by ROS and CAMPs. (b) Neutrophils in
the anaerobic milieu in the mucus layer overlaying the respiratory
epithelium in individuals with CF are devoid of ROS, leading to intraneu-
trophil survival of S. aureus (blue), which produces a protective exopoly-
saccharide coat (red), and B. cepacia (green), which is intrinsically
resistant to nonoxidative killing (adapted from Döring & Gulbins, 2009).
Pathophysiology of COPD
The pathophysiology of COPD is characterized by chronic
inflammation of airways as a result of active and passive
smoking (Barnes, 2000; Celli & MacNee, 2004). The indices
FEMS Microbiol Rev 35 (2011) 124–146
127
of airway inflammation – increased neutrophils, IL-8, TNF-
a and neutrophil elastase – are all increased compared with
healthy controls (Keatings et al., 1996; Hill et al., 1999a b;
Barnes et al., 2003). Chronic colonization of the airways
contributes to this airway inflammation (Hill et al., 2000;
Parameswaran et al., 2009). However, the degree of airway
inflammation does not reach the threshold of patient-
perceived symptoms. When airway inflammation crosses
this threshold for short episodes, these are called exacerba-
tions (Hill et al., 1999a, b). The course of COPD is punc-
tuated by episodes of acute exacerbations, which are
associated with increased airway inflammation compared
with baseline levels (Hill et al., 1999a, b; Sethi et al., 2008).
Goblet cell hyperplasia and mucus hypersecretion are pre-
sent in the airways of most COPD patients, especially in
those with clinical features of chronic bronchitis (Barnes,
2000). Although mucus viscosity is considerably lower in
COPD compared with CF, mucociliary clearance of bacterial
pathogens may be impaired by the fact that respiratory
epithelial cells lose cilia and undergo squamous metaplasia
(Hogg & Timens, 2009). Lung inflammation leads to the
activation of innate immune cells, particularly macro-
phages, which have a profound effect on immune cell
recruitment into the airways and tissue remodelling (Barnes,
2004). In the small airway walls, infiltrating neutrophils and
lymphocytes cause airway thickening and remodelling,
resulting in reduced airway diameter and airflow (Hogg
et al., 2004). Inflammatory cellular infiltration of alveolar
walls is associated with the destruction of alveoli and
enlargement of air spaces, i.e. emphysema (Cosio et al.,
2009). Infiltration especially by CD81 lymphocytes occurs
in pulmonary vessel walls that are thickened due to in-
creased collagen deposition (Cosio et al., 2009). As in CF,
chronic lung inflammation is accompanied by an imbalance
between proteases and antiproteases in COPD (Stockley,
1999). Furthermore, oxidative stress is persistently enhanced
(Rahman & MacNee, 1996) as well as a number of other
innate immune functions that lead to tissue remodelling
(Barnes, 2000; Cosio et al., 2009; Hogg & Timens, 2009).
Alveolar macrophages from COPD patients have decreased
sensitivity to nontypeable Haemophilus influenzae (NTHI)
antigens and decreased ability to phagocytose NTHI, com-
pared with normal controls (Berenson et al., 2006a, b). The
structural abnormalities in airways, alveoli and pulmonary
vasculature vary among COPD patients (Hogg & Timens,
2009).
Bacterial colonization of the CF lung
Chronic bacterial pulmonary infection leading to an irre-
versible decline in lung function is the main cause of
mortality and morbidity in CF patients, with 4 95% of
deaths due to respiratory failure (Dodge et al., 2007).

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128
Staphylococcus aureus is the most prevalent opportunistic
bacterial pathogen, which chronically infects CF airways in
the age group of 0–9 years (55%), while P. aeruginosa is the
most prevalent pathogen thereafter (81%) (Høiby & Freder-
iksen, 2000). At a much lower prevalence, H. influenzae, S.
pneumoniae, Escherichia coli, strains of the Burkholderia
cepacia complex (Bcc), comprising 10 genomovars, and
fast-growing mycobacteria can be present in airway speci-
mens of CF patients (Høiby & Frederiksen, 2000).
In addition, a large number of strict anaerobes have been
detected, which may be surprising for an air-filled organ such
as the lung. However, the viscous mucus plugs, in which all
these bacteria thrive, have been shown to create microaerobic/
anaerobic conditions (Worlitzsch et al., 2002), which favour
the growth of anaerobes. Anaerobic bacteria have been
detected in significant numbers (4105 CFU mL1) in the
lungs of CF patients using classic culturing methods, in-
dicating that a highly diverse bacterial community is present
within the CF lung (Brook & Fink, 1983; Jewes & Spencer,
1990; Tunney et al., 2008; Worlitzsch et al., 2009). These
include diverse species belonging to the genera Prevotella,
Bifidobacterium, Veillonella, Peptostreptococcus and Fusobac-
terium. Single obligate anaerobic species persisted for up to
11 months in sputum plugs in vivo (Worlitzsch et al., 2009).
Whether these bacteria contribute to the pathophysiology of
CF lung disease is still an open question. CF patients with
and without obligate anaerobes in sputum specimens did
not differ in lung function (Worlitzsch et al., 2009).
Culture-independent profiling methodologies (Nocker
et al., 2007) such as terminal restriction fragment length
polymorphism have considerably advanced our knowledge
of the polymicrobial nature of CF lung disease (Rogers et al.,
2003, 2004, 2006, 2009; Harris et al., 2007; Bittar et al., 2008;
Sibley et al., 2008, 2009; Klepac-Ceraj et al., 2010). For
instance, it has been shown by culture-independent meth-
ods that the Streptococcus milleri group of viridans strepto-
cocci, comprising Streptococcus constellatus, Streptococcus
intermedius and Streptococcus anginosus, is present in CF
sputum specimens and related to the pathophysiology of CF
lung disease (Sibley et al., 2008).
During acute infections, which may resolve after several
days, microorganisms reversibly regulate gene expression by
responding to environmental signals, and thereby adapt
their phenotypes accordingly (Green & Paget, 2004; Gilles-
Gonzalez & Gonzalez, 2005). During the course of chronic
infections, which may last 4 10 years, however, mutants
can be selected that differ genotypically and phenotypically
from the originally infecting strain. Given the large micro-
biota in the airways of infected CF patients, complex
interactions not only occur between microorganisms and
the environment but also among members of the commu-
nity that use primary or secondary metabolites in a multi-
tude of chemical interactions (Duan et al., 2009). A

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
fascinating view is that ‘cheater’ cells that refrain from
producing ‘expensive’ metabolites benefit in such commu-
nities from those produced by the rest of the population
(Keller & Surette, 2006; Duan et al., 2009). The chemical and
physical nature of the mucus plugs in CF airways, in which
the bacterial pathogens multiply, favours such a microevo-
lution during the course of chronic infection. Interestingly,
it has been found that the number of taxa and the phyloge-
netic diversity and evenness within the microbiota in the
airways of CF patients decrease with age, which has been
attributed to either repeated antibiotic therapy courses or
invasion of P. aeruginosa into the CF airways (Klepac-Ceraj
et al., 2010). Microevolution processes have been studied
most widely in P. aeruginosa.
Pseudomonas aeruginosa is the most prevalent
pathogen
The capacity of P. aeruginosa to cause infections in CF
patients and other hosts can be traced back to its consider-
able adaptability to specific environmental conditions and
the production of numerous virulence factors (Lee et al.,
2006) (see Box 1). This adaptability is linked to a large
number of regulatory genes. For example, in the 6.3 Mb
genome of P. aeruginosa PAO1, which contains 5570 pre-
dicted ORFs, 521 known or putative regulatory genes have
been identified, demonstrating a highly complex gene
regulation (Stover et al., 2000). The P. aeruginosa chromo-
some contains a core genome consisting of the sequences
that are common to all strains of the taxon and variable
amounts of accessory DNA segments. The accessory genome
that can be horizontally transferred among strains repre-
sents the flexible gene pool that frequently undergoes
acquisition and loss of genetic information and hence plays
an important role in the adaptive evolution of bacteria. The
flexible gene pool is made up of elements such as bacter-
iophages, plasmids, insertion elements, transposons, con-
jugative transposons, integrons and genomic islands.
Isolates from CF patients and environment are endowed
with a variable repertoire of genomic islands (Klockgether
et al., 2007; Wiehlmann et al., 2007). The genes required for
pathogenicity in one strain of P. aeruginosa are neither
required for nor predictive of virulence in other strains
(Lee et al., 2006; Bragonzi et al., 2009), indicating a broad
intraclonal diversity of pathogenicity of this organism, yet
no difference in virulence. Despite the high overall genome
similarity of P. aeruginosa of o 0.5% sequence diversity in
the core genome, comprising 80% of all genes, differences in
phenotypes can be striking even at the intraclonal level of
genetically highly related strains (Wehmhöner et al., 2003;
Salunkhe et al., 2005a, b).
Various morphotypes that are not normally present in
environmental P. aeruginosa strains have been described in
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD 129

Box 1. Virulence factors of Pseudomonas aeruginosa and their regulation by quorum sensing
Pseudomonas aeruginosa produces many virulence factors, which can be divided into cell-associated and extracellular virulence factors. Many
virulence factors are regulated by the quorum-sensing circuit and other regulatory genes, which have been demonstrated in vitro and in vivo. During
the chronic course of lung infections in patients with CF, many mutants have been characterized that are defective in virulence factor expression and
quorum sensing (D’Argenio, 2004; Hogardt et al., 2004, 2007; van Delden, 2004; Ramsey & Wozniak, 2005; Lee et al., 2006; Williams & Camara,
2009; Winstanley & Fothergill, 2009).
Cell-associated virulence factors
Flagella, type IV pili, lipopolysaccharide and three different exopolysaccharides, encoded by the alg, psl and pel clusters, contribute to adhesion of the
microorganism to various surfaces. On epithelial cells, flagellar proteins bind to toll-like receptor (TLR)-5 and lipopolysaccharide to TLR-4, leading to
the stimulation of innate immune functions and inflammation. The exopolysaccharides have been implicated in biofilm formation. Flagella,
lipopolysaccharide and alginate antibodies have been detected in the sera of infected CF patients.
Extracellular virulence factors
Type I, II and III secretion systems secrete protein toxins and provide contact with eukaryotic cells. Alkaline protease is secreted by the type I secretion
system, while the LasB and LasA elastases, protease IV, exotoxin A, phospholipase C and lipase are secreted by the type II secretion system. The type III
secretion system injects the exoenzymes S, T, Y and U into eukaryotic cells. Other extracellular virulence factors include the secondary metabolites
pyocyanin and other phenazine pigments, siderophores, hydrogen cyanide and rhamnolipids. Most exoenzymes and secondary metabolites have been
shown to be cytotoxic in in vitro and in animal models, and antibodies to many of these factors have been detected in the sera of infected CF patients.
Quorum-sensing circuit
Several virulence factors are regulated in a cell-density-dependent manner by the quorum sensing circuit composed of lasRI, rhlRI and pqs systems.
Pseudomonas aeruginosa produces several diffusible signal molecules with autoinducer properties such as N-3-oxo-dodecanoyl homoserine lactone
(produced by the LasI enzyme) and N-butanoyl homoserine lactone (produced by theRhlI enzyme), which bind at critical concentrations to the
corresponding regulators LasR and RhlR. The lasRI system mainly regulates lasB, lasA, exoA, xcpP and xcpR expression. The rhlRI system regulates the
expression of rpoS, rhamnolipid, LasB and LasA elastase, hydrogen cyanide, pyocyanin, lipase and alkaline protease. The Pseudomonas quinolone signal
system potentiates the effects of the rhlRI system. Quorum sensing controls not only virulence in P. aeruginosa but also many other genes involved in
cellular processes, including chemotaxis and biofilm formation, comprising altogether about 10% of the P. aeruginosa genome. In addition, quorum-
sensing molecules also modulate host responses.

P. aeruginosa strains, isolated from chronically infected CF
patients, including strains that are mucoid, smooth, rough,
dwarf, colourless or present as small colony variants (SCV)
(size: 1–3 mm). The latter may contribute to the progressive
pulmonary damage in CF and persistence in CF airways
(Häussler et al., 1999, 2003; Lory et al., 2009; Starkey et al.,
2009). Additionally, ‘rugose SCVs’ have been detected that
have characteristics of enhanced biofilm formation and
display increased expression of the pel and psl polysacchar-
ide gene clusters, decreased expression of motility functions
and a defect in growth on some amino acid and tricarboxylic
acid cycle intermediates as sole carbon sources (Starkey
et al., 2009). The psl (polysaccharide synthesis locus) genes
are responsible for the production of mannose-rich exopo-
lysaccharides and the pel locus for pellicle formation.
A consistent finding in CF airways is the presence of
mucoid P. aeruginosa strains that overproduce the exo-
polysaccharide alginate, a negatively charged polymer of
b-1-4-linked D-mannuronate, and its C5 epimer, L-gulur-
onate (Deretic et al., 1995; Boucher et al., 1997). Alginate
expression is upregulated by microaerobic environmental
conditions that are present in the viscous mucus of CF
patients (Worlitzsch et al., 2002). Alginate expression is
considered to be an important factor for the persistence of
P. aeruginosa in the airways of CF patients as alginate
protects the pathogen not only against innate immune
functions but also against opsonophagocytosis involving
specific antibodies, produced by the adaptive immune
system. A key element in alginate regulation is the alter-
native sigma factor AlgT, also known as AlgU, RpoE or s22,
which induces the expression of algD and increases the
expression of regulatory proteins that enhance algD tran-
scription (Ramsey & Wozniak, 2005). The anti-sigma factor
MucA inhibits AlgT; thus, inactivation of mucA leads to
upregulation of AlgT and conversion to mucoidy. Over-
production of alginate has been linked to biofilm formation
in P. aeruginosa – a phenotype that favours chronic persis-
tence of the pathogen in murine and CF lungs (Hoffmann
et al., 2005). However, both mucoid and nonmucoid
P. aeruginosa clonal strains occur in the airways of CF
patients simultaneously (Bragonzi et al., 2009). Further-
more, the finding that a high number of nonmucoid strains
from CF patients carry a mucA mutation (Bragonzi et al.,
2006) suggests that a mucoid morphotype, intermittently
selected in the CF lungs, can be suppressed at a later stage
due to other, secondary site mutations (Boles et al., 2004),
including mutations in algU (Ciofu et al., 2008). Interest-
ingly, P. aeruginosa rpoS mutants reveal a significantly
reduced production of alginate (Suh et al., 1999). Mucoid
P. aeruginosa strains from CF patients also contain increased
levels of G6PDH activity and expression of the correspond-
ing zwf gene. This allows the bacterium to supply sufficient

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c2010 Federation of European Microbiological Societies
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130
amounts of precursor for the alginate pathway (Silo-Suh
et al., 2005).
Other phenotypic changes of P. aeruginosa strains occur-
ring during the course of chronic CF lung infection include
loss of motility (Luzar et al., 1985; Rau et al., 2010),
decreased exotoxin A expression (Suh et al., 1999), loss of
O antigen in lipopolysaccharide (Hancock et al., 1983) and
altered lipid moieties (Ernst et al., 1999; Sabra et al., 2003).
The latter change confers enhanced resistance to aminogly-
coside antibiotics, cationic antimicrobials such as polymyx-
in E (colistin) and potentially to cationic peptides produced
by the host (defensins). Mutations in nfxB, the repressor of
the MexCD-OprJ operon, results in increased resistance to
fluoroquinolones (Rau et al., 2010). Furthermore, increased
auxotrophy (Taylor et al., 1993; Thomas et al., 2000), defects
in type II (Woods et al., 1986) and III secretion (Jain et al.,
2004; Lee et al., 2005; Hogardt et al., 2007; Rau et al., 2010),
reduced production of proteases and phospholipase C, loss
of pyoverdine, pyocins and elastase expression (Hogardt
et al., 2007), altered metabolic activities (Silo-Suh et al.,
2005) and antibiotic resistance (Thomassen et al., 1979;
Oliver et al., 2000; Smith et al., 2006; Hogardt et al., 2007)
have been described in P. aeruginosa strains from CF
patients. Particularly during acute exacerbations, methio-
nine-dependent auxotrophic mutants of P. aeruginosa have
been isolated from CF patients (Taylor et al., 1993). In
conclusion, chronic exposure to the CF environment selects
for a variety of mutations in P. aeruginosa strains.
The frequency of mutations is increased in hypermutable or
mutator strains that reveal defects in genes involved in the
DNA repair or error avoidance systems, particularly in the
methyl-directed mismatch repair system (Oliver et al., 2002;
Ciofu et al., 2005, 2010; Hogardt et al., 2007; Montanari et al.,
2007). The percentage of CF patients (60%) carrying at least
one hypermutable strain is remarkably high (Montanari et al.,
2007). Analysis of the genetic background of the P. aeruginosa
mutator phenotypes from CF airways showed that mutS was
the most commonly affected gene, followed by mutL (Ciofu
et al., 2010). A sequential analysis of CF isolates revealed that
mutations in mucA, leading to the mucoid phenotype and
lasR, leading to loss of quorum sensing (Box 1), occurred
earlier than mutations in the mut genes, showing that hyper-
mutability is not a prerequisite for the acquisition of mucoidy
and loss of quorum sensing (Ciofu et al., 2010). Accumulation
of mutators in the CF lung is associated especially with
multidrug resistance development, thus suggesting that the
intensive antibiotic treatment is one of the main selective
pressures for the maintenance and amplification of mutators
(Oliver et al., 2000; Ciofu et al., 2010). The acquisition of a
stable mutator phenotype may confer a selective advantage for
bacteria, particularly in stressful and/or fluctuating environ-
ments because this may increase the probability of generation
of adaptive variants (Taddei et al., 1997).

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
The genetic analysis of clonal isolates, obtained from the
CF lung of single patients over several years, revealed the
presence of mutations in many genes (Mahenthiralingam
et al., 1994; Anthony et al., 2002; Nguyen & Singh, 2006;
Smith et al., 2006; Tümmler, 2006; Hogardt et al., 2007;
Hoboth et al., 2009; Mandsberg et al., 2009; Ciofu et al.,
2010; Rau et al., 2010). When a P. aeruginosa isolate was
compared with its clonal variants, isolated 90 months later
from a single CF patient, revealing 68 mutations, the
analysis suggested a reduced virulence of the latter strains
with regard to their ability to induce acute infections, based
on mutations in many virulence genes including type III
secretion, quorum sensing and motility (Smith et al., 2006)
(Table 1). The progressive microevolution of P. aeruginosa in
CF patients, known as adaptive radiation, has been inter-
preted as an in vivo selection process, resulting in less
virulent variants that consequently do less harm to their
host than the original colonizing strain (Smith et al., 2006).
Although this notion may actually be correct, the defini-
tion of virulence also includes the capacity of the pathogen
to persist in a given host, causing chronic infection. To test
this hypothesis, Bragonzi et al. (2009) isolated P. aeruginosa
clones from airways of six CF patients during a period of up
to 16.3 years and assessed the virulence of clonal variants in
mice by monitoring acute and chronic lung infection.
Because the definition of virulence is also dependent on a
specific host, they tested the clonal variants in mice with
different genetic backgrounds, including CF mice. The data
demonstrate that early and intermediate P. aeruginosa CF
isolates are more virulent to cause acute infections (e.g.
bacteraemia) in mouse lungs than late isolates; however,
early, intermediate and late isolates do not differ in their
capacity to cause chronic infection (Fig. 3). These findings
suggest that clonal expansion of P. aeruginosa strains during
microevolution within CF lungs leads to populations with
altered, but not reduced virulence in mouse lung models.
Thus, it may be questionable to define late isolates as less
virulent than early isolates or to describe the pathogenicity
of CF lung disease as a process leading to an increasingly
benign situation. However, the animal experiments used to
determine virulence do not reflect a natural colonization
process with initially small numbers of bacteria.
Pseudomonas aeruginosa proteins are upregulated in the
CF lung, including the outer membrane protein OprF and
enzymes of the arginine deiminase pathway (Hoboth et al.,
2009) (Table 2). These changes indicate an adaptive shift
towards constitutive expression of genes required for growth
under the nutritional and microaerobic conditions present
in the sputum of CF patients. The high viscosity of the CF
mucus also leads to a limited diffusion of siderophore
molecules and therefore to an increased fitness of side-
rophore-producing cells (Cornelis et al., 2009; Kummerli
et al., 2009).
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD 131

Table 1. Gene mutations present in Pseudomonas aeruginosa cystic fibrosis isolates

Name Annotation Function References
mexZ w
PA2020 Transcriptional regulator of multidrug efflux 1
lasRw PA1430 Transcriptional regulator of quorum sensing 1
PA0313 Probable permease of the ABC transporter 1
mexAw PA0425 RND multidrug efflux membrane fusion protein 1
accCw PA4848 Biotin carboxylase 1
vfrw PA0652 Transcriptional regulator of lasR 1
mexSw PA2491 Probable oxidoreductase 1
exsAw PA1713 Transcriptional regulator of type III secretion 1
PA0506 Probable acyl-CoA dehydrogenase 1
wspF PA3703 Probable methylesterase involved in chemosensory signal 1
transduction
rpoN PA4462 RNA polymerase s-54 factor 1
fleQ PA1097 Transcriptional regulator of flagella synthesis 1
mexT PA2492 Transcriptional regulator of multidrug efflux 1
nalD PA3574 Probable transcriptional regulator 1
ampD PA4522 b-Lactamase expression regulator 1
PA0366 Probable aldehyde dehydrogenase 1
cyaB PA3217 Adenylate cyclase 1
pilB PA4526 Type 4 fimbrial biogenesis protein 1
PA1333 Hypothetical protein 1
PA3565 Probable transcriptional regulator 1
PA2312 Probable transcriptional regulator 1
anr PA1544 Transcriptional regulator of anaerobic metabolism 1
rhlR PA3477 Transcriptional regulator of quorum sensing 1
phoP PA1179 Two-component response regulator 1
rhlI PA3476 Autoinducer synthesis protein 1
PA4796 Hypothetical protein 1
pqsB PA0997 Homologous to b-keto-acyl-acyl carrier 1
protein synthase 1
toxR PA0707 Transcriptional regulator 1
PA2435 Probable cation-transporting P-type ATPase 1
PA4420 Hypothetical protein 1
PA2121 Hypothetical protein 1
mutS PA3620 DNA mismatch repair protein MutS 2–4
mutL PA4946 DNA mismatch repair protein MutL 3, 4
mutY PA5147 A/G specific adenine glycosylase 4, 5
uvrD PA5443 DNA helicase II 3, 4
mucA PA0763 Anti-sigma factor MucA 4, 6
nfxB PA4600 Transcriptional regulator 7
Numbers relate to the genome of PAO1. References 1, Smith et al. (2006); 2, Hogardt et al. (2007); 3, Montanari et al. (2007); 4, Ciofu et al. (2010); 5,
Mandsberg et al. (2009); 6, Anthony et al. (2002); 7, Rau et al. (2010).
w
Genes that are mutated in 4 5% of isolates.

Other bacterial pathogens in CF
Staphylococcus aureus also changes its phenotype under
microaerobic/anaerobic environmental conditions in vitro
and in the CF mucus, forming biofilm-like aggregates
(McKenney et al., 1999; Cramton et al., 2001; Ulrich et al.,
2007), which differ genotypically and phenotypically from
environmental strains and strains from the nasal habitat
(Goerke & Wolz, 2004). The exopolysaccharide present in
S. aureus biofilms (Cramton et al., 1999), initially named
polysaccharide intracellular adhesin and later poly-N-acetyl
glucosamine (PNAG), differs structurally from the S. aureus
capsule polysaccharides, which are lost during growth in the
CF airways due to elevated pCO2 (4%) (Herbert et al.,
1997). The PNAG-encased cells confer resistance to nonox-
idative killing (Ulrich et al., 2007). Phage mobilization,
possibly via transduction, contributes significantly to gen-
ome alteration in S. aureus during infection in CF patients
(Goerke et al., 2004), and agr mutants are frequently isolated
from these specimens (Goerke et al., 2000). Agr (for
accessory gene regulator) is a well-characterized global
regulatory locus that is expressed from two promoters: P2

FEMS Microbiol Rev 35 (2011) 124–146 

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
132
Clinical strains
100
80
60
% Mortality, survival, chronic
infection and clearance
40
20
0 et al., 2006; Biswas et al., 2009). Molecular characterization
100
80
60
40
20
0 biotic treatment. Without optimized anti-staphylococcal
Fig. 3. Virulence of Pseudomonas aeruginosa strains in a murine
model of airways infection. C57Bl/6NCrlBR mice were infected with
1–2  106 CFU per lung of 32 P. aeruginosa different strains embedded in
agar beads. Mortality induced by bacteraemia (red) and survival (grey)
were evaluated on challenged mice. Clearance (white) and capacity to
establish chronic airways infection (green) after 14 days from challenge
were determined on surviving mice. The data show the percentage of
mice infected with P. aeruginosa reference strains (PAO1, n = 66; PA14,
n = 14), a pool of environmental strains (n = 65) and a pool of clinical
strains (n = 274), isolated at the onset (n = 91), during the intermediate
phase of colonization (n = 65) or later during chronic infection (n = 118)
(adapted from Bragonzi et al., 2009).
and P3 (Novick et al., 1993, 1995). Agr regulates staphylo-
coccal virulence and other accessory gene functions, includ-
ing the expression of haemolysins. While the P2 RNAII
transcript codes for the quorum-sensing elements AgrA,
AgrC, AgrB and AgrD, P3 activation, during the late
exponential growth phase, leads to the transcription of
RNAIII, resulting in the repression of surface proteins such
as adhesins or protein A and in the enhanced expression of
exoproteins such as haemolysins (Novick, 2003). Agr mu-
tants show enhanced biofilm formation in S. aureus, as Agr
activation results in the expression of extracellular proteases
(Boles & Horswill, 2008) and membrane-active molecules
(Kong et al., 2006), such as the d-toxin, which contribute to
the dispersal of biofilms.
Often SCVs of S. aureus, which may persist in respiratory
epithelial cells, are detected in CF lung specimens (Sadowska
et al., 2002; von Eiff, 2008). SCVs are mostly nonpigmented
and nonhaemolytic and their colonies are 10 times smaller
than those of the parental strain. In general, SCVs are
auxotrophic for haemin and menadione, compounds in-
volved in the biosynthesis of electron transport chain
components. Most SCVs of S. aureus, isolated from CF
patients, are thymidine-auxotrophic due to long-term tri-
methoprim-sulphomethoxazol treatment (Schneider et al.,

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
2008). Treatment of CF lung infections using the amino-
glycoside tobramycin may result in local subinhibitory
concentrations that favour the emergence of aminoglyco-
side-resistant SCVs (Mitchell et al., 2010), involving the
global regulator SigB (Biswas et al., 2009). Besides antibio-
tics, coinfecting P. aeruginosa can also promote the forma-
tion of S. aureus SCVs by producing respiration inhibitors
such as 4-hydroxy-2-heptylquinoline-N-oxide (Hoffman
of SCVs from CF patients also revealed that they may be
defective in the expression of the agr system (Novick et al.,
1993). Taken together, the adaptation of S. aureus to the CF
lung leads to biofilm formation and SCVs, both of which
favour chronic infection states as a result of increased
resistance to innate immune defence mechanisms and anti-
treatment strategies that reduce the bacterial load in the
airways (Döring et al., 2004), chronic S. aureus lung infec-
tions may be considerably harmful for the CF patient.
Indeed, before the introduction of anti-staphylococcal
agents, virtually all CF patients were infected with this
opportunistic pathogen (Andersen, 1938), which was held
responsible for the widespread pulmonary changes in CF
(di Sant’Agnese & Talamo, 1967).
The Bcc is a group of genetically distinct, but phenotypi-
cally similar bacteria that are divided into at least nine
species (Coenye et al., 2001). All group members can cause
infections in CF patients, albeit with a low prevalence
(2–3%). Bcc strains are extremely sensitive towards ROS;
this is reflected by the minimal mortality of neutropenic
mice when infected with this opportunistic pathogen (Chu
et al., 2002). However, in sputum plugs in airways of CF
patients where ROS are essentially absent due to the
anaerobic/microaerobic environment and in patients with
the hereditary disorder chronic granulomatous disease
(CGD) who suffer from mutations in NADPH oxidase, this
opportunistic pathogen is highly virulent due to its intrinsic
resistance to nonoxidative killing during phagocytosis by
professional phagocytes (Speert et al., 1994; Pollock et al.,
1995; Baird et al., 1999; Sahly et al., 2003; Segal et al., 2003;
Sousa et al., 2007). Resistance to cationic antimicrobial
peptides (CAMPs) has been linked to the presence of 4-
amino-4-deoxyarabinose moieties, which are attached to the
phosphate residues in the lipid A backbone of Bcc lipopoly-
saccharides (Cox & Wilkinson, 1991; Vinion-Dubiel &
Goldberg, 2003).
While some strains may cause long-term asymptomatic
airway colonization, others cause a rapid decline of lung
function in CF patients and are highly transmissible, result-
ing in patient-to-patient spread within and between CF
centres (Govan et al., 1993; Mahenthiralingam et al., 2005).
For instance, Burkholderia cenocepacia strains of the multi-
locus enzyme electrophoresis type 12 lineage can cause a
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD 133

Table 2. Metabolic adaptation of Pseudomonas aeruginosa strains during chronic lung infection in cystic fibrosis patients based on protein expression
Name Annotation Function
rbsB PA1946 Binding protein RbsB of ABC ribose transporter
aotJ PA0888 Arginine-ornithine-binding protein
PA5153 Probable lysine-arginine-ornithine-binding periplasmic protein
PA1260 Probable lysine-arginine-ornithine-binding periplasmic protein
braC PA1074 Periplasmic branched-chain amino acid transport protein
spuD PA0300 Polyamine transport protein
oprFw PA1777 Outer membrane porin OprF
oprD PA0958 Outer membrane porin OprD
fda PA0555 Fuctose-1,6-bisphosphate aldolase
adk PA3686 Adenylate kinase
azuw PA4922 Azurin precursor
ccpRw PA4587 Cytochrome c551 peroxidase precursor
leuD PA3120 3-Isopropylmalate dehydratase small subunit
hisF1 PA5140 Imidazoleglycerol-phosphate synthase, cyclase subunit
arcAw PA5171 Arginine deiminase
arcCw PA5173 Carbamate kinase
PA2888 Probable biotin-dependent carboxylase
acpP PA2966 Acyl carrier protein
accB PA4847 Acetyl-CoA carboxylase
ppaw PA4031 Inorganic pyrophosphatase
trxA PA5240 Thioredoxin
sucC PA1588 Succinyl-CoA synthetase (b-chain)
ansB PA1337 Glutaminase-asparaginase
lpd3 PA4829 Dihydrolipoamid dehydrogenase 3
aceBw PA0482 Malate synthase G (glyoxylate shunt)
atpF PA5558 ATP synthase B chain
atpH PA5557 ATP synthase H chain
pdxH PA1049 Pyridoxamin 5-phosphate oxidase
glnK PA5288 Nitrogen-regulatory protein P-II 2
atoD PA1999 Probable CoA-transferase, subunit A
atoB PA2001 Acetyl-CoA acetyltransferase
PA0318 Hypothetical protein
PA2575 Hypothetical protein
PA0388 Hypothetical protein
PA3440 Hypothetical protein
PA1677 Hypothetical protein
PA5178 Hypothetical protein
PA3309 Hypothetical protein
PA5339 Hypothetical protein
btuEw PA0838 Glutathione peroxidase
sodB PA4366 Superoxide dismutase
kynB PA2081 Kynurenine formamidase
bfrA PA4235 Bacterioferritin A
katA PA4236 Katalase
ahpC PA0139 Alkyl hydroperoxide reductase subunit C
tsaA PA3529 Probable peroxidase
ibpA PA3126 Heat-shock protein IbpA
ppiB PA1793 Peptidyl-prolyl cis-trans isomerase B
groEL PA4385 GroEL
rpsF PA4935 30S ribosomal protein S6
PA4671 Probable ribosomal protein L25
greA PA4755 Transcription elongation factor GreA
rplL PA4271 50S ribosomal protein L7/L12
tsf PA3655 Elongation factor
tufA PA4265 Elongation factor Tu
guaB PA3770 Inosine-5 0 -monophosphate dehydrogenase
pilH PA0409 Twitching motility protein PilH

FEMS Microbiol Rev 35 (2011) 124–146 

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
134
Table 2. Continued.
Name Annotation
fliC PA1092
murF PA4416
ftsZ PA4407
Adapted from Hoboth et al. (2009).
w
Genes whose expression tends to be strongly upregulated during late chronic infection.
fulminate, necrotizing pneumonia, which may occasionally
be complicated by rapidly fatal septicaemia termed the
cepacia syndrome in CF patients (Corey & Farewell, 1996;
Jones et al., 2001).
As with other opportunistic pathogens, Bcc virulence in
CF and CGD patients is multifactorial (for a comprehensive
review of Bcc virulence factors, see Mahenthiralingam et al.,
2005). Besides lipopolysaccharides, which cause significantly
more inflammation than does P. aeruginosa lipooligosac-
charides (Mahenthiralingam et al., 2005), biofilm formation
has been implicated to play a role in the pathophysiology of
CF lung disease (Mahenthiralingam et al., 2005). Many Bcc
strains can produce biofilms in vitro (Ferreira et al., 2010),
associated with the ability to produce N-acyl-homoserine
lactones (Conway et al., 2002), which may protect single
cells from antibiotic killing (Desai et al., 1998). Also, the
ability of Bcc strains to form mixed biofilms with
P. aeruginosa has been demonstrated (Riedel et al., 2001).
In a CGD mouse model of Bcc lung infection (Sousa et al.,
2007), survival and death of the infected animals was
correlated to the growth rate of the different Bcc strains.
The highest growth rate was found for the epidemic strain
J2315, which multiplied from 103 CFU–108 CFU within 3
days, whereas for example a Burkholderia vietnamensis strain
never killed any mouse and reached only 104 CFU after 12
days of infection. Thus, genes active in metabolism may be
important for Bcc virulence in CF and CGD. Whether Bcc
strains adapt genotypically or phenotypically to the CF
niche is not known.
As in P. aeruginosa, hypermutable phenotypes have been
observed repeatedly in S. aureus (Prunier et al., 2003), H.
influenzae (Roman et al., 2004; Watson et al., 2004) and Bcc
strains from CF patients (Burns, 2005), suggesting that this
mechanism may play a crucial role in the pathogenesis of
chronic lung infection.
Bacteria colonization in COPD
In contrast to CF, the role of pathogenic bacteria in the
progression of COPD is an area of uncertainty and the
subject of active research. Opportunistic pathogenic bacteria
including NTHI, M. catarrhalis, S. pneumoniae and P.

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
Function
Flagellin type B
UDP-N-acetylmuramoylalanyl-D-glutamyl-2,
6-diaminopimelate-D-alanyl-D-alanyl ligase
Cell division protein FtsZ
aeruginosa are detectable in airways of approximately
25–50% of clinically stable COPD patients (Sethi & Murphy,
2008). Pathogenic bacteria in the lower airways induce
increased airway inflammation, a hallmark of COPD, even
in the absence of symptoms of acute exacerbation (Soler
et al., 1999; Hill et al., 2000), supporting the ‘vicious circle’
hypothesis that proposes that persistent bacterial infections
cause progressive lung damage in COPD. This notion is
further substantiated by the finding that the frequency of
positive cultures increases in patients with more severe
COPD (Eller et al., 1998). Thus, chronic bacterial and/or
viral infections may play a role in the development and
progression of COPD (Sethi & Murphy, 2008). However,
conclusive evidence for this hypothesis is lacking at present.
A key question on which research effort should be focused is
whether or not chronic infection of the airways in COPD
accelerates the progressive loss of lung function. Under-
standing the role of chronic infection in the course of the
disease would serve as an important guide in directing
research in the field. For example, if chronic infection were
shown to cause increased progression of the disease, then the
priority should be to develop methods such as vaccines or
immunomodulators to eradicate chronic colonization.
The causative role of pathogenic microorganisms in acute
exacerbations of COPD, however, is well established (Sethi
& Murphy, 2008). Opportunistic pathogenic bacteria are
responsible for approximately 50% of exacerbations, while
virus-induced exacerbations caused by rhinovirus, respira-
tory syncytial virus, adenovirus, influenza virus and human
metapneumovirus (Greenberg et al., 2000; Seemungal et al.,
2001) are approximately 30% (Sethi & Murphy, 2008).
Coinfections are frequent. Table 3 shows the relative dis-
tribution of bacterial pathogens that caused exacerbations in
a 10-year prospective study (Murphy & Parameswaran,
2009). Studies of COPD patients using bronchoscopy and
protected specimen brush sampling of distal airways, avoid-
ing upper airway contamination, have consistently shown
pathogenic bacteria in quantities indicative of infection
(Monso et al., 1995; Soler et al., 1999). Moreover, a
comparison of exacerbation and stable state patients, using
the same technique, showed significantly higher quantities
of these bacteria during exacerbation (Fagon et al., 1990;
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD
Table 3. Relative distribution of bacterial pathogens in COPD
Bacterial species Positive culture Exacerbation w
Haemophilus influenzae 707 84
Moraxella catarrhalis 310 68
Streptococcus pneumoniae 219 11
Pseudomonas aeruginosa 230 15
Number of positive cultures for the pathogen, isolated from the sputum
of adults with COPD, followed in a 10-year prospective study in Buffalo,
NY.
w
Number of exacerbations in the same 10-year prospective study as
defined by the acquisition of a new bacterial strain (based on molecular
typing of cultured isolates) simultaneous with symptoms of exacerbation
(adapted from Murphy & Parameswaran, 2009).
Soler et al., 1998). Longitudinal molecular typing of patho-
genic bacteria from sputum cultures showed that acquisition
of a new strain was associated with a twofold increase in the
incidence of exacerbations (Sethi et al., 2002). Purulent
sputum, a marker of exacerbation, is strongly associated
with the presence of pathogenic bacteria in sputum (White
et al., 2003; Soler et al., 2007).
NTHI and M. catarrhalis are prevalent pathogens
Haemophilus influenzae strains may produce antigenically
distinct polysaccharide capsules, which distinguishes strain
types ‘a’ through ‘f’. In contrast, strains of H. influenzae that
colonize and infect the airways in COPD are nonencapsu-
lated as demonstrated by a lack of reactivity with typing sera
for each of the six known capsular serotypes. Thus, these
strains are referred to as nontypeable, abbreviated NTHI.
Haemophilus influenzae strains use several genetic mechan-
isms that alter either the gene expression or the gene content
that allow rapid adaptation and improved fitness to chan-
ging environments including phase variation based on
slipped-strand mispairing, mediated by short DNA repeats
in either the coding regions or the upstream promoter
regions of virulence genes (Gilsdorf et al., 2004; Srikhanta
et al., 2010). Phase variation results in a large genetic
diversity among H. influenzae strains present in different
environments, which complicates the interpretation of viru-
lence-related phenotypes. The multifactorial virulence of H.
influenzae strains includes factors such as lipooligosacchar-
ides (Schweda et al., 2007), fimbriae encoded by the hif
locus, IgA1 protease(s) (Erwin & Smith, 2007) and the
ability to form biofilms (Starner et al., 2006).
NTHI strains show a highly variable pattern of carriage
among COPD patients. Some strains of NTHI, causing
exacerbations, can also persist for longer periods of time
than is apparent from sputum cultures. Molecular analysis
shows that a strain is present in COPD airways during
periods when it cannot be recovered by routine culture,
FEMS Microbiol Rev 35 (2011) 124–146
135
indicating that selected strains persist in the airways for
months to years. A 7-year prospective study (Murphy et al.,
2004) including 104 COPD patients observed 17 episodes of
prolonged ( 4 6 months) periods of negative sputum cul-
tures preceded and followed by genotypically identical
strains of NTHI. Clearing and reacquisition of the identical
strain by the patients was ruled out, because NTHI strains
are genetically highly heterogeneous, making the reacquisi-
tion of the same strain circulating in the community a highly
unlikely event. Furthermore, H. influenzae does not remain
viable on inanimate objects in the environment even for
minutes. Finally, analysis of sputum samples yielding nega-
tive cultures revealed the presence of strain-specific NTHI
genes, further establishing persistent colonization in COPD
patients. Newly acquired NTHI strains isolated from COPD
patients during exacerbations, when compared with persist-
ing strains, induce more airway inflammation, including
neutrophil influx and IL-8 secretion from respiratory
epithelial cells, and adhere more efficiently to the epithelial
cells in vitro (Chin et al., 2005). These data suggest that, as
with P. aeruginosa in chronic CF lung infection, adaptive
processes may occur that modulate the virulence of this
pathogen. However, similar molecular studies as mentioned
above for P. aeruginosa (Smith et al., 2006; Bragonzi et al.,
2009) have not yet been carried out for sequential NTHI
isolates from single COPD patients to substantiate this
notion. Analysis of the genome content of strains of NTHI
that persist in the airways of COPD patients in comparison
with those that are cleared quickly will help elucidate the
relative roles of the host and pathogen in the dynamics of
infection in COPD airways.
Also, M. catarrhalis is regarded as an important human
mucosal pathogen in COPD patients (de Vries et al., 2009;
Murphy & Parameswaran, 2009). Virulence factors include a
family of ubiquitous surface proteins, including the ubiqui-
tous surface protein A1 (UspA1) and UspA2, which can bind
a1-antichymotrypsin, thereby potentially increasing the
protease activity in vivo, which may cause excessive inflam-
mation in COPD patients (Perez Vidakovics & Riesbeck,
2009). In contrast, UspA1 has been shown to bind to the
human-specific extracellular immunoglobulin V (IgV)-like
amino-terminal domain of carcinoembryonic antigen-re-
lated cell adhesion molecule 1, resulting in reduced toll-like
receptor 2-initiated transcription factor nuclear factor-kB-
dependent inflammation (Slevogt et al., 2008). Which of
these mechanisms dominate during M. catarrhalis infection
in COPD patients is unclear.
COPD patients who acquire new strains of M. catarrhalis
usually clear this species efficiently from their airways in
approximately 1 month. In a study including 104 COPD
patients who experienced 560 exacerbations, molecular
typing identified 120 episodes of acquisition and clearance
of M. catarrhalis in 50 patients (Murphy et al., 2005).

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136
Reacquisition of the same strain was rare. The rapid estab-
lishment of protective antibody responses against the patho-
gen in these patients suggested that the clearance of
M. catarrhalis is a function of a functional immune system
(Murphy et al., 2005). The findings that 57 (47.5%) of
M. catarrhalis acquisitions were associated with clinical
exacerbations (Murphy et al., 2005), and that increased
inflammation and tilting of the protease–antiprotease bal-
ance in favour of protease activity occurred in airways of
COPD patients during exacerbation as well as colonization
with M. catarrhalis (Parameswaran et al., 2009), suggest that
this pathogen contributes to the long-term decrease of lung
function in COPD patients. Calculations revealed that M.
catarrhalis likely causes 10% of exacerbations of COPD.
Pseudomonas aeruginosa does not commonly
show persistence in COPD
In a chronic lung disease such as COPD, which is character-
ized by chronic inflammation, progressive tissue destruction
and increased mucus production, one would expect persist-
ing P. aeruginosa clones undergoing a similar microevolu-
tion as described above for the P. aeruginosa strains in CF
airways. Indeed, single clones of P. aeruginosa have been
identified in COPD patients that persisted over several years
and developed an increasing number of mutations during
the chronic course of the disease, resulting in reduced
motility, decreased protease production and increased anti-
biotic resistance as also recognized in patients with CF
(Martinez-Solano et al., 2008). Furthermore, different mor-
photypes of P. aeruginosa, including SCVs and mucoid
phenotypes, were observed in sputum isolates from a single
COPD patient, suggesting that in this disease diversification
also plays an important role in bacterial persistence. Addi-
tionally, strains that persisted over long periods of time
tended to be hypermutable as in CF (Martinez-Solano et al.,
2008).
However, the similarity to CF reduces when larger patient
groups are investigated longitudinally. In a 10-year study
involving 126 COPD patients, only 13 strains persisted for
4 6 months and only four of these were mucoid (Murphy
et al., 2008). These data suggest that most COPD patients
had sporadic and intermittent infection of airways with P.
aeruginosa, which were repeatedly cleared. Strains from one
clone were generally cleared by the patients’ immune system
and a gap in time existed before the acquisition of a strain
from a different clone (Rakhimova et al., 2009). Chronic
carriage of a clone was observed in a minority of investigated
patients in this study. When a clone chronically persisted in
COPD-affected lungs for an extended period of time, the
clone often altered the repertoire of its accessory genome.
Hence, intraclonal microevolution and the frequent turn-
over or loss of clones are typical of infections with P.

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
aeruginosa in COPD. This epidemiological signature is
divergent from that of the chronic P. aeruginosa infections
in patients with CF, the latter being characterized by no or
slow turnover of clones (Rakhimova et al., 2009).
Circumstantial evidence suggests that chronic infection of
COPD airways by P. aeruginosa occurs in those with more
severe COPD, particularly among patients who require
mechanical ventilation for severe exacerbations (Miravitlles
et al., 1999; Ferrer et al., 2005; Rosell et al., 2005; Nseir et al.,
2006), or in other words, patients with more severe reduc-
tion in FEV1, when compared with those with milder
disease, are more likely to harbour P. aeruginosa in their
lower airways (Eller et al., 1998). Patients with more severe
COPD also experience a higher number of acute exacerba-
tions and there is increasing recognition that P aeruginosa is
responsible for such exacerbations (Murphy et al., 2008).
Whether chronic P aeruginosa infection has a negative
impact on lung function and the patients’ prognosis requires
further study.
The role of P. aeruginosa in the clinical evolution of
COPD is not well understood. Unlike in CF, no clear
evidence of worsening of lung infection and increased
mortality is correlated to chronic P. aeruginosa infection in
COPD. Furthermore, unlike in CF, eradication of colonizing
pathogenic bacteria, in the absence of exacerbation symp-
toms, has not been shown to be beneficial in the clinical
course of COPD. The questions as to why only some patients
develop these chronic infections and why only selected
bacterial pathogens establish these infections in COPD
remain unanswered at present. Multiple host factors includ-
ing the severity of COPD, genetic polymorphisms as well as
bacterial virulence factors in specific clones may play a role
in this context. However, a comparison of P. aeruginosa
clones in the airways of patients with COPD with those in
the airways of chronically infected patients with CF revealed
that despite the disparate geographic origin of the isolates,
the dominant P. aeruginosa clones were found with similar
frequencies in acutely and chronically infected respiratory
tracts irrespective of the aetiology of the disease (Rakhimova
et al., 2009) (Fig. 4).
Antimicrobial treatment for CF patients
For chronic P. aeruginosa lung infection in CF patients,
antibiotic therapy has been a cornerstone, allowing a largely
improved life expectancy of the affected patients due to
stabilization of lung function; by contrast, lung function
rapidly decreases in untreated CF patients. Commonly
applied antibiotics include the aminoglycoside tobramycin,
the b-lactam antibiotic ceftazidime, the fluoroquinolone
ciprofloxacine and the polymyxin E colistin (Döring et al.,
2000, 2004). However, this therapy regimen, even when
applied as recommended (Döring et al., 2000, 2004), i.e.,
FEMS Microbiol Rev 35 (2011) 124–146
Bacterial infections in CF and COPD
Fig. 4. Distribution of Pseudomonas aeruginosa strains isolated from
patients with CF and COPD. (a) Venn diagram of the number of P.
aeruginosa clonal complexes obtained from the airways of patients with
COPD and CF, respectively. Clonal complexes were calculated from the
15-marker genotype of the core genome using the program EBURST
(Maiden, 2006). The intersections represent the clonal complexes that
had been identified in both diseases. (b) Clonal complex structure of P.
aeruginosa strains collected from various different habitats and geo-
graphic origins. The EBURST analysis was carried out with the informative
SNPs of a microassay of the core genome. Coloured clonal complexes
contain airway isolates from patients with COPD (yellow), from patients
with CF (blue) and from both patient groups (green). The size of the
symbol indicates the number of typed strains with an identical genotype.
Clones C and PA14 are the most abundant clones (Wiehlmann et al.,
2007). Genotypes only observed in other habitats are depicted in grey.
(Figure adapted from Rakhimova et al., 2009.)
aggressively every 3 months with high doses of inhaled
antibiotics, does not eradicate the chronically persisting P.
aeruginosa from the airways of CF patients. The highly
viscous mucus plugs and the biofilm-like macrocolonies of
P. aeruginosa within these plugs, which impede the penetra-
tion of antibiotics, are assumed to be responsible for this
negative outcome (Xu et al., 1998; Wimpenny et al., 2000;
Worlitzsch et al., 2002; Döring et al., 2004). As mentioned
above, the stressful environment in the CF lung also favours
the emergence of hypermutable (or mutator) strains of P.
aeruginosa, which are deficient in the DNA mismatch repair
system. Mutator strains produce antibiotic-resistant mu-
tants at elevated rates (Oliver et al., 2000; Hogardt et al.,
2007). Combination therapy using antibiotics having differ-
ent modes of action against P. aeruginosa could be superior
to monotherapy in this situation. Recently, the combination
of the fluoroquinolone ciprofloxacin and the amphipathic
oligopeptide polymyxin E (colistin) has yielded promising
results in killing P. aeruginosa biofilms in vitro, based on the
concept of combining antibiotics that have different targets
in the biofilm cells. While biofilm cells exhibiting low
metabolic activity were killed by colistin, ciprofloxacin was
found to specifically kill the subpopulation of metabolically
active biofilm cells (Pamp et al., 2008). Whether this strategy
is successful in CF patients still needs to be tested.
To avoid the development of resistance and in an attempt
to eradicate P. aeruginosa early, CF patients were treated
FEMS Microbiol Rev 35 (2011) 124–146
137
with antibiotics immediately after the first detection of the
pathogen. In this case, reported eradication rates were above
95% (reviewed in Döring et al., 2000, 2004).
In addition to antibiotic therapy, other treatment strate-
gies may also be successfully used in CF patients to fight
bacterial lung infection. Based on the abnormal accumula-
tion of ceramide in CF airways, leading to DNA deposits on
the respiratory epithelium, the sphingomyelinase blocker
amitriptyline is currently under investigation in clinical
trials (Riethmüller et al., 2009). Furthermore, recombinant
human DNAse may be helpful in this context, as evidenced
in experimental mouse models of P. aeruginosa lung infec-
tion (Teichgräber et al., 2008) and in CF patients with regard
to S. aureus (Frederiksen et al., 2006).
An important question is whether antibiotic therapy
directed against P. aeruginosa is also effective against the
large number of strict anaerobes present in the mucus plugs
within the airways of CF patients. Two studies suggest that
this is not the case (Tunney et al., 2008; Worlitzsch et al.,
2009). If strict anaerobes actually did contribute to lung
disease in CF patients, additional antibiotics, specifically
active against these pathogens, should be administered to CF
patients.
In the case of Bcc infections, there is no doubt that these
microorganisms cause lung disease in CF patients. Because
of the intrinsic resistance of Bcc strains to many antibiotics,
combination therapy with two to three different antibiotics
according to minimal inhibition concentration assessment is
recommended (Döring et al., 2004).
Antimicrobial treatment for COPD
patients
Antibiotic therapy regimens in COPD have focused on the
treatment of exacerbations. However, only approximately
50% of acute exacerbations in COPD patients are caused by
bacterial pathogens, a situation that complicates the inter-
pretation of results in such studies. Nevertheless, the data
available strongly suggest that antibiotic therapy for acute
exacerbations in COPD patients reduces complications and
hastens recovery. In a placebo-controlled study, including
173 COPD patients who suffered from 362 acute exacerba-
tions during the study period, the success rate was 68.1% in
the antibiotic group vs. 55% in the placebo group (Antho-
nisen et al., 1987). Most of the success was seen in patients
with more severe exacerbations, indicating that patients
with more severe exacerbations benefit more from antibiotic
therapy. A systematic review analysed 11 placebo-controlled
trials (917 patients) of antibiotics in exacerbations of COPD.
Combining the data from three trials that used objective
definitions of COPD to qualify for inclusion showed a
reduction in mortality with antibiotic use and reduction in
the rates of treatment failure (Ram et al., 2006). Also, other

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
138
meta-analyses of placebo-controlled trials revealed a sub-
stantial benefit for the antibiotic treatment of acute exacer-
bations in COPD patients, emphasizing the pathogenic role
of bacteria in the course of this disease (Quon et al., 2008;
Roede et al., 2009).
Another goal of antimicrobial therapy is to slow or to
prevent the worsening of pulmonary function. The use of
antibiotics in stable COPD patients to prevent progressive
worsening of lung function in the absence of exacerbations
has yielded unclear results due to the lack of large studies
with stratified COPD patients. Nevertheless, a recent meta-
analysis of data from trials conducted in 1950s and 1960s
showed that ‘prophylactic antibiotics’ produced a relative
risk reduction of 9% for the occurrence of exacerbations
(Black et al., 2003). However, even this modest gain should
be interpreted with caution because the trials varied mark-
edly in methodology and duration and many of the anti-
biotics used in these trials would not be expected to be
effective with the current susceptibility patterns of bacteria.
In a randomized trial, prophylactic erythromycin reduced
the frequency of exacerbations in a group of COPD patients
and increased the mean interval between exacerbations
(Seemungal et al., 2008). Whether the benefits are due to
antimicrobial, anti-inflammatory or both effects of erythro-
mycin is not clear. In a recent pilot study involving 13 COPD
patients, colonized with multi-drug-resistant P. aeruginosa,
inhalation of tobramycin (300 mg twice daily for 2 weeks)
reduced the sputum density of P. aeruginosa and the exacer-
bation rate (Dal Negro et al., 2008). This small uncontrolled
study is promising, but needs to be replicated in larger
controlled trials that examine the impact of such a treatment
on lung function and clinical outcomes.
The success of early treatment with antibiotics in redu-
cing the incidence of airway infection in CF raises the
question of whether a similar approach might be effective
in adults with COPD, particularly with regard to P. aerugi-
nosa infection. The majority of adults with COPD who
acquire P. aeruginosa clear the organism without antibiotic
therapy (Murphy et al., 2008). However, patients with more
advanced COPD, persistent infection with P. aeruginosa and
severe exacerbations are more likely to benefit from anti-
biotic therapy. Therefore, COPD patients hospitalized with
exacerbations, particularly with severe exacerbations reveal-
ing purulent sputum masses, and patients with severe
underlying COPD having exacerbations should receive anti-
biotics, according to a decision-making algorithm (Sethi &
Murphy, 2008).
It is not known whether the subset of COPD patients who
have persistently positive sputum cultures for P. aeruginosa
experience adverse consequences of chronic infection, ana-
logous to patients with CF. This is an important question
that needs to be addressed in carefully designed clinical
trials. The results will have important implications in under-

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
G. Döring et al.
standing the pathogenesis of P. aeruginosa infection in
COPD and in the clinical management of these patients.
Concluding remarks
The pathophysiology of CF and COPD lung disease is
complex. While chronic pulmonary inflammation triggered
by opportunistic pathogens is the cause of the more or less
rapidly decreasing lung function in CF, the role of bacterial
infection in the pathogenesis of COPD is less well under-
stood. Chronic P. aeruginosa infection plays a critical role in
the course of CF, whereas only a small minority of adults
with COPD experience chronic P. aeruginosa infection. In
COPD, the predominant pathogens are H. influenzae and
M. catarrhalis. While in CF airways single P. aeruginosa
clones, which adapt genotypically and phenotypically to the
CF niche, persist in the majority of patients due to a heavily
impaired innate immune system, a frequent turnover and
loss of clones are typical of infections with P. aeruginosa in
COPD. However, once the extensive tissue remodelling and
tissue destruction has reached a certain threshold (which has
not yet been determined), opportunistic pathogens such as
P. aeruginosa may persist and develop epidemiological
signatures similar to those in CF. To prevent this point of
no return is a challenge for microbiologists and clinicians.
Acknowledgements
The authors want to thank Peter Michael Weber, Children’s
Clinic, University of Tübingen, Germany, for excellent
graphic art.
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