Download Microbiology of non- CF bronchiectasis

Chapter 6
Microbiology of nonCF bronchiectasis
J.E. Foweraker* and D. Wat#
MICROBIOLOGY
Summary
Non-cystic fibrosis (CF) bronchiectasis is a complex disorder
characterised by recurrent chest infections and poorly regulated
respiratory innate and adaptive immunity. These lead to a
‘‘vicious cycle’’ of impaired mucociliary clearance, chronic
infection, bronchial inflammation and progressive lung injury.
The most prevalent pathogenic bacteria are Haemophilus
influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae,
Staphylococcus aureus and Moraxella catarrhalis although
variations in sampling techniques and detection methods have
influenced their isolation rates. These organisms can inhibit
mucociliary clearance, destroy respiratory epithelium and
produce biofilms that promote persistent infection by blocking
innate immune defences and increasing antibiotic resistance.
While numerous studies have examined the role of different
bacteria in CF and chronic obstructive pulmonary disease, little
is known about how they contribute to the pathogenesis of nonCF bronchiectasis. There is also a paucity of data regarding the
role of respiratory viruses in this condition. This chapter
describes the microbiology of non-CF bronchiectasis, defines
the bacterial mechanisms that may contribute to persistent
infection and airway damage and discusses the potential role for
respiratory viruses in this condition. Understanding the
pathogenic properties of these microorganisms may allow the
development of novel therapies for the management of
respiratory exacerbations.
Keywords: Anaerobes, Haemophilus, Moraxella, Pseudomonas,
Streptococcus, viruses
P
*Dept of Microbiology, and
#
Lung Defence Unit, Papworth
Hospital, Cambridge, UK.
Correspondence: J.E. Foweraker,
Dept of Microbiology, Papworth
Hospital, Papworth Everard,
Cambridge, CB23 3RE, UK, Email
[email protected]
Eur Respir Mon 2011. 52, 68–96.
Printed in UK – all rights reserved.
Copyright ERS 2011.
European Respiratory Monograph;
ISSN: 1025-448x.
DOI: 10.1183/1025448x.10003610
68
atients with bronchiectasis are commonly colonised with potentially pathogenic microorganisms in the airways [1]. These microorganisms can cause lung infections and may produce a
number of inflammatory mediators that can lead to progressive tissue damage and bronchial
obstruction. The phenomenon of chronic infection, bronchial inflammation and progressive lung
injury is a ‘‘vicious cycle’’ and is also the reason why prompt evaluation of infection is important [2].
Being able to identify the causative bacterium may allow appropriate antibiotic administration to
break this vicious cycle.
The most prevalent microorganisms found in non-cystic fibrosis (CF) bronchiectasis are discussed
in this chapter and we have included the role of viruses, as well as some recent studies that have
investigated microorganisms that are not usually considered to be pathogens in the respiratory
tract. Surprisingly, there is little published data on the epidemiology and pathogenesis of infections
in non-CF bronchiectasis. However, there are similarities with infections in CF bronchiectasis and
chronic obstructive pulmonary disease (COPD). Where the literature for non-CF bronchiectasis is
sparse, studies in CF and COPD have been drawn upon as these may aid the understanding of the
microbiology; in particular the adaptations that take place to enable microorganisms to establish
and maintain chronic infection and the role taken in the development of exacerbations.
Fungal infections, including allergic bronchopulmonary aspergillosis (ABPA), are discussed further
by HILVERLING et al. [3], while nontuberculous mycobacteria infections are discussed further by
DALEY [4] in this Monograph.
Several studies have reviewed the bacteria found in patients with non-CF bronchiectasis (table 1).
A similar range of organisms is found in most studies, but the prevalence of each varies. Age,
ethnicity, the underlying causes of bronchiectasis and the proportion of patients that were stable
or had cultures taken during an exacerbation varies between the different studies and would be
expected to affect the microbial flora found. The pattern of antibiotic usage, including long-term
prophylaxis, may vary between different centres and could also have an affect on the type of
microorganisms cultured. The type of respiratory specimen tested may also determine the rate of
positive cultures found. The use of a protected specimen brush to take samples at bronchoscopy
yielded the highest positivity rate when compared with sputum specimens in one study [7]. Finally
the methodology used for analysis (quantification, culture and identification techniques) will vary
between centres and could also affect the result.
Haemophilus influenzae and Pseudomonas aeruginosa were the most common bacteria found in the
majority of the studies and the most likely to cause long-term colonisation [12]. No potentially
pathogenic microorganisms were cultured from 18–24% of the patients investigated and an
absence of a potentially pathogenic microorganism was associated with the milder disease [9, 13].
Haemophilus influenzae
J.E. FOWERAKER AND D. WAT
Range of bacteria in patients with non-CF bronchiectasis
H. influenzae has been reported in 14–52% of patients with non-CF bronchiectasis. It is a Gramnegative coccobacillus with specific growth requirements, which can be difficult to isolate in the
laboratory if mixed with other flora. Some H. influenzae possess a polysaccharide capsule and can
be typed using type-specific anticapsule antisera. Those with the type B capsule (Hib) can cause
invasive infection with bacteraemia, and are most familiar as a cause of meningitis or epiglottitis.
The use of Hib vaccine has greatly reduced the incidence of these life-threatening conditions.
H. influenzae with capsule types other than type B are relatively rare and are far less pathogenic.
The nonencapsulated strains, referred to as nontypeable H. influenzae (NTHi), are also less
pathogenic than Hib and only rarely cause bacteraemia. They live as commensals in the human
upper respiratory tract but can cause otitis media, sinusitis and conjunctivitis, often following a
primary viral infection. NTHi are a common cause of lower respiratory infection in patients with
underlying respiratory abnormalities including non-CF bronchiectasis [9]. The Hib vaccine does
not prevent infection with NTHi as it only contains the H. influenzae type B capsule antigen.
69
NTHi could be an oral contaminant in expectorated sputum; however, studies using a protected
specimen brush (PSB) at bronchoscopy found NTHi in significant numbers in non-CF
bronchiectasis, confirming its presence in the lower respiratory tract [7]. In contrast
15 (10)
9 (6)
47 (33)
47 (33)
Colonised
subgroup"
13 (9)
21 (14)
39 (27)
30 (20)
39 (27)
20 (13)
42 (30)
46 (31)
62 (43)
52 (35)
75 (52)
ND
ND
Sputum
Sputum
PASTEUR [10]
MACFARLANE [11]
UK
UK
150
143
60.6
(16–90)
3 (4)
7 (8)
6 (7)
11 (12)
42 (47)
Stable
Sputum
89
KING [9]
Australia
57¡14
9 (7)
3 (2)
13 (11)
38 (31)
37 (30)
ND
Sputum
123
USA
NICOTRA [8]
57.2¡16.7
2 (3)
3 (4)
6 (8)
12 (16)
24 (32)
Stable
58 (16–76)
PSB
75
Spain
ANGRILL [7]
Data are presented as mean (range), mean¡SD or n (%), unless otherwise stated. RTFlora: upper respiratory tract flora; PPM: potentially pathogenic microorganisms; ND: not described; K. pneumoniae: Klebsiella
pneumoniae; GNB: Gram-negative bacilli; GPB: Gram-positive bacilli; PSB: protected specimen brush; A. xylosoxidans: Achromobacter xylosoxidans: E. coli: Escherichia coli; S. maltophilia: Stenotrophomonas
maltophilia; #: some had more than one PPM cultured; ": bacteria were isolated on at least two occasions, 3 months apart, in 1 year.
7 (14) K. pneumonia
7 (14) GNB
2 (4) GPB
18 (24) RTFlora 1 (1) A. xylosoxidans
1 (1) E. coli
ND
16 (13) GNB
2 (3) Anaerobes
19 (21) no PPM
2 (2) E. coli
1 (1) A. xylosoxidans
ND
28 (20)
12 (8) S. maltophilia
4 (3) A. xylosoxidans
42 (30) Coliforms
2 (1) S. maltophilia
2 (1) A. xylosoxidans
13 (9) Coliforms
Sputum
Sputum
92
50
Ireland
Thailand
ZAID [5]
PALWATWICHAI [6]
,18
58 (30–85)
ND
ND
50 (46)
7 (14)
8 (9)
10 (20)
34 (37)
3 (6)
9 (10)
2 (4)
14 (15)
ND
9 (18) RTFlora
Other
RTFlora/
no PPM
Haemophilus Pseudomonas Streptococcus Moraxella Staphylococcus
influenzae
aeruginosa
pneumoniae catarrhalis
aureus
Patients with organisms cultured from respiratory tract#
Stable or at
exacerbation
Age yrs
Sample
Subjects
n
Country
First author
[ref.]
Table 1. Range of microorganisms cultured from patients with non-cystic fibrosis bronchiectasis
MICROBIOLOGY
70
Haemophilus parainfluenzae, a common commensal organism found in
the upper respiratory tract, may be
cultured from sputum but was not
found in PSB samples. In patients
with COPD the presence of NTHi in
sputum was associated with raised
inflammatory cytokines, whereas patients with H. parainfluenzae in sputum had similar levels of cytokines to
those who had no microorganisms
cultured from their sputum, suggesting that even if present in the lower
tract it does not have a direct pathogenic role [14, 15].
There is little published data on the
epidemiology of H. influenzae in
non-CF bronchiectasis. It may be
cultured repeatedly from the same
patient over several years, but without typing data it is not known if
this is the persistence of a single
strain or repeated episodes of infection [9]. In COPD patients, NTHi
were found in higher numbers (.106
colony forming units (CFU)?mL-1)
during exacerbation compared with
when the patient was stable, and
exacerbations in COPD may be associated with the appearance of a new
strain [16, 17]. A prospective study in
COPD using molecular typing of
H. influenzae and direct analysis of
amplified DNA from sputum showed
persistence of the same strain over
prolonged periods [18]. This suggests
either long-term colonising infection
in the lung or persistence in the
upper respiratory tract with repeated
deposition followed by clearance from
the lung. In CF, sequential infection
with different strains of H. influenzae
was found in some patients and persistence of the same clone in other
individuals [19].
NTHi have various properties that
can help explain their pathogenicity
and ability to persist in the lung. They
can adhere to mucus and to various
cell types in the human respiratory
tract using pili and other adhesion
molecules. Virulence factors include
the endotoxin lipo-oligosaccharides
(LOS), and immunoglobulin (Ig)-A protease [20]. NTHi possess mechanisms to vary the structure
and activity of LOS and these may explain variations in the pathogenicity of different isolates [21].
Studies comparing the proteome of H. influenzae grown in vitro, either in pooled sputum or
chemically defined media, have shown that the organism is able to adapt to oxidative stress and
limited nutrients [22]. This is thought to be because H. influenzae has the ability to generate a diverse
population that allows rapid adaptation to changes in the environment by expansion of the clonal
members that express the phenotypic characteristics needed to survive. Mechanisms used by H.
influenzae to generate phenotypic diversity include altered gene expression, e.g. by phase variation,
and altered gene content by mutation or by horizontal gene transfer, e.g. direct DNA uptaketransformation, or via bacteriophage [23].
H. influenzae (along with other bacteria infecting a bronchiectatic lung) may exist in biofilms in the
respiratory tract. These are co-operative populations of bacteria surrounded by an amorphous matrix
and could help the organism to survive in a hostile environment by resisting both host defences and
antibiotics. The antibiotic resistance observed for bacteria growing in biofilms is in part attributable to
its electrolyte content but also by reduced bacterial growth or even dormancy within the biofilm
matrix. NTHi from patients with COPD can form biofilms in vitro, and NTHi biofilms were seen in
the chinchilla model of otitis media [28, 29]. NTHi cultured from CF patients could form biofilms in
vitro and on the surface of cultured airway epithelial cells. Structures consistent with biofilms
containing H. influenzae were also found in bronchoalveolar lavage (BAL) samples from children with
CF [30]. NTHi in biofilms were more resistant to antibiotics in vitro. Sub-inhibitory concentrations of
azithromycin were found to reduce the size of both growing and established biofilms [31].
The prevalence of antibiotic resistant NTHi increases over time in patients with non-CF
bronchiectasis [9]. Many are resistant to amidopenicillins (e.g. amoxicillin, ampicillin) either due
to production of b-lactamase or alteration of penicillin binding proteins. Quinolone resistance is
now recognised and resistance rates to trimethoprim and tetracycline are rising.
J.E. FOWERAKER AND D. WAT
NTHi may be able to evade the immune response by varying its surface antigens. Mechanisms
include phase variation of LOS [24], and changes to outer membrane proteins (OMP) either by
horizontal gene transfer or point mutations of the immuno-dominant OMP, i.e. P2. Antigenic
drift, resulting from change in the P2 gene, has been observed in persistent infections in patients
with COPD [25]. NTHi could be protected within host cells as they have been found inside
macrophages in the chinchilla otitis media model and in macrophage-like cells in human
adenoids. NTHi were able to enter cultured nonciliated respiratory epithelial cells and cross the
respiratory epithelium [26]. Using in situ hybridisation, NTHi were identified inside cells in
bronchial biopsies taken from patients with COPD [27].
Antibiotic resistance may occur by horizontal transfer of genetic material from other organisms in the
complex polymicrobial environment of the mouth and upper respiratory tract. It may also take place
in the lower respiratory tract, which may be polymicrobial in non-CF bronchiectasis. Alternatively,
resistance may result from gene mutation. Some NTHi have a higher than usual mutation rate due to
a mutation in mutS, which is one of the methyl-directed mismatch repair genes (MMR) that corrects
errors in DNA. This hypermutability is not usually thought to be advantageous, as many random
mutations can reduce bacterial fitness. However, if mutations lead to antibiotic resistance, the
hypermutable state may become beneficial to the bacterial population. Hypermutators are generally
rare in acute infection but hypermutable H. influenzae have been found in patients with CF and these
strains have more resistance to antibiotics compared with normo-mutators [19, 32]. Hypermutability
is seen in other species causing chronic infection (as is discussed later in this chapter) and may be a
general adaptation to long-term survival in the lung. The prevalence and role of hypermutable
H. influenzae in non-CF bronchiectasis has yet to be assessed.
Pseudomonas aeruginosa
71
P. aeruginosa is a versatile nonfermentative Gram-negative bacillus that is found in a range of
environments. It is an opportunistic human pathogen that can cause severe, acute and invasive
infections, such as necrotising ventilator-associated pneumonia and infections in immunocompromised patients often with bacteraemia [33]. It is one of the most common causes of
infection in non-CF bronchiectasis and other chronic lung diseases, most notably CF, but may also
be important in severe COPD. The epidemiology of P. aeruginosa, the mechanisms of
pathogenicity and the genotypic and phenotypic changes in chronic infection have been
extensively studied in CF, with fewer publications in non-CF bronchiectasis and COPD. There are
many similarities between the infections in these different conditions, suggesting a common route
of adaptation to chronic infection in the lung.
MICROBIOLOGY
P. aeruginosa in CF
Early infections in CF are caused by genotypically distinct isolates, suggesting repeated episodes of
acquisition. These early P. aeruginosa have the typical phenotype of isolates causing acute
infections and environmental strains [34]. As chronic infection with P. aeruginosa can lead to an
accelerated deterioration in lung function, antibiotic treatment regimens were developed to clear
early infection and delay the onset of chronic infection [35]. CF patients eventually developed a
persistent infection that seldom cleared despite aggressive antibiotic therapy. While there are some
mixed infections, most CF patients carry a single genotype of P. aeruginosa, often for many
decades [36, 37], and exacerbations do not appear to be due to the acquisition of a new strain of
P. aeruginosa [38]. Early studies in CF showed that individual patients were infected with distinct
strains that were thought to have been acquired from the environment. Some siblings shared
strains but it was not known whether this was cross-infection or exposure to a common
environmental source. More recently there have been reports in several countries of crossinfection between CF patients with what are termed ‘‘epidemic’’ strains. Some, in particular the
Liverpool epidemic strain (LES), have been associated with increased morbidity [39, 40]. LES is
now the most common epidemic strain in the UK affecting as many as 11% of patients in England
and Wales [41].
P. aeruginosa in COPD
P. aeruginosa has been cultured from 4–15% of patients with COPD and was more prevalent in
patients with advanced disease, particularly those requiring mechanical ventilation for severe
exacerbations. P. aeruginosa infection was associated with steroid use, prior antibiotics and a low
forced expiratory volume in 1 second (FEV1) [42]. In a study of 126 patients with moderate-tosevere COPD over an 11-year period, 39 patients grew P. aeruginosa from one or more sputum
culture. There was a significant association with the culture of a new strain of P. aeruginosa and
symptoms of an exacerbation. However, of interest, two-thirds of new infections that later
cleared from the sputum, did so without the use of specific antibiotic treatment [43]. Only
13 patients had carriage of the same clone for more than 6 months with four patients infected
with mucoid strains. Chronic infection is therefore rare in COPD, but when it does occur
P. aeruginosa has a range of colony forms (morphotypes) and adaptations including increased
mutability, reduced motility, reduced protease production and increased antibiotic resistance,
similar to those seen in CF [44].
P. aeruginosa in non-CF bronchiectasis
P. aeruginosa is one of the most common isolates found in 12–43% of non-CF bronchiectasis
patients (table 1). Stable patients with P. aeruginosa have poorer lung function and more sputum
production when compared with patients with other potentially pathogenic microorganisms
(PPM) [45] and it has been associated with a poorer quality of life and more frequent hospital
admissions [46]. There is debate over whether infection with P. aeruginosa leads to a faster decline
in lung function as is seen in CF, or whether it is a marker of more damaged lungs [47, 48].
72
A recent study compared long-term colonisation with P. aeruginosa in 21 patients, of which six
had CF, 10 had non-CF bronchiectasis and five had COPD. The authors typed 125 sequential
isolates from sputa taken at least 1 month apart. The authors found a similar pattern of
colonisation in all three diseases, with a dominant persistent clone, showing that the pattern of
infection found in CF could also be shown in other conditions [49].
There have been no studies using genomic typing methods to investigate whether patients with nonCF bronchiectasis share strains of P. aeruginosa, but there has been one report of a patient with nonCF bronchiectasis acquiring LES from a relative with CF [50]. Interestingly, early studies using
pyocin typing and examining mucinophilic and chemotactic properties of P. aeruginosa suggest that
specific subpopulations may have a predilection to infect bronchiectatic lungs [51, 52].
Pathogenicity of P. aeruginosa
P. aeruginosa possesses a range of virulence factors, although their expression may differ between
isolates that cause acute infection and those responsible for chronic infection. Flagella, type IV pili,
lipopolysaccharide and exopolysaccharides contribute to the adherence to cells and surfaces. Type
I and type II secretion systems export protein toxins, such as alkaline protease, elastase, exotoxin A
and phospholipase C, while type III secretion systems inject exoenzymes directly into eukaryotic
cells. Other extra-cellular virulence factors include rhamnolipids, pyocyanin and hydrogen cyanide
[53, 54]. Another pathogenicity factor is the ability to form alginate-enhanced biofilms [55], which
contributes to the persistence of the organism rather than acute tissue damage and, together with
other adaptations, promotes chronic infection (refer to later section).
P. aeruginosa is intrinsically resistant to many commonly used antibiotics and easily acquires
resistance by chromosomal mutation or the acquisition of new genes from other microorganisms
by horizontal transfer [56]. In addition, the biofilm mode of growth also protects P. aeruginosa
from antibiotics by a variety of mechanisms [57].
Little has been published specifically on antibiotic susceptibility of P. aeruginosa from patients
with non-CF bronchiectasis. In CF the prevalence of resistant P. aeruginosa is increasing as a result
of repeated antibiotic courses. Resistance rates are significantly higher than for strains originating
from patients without CF [58] and pan-resistant bacteria that are resistant to all antibiotics other
than the polymixins have been described.
P. aeruginosa can develop resistance by either: 1) producing enzymes that destroy the antibiotic,
such as AmpC b-lactamase, carbapenemases or aminoglycoside modifying enzymes; 2) modifying
the antibiotic target, such as gyrA for quinolone resistance; or 3) reducing exposure either by a
decrease in permeability or increased removal of the antibiotic from the bacterial cell (efflux).
Efflux mechanisms often affect more that one class of antibiotics and therefore contribute to
multi-drug resistance [56].
J.E. FOWERAKER AND D. WAT
Antibiotic resistance
Antibiotic resistance and its regulation can be complex in P. aeruginosa and various mechanisms
that affect resistance to a single antibiotic may be present in the same organism. For example, low
level resistance to meropenem may be due to reduced permeability following changes to the
membrane porin OprD. More resistance can result from an increase in an efflux pump that can
remove the meropenem from the cell. Both mechanisms may be present and additive, leading to
high-level resistance. Enzymes that can destroy meropenem (penemases such as VIM) do occur
but are currently rare [56].
73
AmpC codes for an inducible cephalosporinase which, when production is increased, can result in
resistance to nearly all b-lactam antibiotics except the penems. Treatment with piperacillin or
ceftazidime can lead to the selection of bacteria that produce the enzyme constitutively rather than
just on induction. These are called derepressed mutants and offer a survival advantage. Imipenem
induces the AmpC b-lactamase, even though it is not affected by the enzyme, and it also induces
genes involved with alginate production [59]. The regulation of ampC is exceedingly complex and
is intimately linked to cell wall recycling [60–62]. Some mutations can reduce biological
competitiveness and more work is needed to assess the link between antimicrobial resistance and
fitness [61]. AmpR does not just regulate ampC but is a global transcriptional regulator that
regulates another b-lactamase PoxB, as well as proteases, quorum sensing and other virulence
factors [63]. Antibiotic resistance may therefore be associated with a change in virulence and/or
fitness. This could explain why some CF patients respond to treatment for acute exacerbation,
even though some of the P. aeruginosa are resistant to the antibiotic used [64].
P. aeruginosa possesses multi-drug efflux pumps that can expel a wide range of antibiotics and are
responsible for much of the organism’s intrinsic resistance to antimicrobials. For example,
substrates for efflux pump MexAB-OprM include ticarcillin, aztreonam, piperacillin, ceftazidime
and tetracycline [65]. MexXY uses the same exit duct OprM and can export aminoglycosides,
cefepime and ciprofloxacin. Antibiotic resistance may arise from an increase in the efflux pump
activity, e.g. MexXY-OprM over-expression may be due to mutation in the regulatory gene mexZ
and/or to mutations in the MexXY translocase genes [66].
MICROBIOLOGY
Conversely, P. aeruginosa may be hyper-susceptible in vitro to some anti-pseudomonal antibiotics
and susceptible to agents such as tetracycline and chloramphenicol to which P. aeruginosa would
normally be intrinsically resistant. This has been described in chronic infection in both CF and
non-CF bronchiectasis [67, 68], but the clinical relevance of these findings has not been
investigated. It was found that 25 out of 46 CF patients had strains hyper-susceptible to ticarcillin
due to deficiencies in MexAB-OprM efflux activity, resulting from various gene defects including
reduced or abnormal expression of MexB and OprM [66]. It is unclear why this phenomenon
exists. Efflux pumps do not just expel antibiotics and therefore reduced efflux may give a selective
advantage under certain physiological conditions in the chronically infected lung.
Adaptations to chronic infection
One of the characteristics of chronic infection with P. aeruginosa is the appearance in vitro of a
variety of colony forms (morphotypes) that differ from those seen in environmental strains or
those causing acute infection (fig. 1).
Several different morphotypes may be found in the same sputum, even though the isolates are
clonally related. These can include colonies lacking the typical pigmentation, mucoid forms, some
that look like coliforms, ‘‘dwarf’’ forms and very slow growing ‘‘small colony variants’’. One of the
most easily recognised is the mucoid morphotype. This results from over-production of the
polysaccharide alginate, due to mutation in the
regulatory genes. Hyper-alginate producers were originally thought unique to CF but they are also found in
non-CF bronchiectasis and COPD [45, 49, 69]. They
are thought to be an adaptation to chronic infection
irrespective of the underlying cause. Alginate may
protect against phagocytosis [70] and contribute to the
formation of biofilms [71, 72]. Small colony variants
(SCVs) have enhanced ability to form biofilms and may
also contribute to persistence [73]. SCVs have only
been described so far in CF but are easily missed unless
cultures are prolonged.
Figure 1. Different pseudomonal morpho-
74
logical types of Pseudomonas aeruginosa
found in a single sputum sample taken from
a chronically infected individual.
The phenotypic changes found in chronic infection
have been studied extensively in CF but not in non-CF
bronchiectasis. They include loss of acute virulence
factors, such as toxin production (e.g. elastase, phospholipase C, pyoverdin, hydrogen cyanide) and type III
secretion [74]. Many virulence factors are regulated by
the quorum sensing (QS) system. These are signalling
molecules that act on the regulators of gene transcription. Some QS molecules depend on
population density, and only have their effect when the number of organisms reaches a critical
concentration (or quorum). P. aeruginosa QS molecules comprise acyl-homoserine lactones and
molecules of the PQS system. They can affect a large number of functions including pathogenicity,
metabolic adaptation and persistence [75]. P. aeruginosa with mutations in QS genes, most
frequently las R, do not respond to QS molecules and are surprisingly common, they were found
in 19 out of 30 CF patients in a study by SMITH et al. [76]. Las R mutants form characteristic
iridescent colonies and have also been cultured from patients with non-CF bronchiectasis (J.E.
Foweraker, Papworth Hospital, Cambridge, UK; personal communication). These mutants do not
produce the toxins elastase, phenazines or hydrogen cyanide. Las R mutants can use a more diverse
range of compounds as a source of carbon, nitrogen, phosphorus or sulphur and have a growth
advantage over the wild type when grown with phenylalanine, isoleucine or tyrosine. They
therefore appear to be less pathogenic but better able to adapt to the local environment. Again,
different phenotypes can co-exist so sputum may contain Las R mutants and organisms without
the mutation.
Longitudinal studies in CF have analysed strains from patients over several years. It is thought that
with time the bacteria adapt to a form that is less virulent but better able to persist in the damaged
lung [76]. Multiple phenotypic variants of the underlying clonal population of P. aeruginosa coexist and form a complex population in the chronically infected lung. This is described as
‘‘adaptive radiation’’ and is thought to give the bacteria an advantage in that they can rapidly
respond to changes in the environment, as individual organisms that have the necessary
adaptation may already be present in the population.
P. aeruginosa is thought to grow in biofilms in chronic infections in both CF and non-CF
bronchiectasis. Biofilm formation is thought to be a general adaptation to a hostile environment
and may allow persistence of infection by protecting the bacteria from the host response and the
effects of antibiotics. Biofilm fragments have been seen in CF sputum [77] and may contain a
mixture of P. aeruginosa plus other bacteria and even fungi, such as Candida spp. The extracellular matrix comprises alginate produced by P. aeruginosa plus proteins and DNA from other
microorganisms and host cells. The biofilm contains a steep oxygen gradient and is anaerobic just
below the surface. Different concentrations of nutrients and waste products will also be found in
different areas of the biofilm. Therefore, the biofilm contains a wide range of physiological
conditions, by which the bacteria possess a variety of adaptations that enable them to survive
within these microniches [78].
J.E. FOWERAKER AND D. WAT
Biofilms
Alginate protects P. aeruginosa in biofilms from interferon (IFN)-c activated macrophages [79].
Neutrophils have been observed immobilised in the extra-cellular matrix, unable to penetrate the
biofilm [80]. It is thought that neutrophils may actually enhance early biofilm formation, as
biofilms formed in vitro in the presence of neutrophils are thicker and contain more bacteria [81].
If P. aeruginosa and neutrophils are combined, the bacteria aggregate around necrotic dying
neutrophils. If neutrophil apoptosis is induced before the bacteria are added, the neutrophils are
intact and the P. aeruginosa remain dispersed. Neutrophils can release DNA and F-actin
complexed with histones and other cations, and these may form the framework for the biofilm.
The combination of DNAse and anionic polymers has a synergistic effect in clearing early
neutrophil-associated biofilms in vitro and is being studied as a potential treatment to prevent or
disrupt early biofilm formation [81]. Neutrophil lysis is thought to be caused by rhamnolipid, a
toxin produced by P. aeruginosa under QS control. Rhamnolipid may therefore help to protect the
biofilm from disruption by neutrophils, especially in the early stages of formation [82].
75
Azithromycin is a macrolide antibiotic that does not directly inhibit or kill P. aeruginosa, but it can
block QS and alginate polymer formation in vitro [83]. It can disrupt early biofilms formed by
nonmucoid strains but has less effect on early biofilms formed by hyper-alginate producers
MICROBIOLOGY
(mucoid strains) or on established biofilms [84]. Azithromycin also has an anti-inflammatory
effect in chronic lung infection and the relative importance of its diverse actions is yet to be
established. Other antibiotics may also influence bacterial virulence. For example ciprofloxacin can
suppress alginate biosynthesis at concentration well below minimum inhibitory concentration
(MIC) [85].
Bacteria cultured in biofilms in vitro are more resistant to most antibiotics than when they are
dispersed (planktonic). Several mechanisms have been proposed to explain this resistance [86]. It
was thought that the extra-cellular matrix formed a physical barrier but there are channels within
the biofilm through which most antibiotics can permeate. Positively charged antibiotics such as
colistin may bind to free anionic DNA and therefore not reach the bacteria, and an anionic
antibiotic, such as an aminoglycoside (e.g. tobramycin) may bind to the alginate. If the AmpC
b-lactamase is over produced by some P. aeruginosa it may form a high local concentration and
protect bacteria that can only produce basal levels of the enzyme. Mutability is increased in
biofilms, partly because of the presence of hypermutators but also because DNA can be damaged
by the increased amounts of reactive oxygen species within the biofilm. The range of metabolic
conditions in the biofilm may affect antibiotic susceptibility. P. aeruginosa can survive in the
anaerobic environment just below the surface of the biofilm by using nitrogen rather than oxygen
as a terminal electron acceptor and aminoglycosides, such as tobramycin, cannot act on organisms
that are metabolising anaerobically. Organisms within a biofilm may become dormant and
therefore resist quinolones and b-lactam antibiotics [87]. These affects have been shown in an in
vitro model of a young biofilm in a flow chamber using live/dead staining. Ciprofloxacin kills
organisms on the surface of the biofilm but cannot kill those deeply set within the biofilm, whereas
colistin can kill the non-dividing cells in the centre [88]. The two antibiotics appear to be very
effective against young biofilms in vitro and may explain why that combination is particularly
effective in eliminating early infection with P. aeruginosa in CF.
Hypermutators
One of the drivers of variability and adaptation seen in persistent infection in bronchiectasis is
thought to be the presence of hypermutator (HM) bacteria [89]. These are P. aeruginosa with a
higher than usual spontaneous mutation rate and are thought to accelerate bacterial evolution.
P. aeruginosa usually mutates at a frequency of one in 108–109, while mutation rates in HM
bacteria can be as high as one in 100. HM P. aeruginosa were found in 37% of chronically infected
CF patients. This was the highest prevalence that had been described for a naturally occurring
population [90]. In comparison a HM prevalence of 1% in Escherichia coli and Salmonella spp. had
previously been considered high [91]. In a longitudinal study of CF patients in Denmark, none of
the bacteria from early infections were found to be HMs but after 20 years of colonisation 65% of
patients were infected with HM P. aeruginosa [92]. HM P. aeruginosa were described in 57% of
chronically infected patients with COPD or non-CF bronchiectasis, suggesting that hypermutability is a general adaptation to long-term survival in the lung [93]. KENNA et al. [94] suggests
that hypermutability is an extremely rare finding in environmental P. aeruginosa and in isolates
from newly infected CF patients.
Most of the information on HM P. aeruginosa comes from work on isolates from CF [95]. Hypermutability usually results from a primary mutation in genes of the MMR system, most commonly
mutS and mutL, or defects in the GO system (mut M, Y and T). The function of these systems is to
detect and repair DNA replication errors and repair oxidative damage. MMR also inhibits
recombination between moderately diverged sequences and therefore reduces the acquisition of
exogenous DNA through horizontal gene transfer [96].
76
HMs are uncommon in most bacterial populations because many of the mutations are deleterious.
HM P. aeruginosa had reduced virulence and fitness both in vitro and in an animal model [97, 98].
However, in changing environments or stressful conditions HM bacteria may be selected because
they have adaptive mutations, such as antimicrobial resistance (referred to as ‘‘hitchhiking’’).
The sequential acquisition of resistance to multiple antibiotics is seen in infection with P. aeruginosa
in CF, and several studies in CF, non-CF bronchiectasis and COPD have shown that HM are more
likely to be antibiotic resistant than isolates with normal mutation rates [93, 99]. In a study of 29 CF
patients over a 5-year period, mutations accumulated at an average mutations rate of three per year
in HM P. aeruginosa compared with 0.25 per year in non-mutators. HM had more mutations
leading to antibiotic resistance but also more mutations in other genes such as lasR [89]. Therefore,
other adaptations may provide a selective advantage for HM isolates, not just antibiotic resistance.
Two recent studies have shown that CF patients with HM had poorer lung function (FEV1
predicted), but longitudinal studies are needed to determine if this was due to infection with a HM
or just an association, both being the result of prolonged infection [99, 100]. Work is needed on
the role of HM in non-CF bronchiectasis.
Chronic P. aeruginosa infection and the clinical microbiology laboratory
One practical implication of the range of phenotypic diversity of P. aeruginosa from non-CF
bronchiectasis is that some isolates may be difficult to identify. Colonies of P. aeruginosa from
chronic infection may lack pigmentation, grow very slowly and may mimic other species.
Another consequence of phenotypic diversity is that a range of antimicrobial susceptibility
patterns can be found in a population of P. aeruginosa in a single sputum sample (fig. 2). Bacteria
with the same morphotype may have different susceptibility and therefore resistant subpopulations may be missed, depending on which colony is picked for testing [68].
In CF, once a chronic infection is established the range in the antibiotic susceptibility of
P. aeruginosa in a single sputum is so diverse that susceptibility testing methods are unreliable
[102, 103]. It is currently unclear whether these findings can equally be applied to chronic infection in non-CF bronchiectasis.
Finally it has been questioned whether current methods used for testing antimicrobial
susceptibility are relevant for bacteria that may be present in biofilms in the chronically infected
lungs. A variety of methods are being developed for testing biofilm susceptibility; however, their
clinical relevance still needs to be determined.
J.E. FOWERAKER AND D. WAT
Commercial identification schemes that use biochemical reactions and assimilation tests are not
reliable in identifying atypical P. aeruginosa and some of the other nonfermenting Gram-negative
bacilli found in chronic infection, and therefore identification methods, such as species-specific
PCR or sequencing of the 16S ribosomal RNA gene may be required [101].
Streptococcus
pneumoniae
Figure 2. Variation in antimicrobial susceptibility testing. The figure
shows the results from testing two bacteria with identical colony
form in sputum taken from a patient with non-cystic fibrosis
bronchiectasis. The same six different antimicrobial discs were
used for both cultures. The susceptibility is proportional to the
diameter of the zone of diffusion around the antibiotic disc.
77
S. pneumoniae is a Gram-positive
coccus appearing in pairs and in
short chains. It may be a harmless
commensal in the oro-pharynx but
can cause severe and invasive disease (pneumonia or meningitis).
It can also cause otitis media or
sinusitis, or lower airway infections
in patients with damaged lungs such
as non-CF bronchiectasis or COPD,
but it is rare in CF. Although
S. pneumoniae can be found in up
to 37% of patients with non-CF
bronchiectasis, very little has been published on its role in this condition. In COPD, S. pneumoniae
has been cultured from both stable patients and those with exacerbation [20, 104]. The patient with
non-CF bronchiectasis due to an underlying antibody deficiency may be particularly susceptible to
recurrent infections with S. pneumoniae [105]. Bronchiectasis in primary and secondary
immunodeficiency patients is discussed further in the chapter by BROWN et al. [106].
S. pneumoniae has a polysaccharide capsule that helps evade opsonisation, and isolates lacking the
capsule are avirulent. There are over 90 capsule types and the capsule type may be one of several factors
that determine the pathogenicity of an individual strain [107]. A polyvalent vaccine containing the
most common serotypes is available and recommended for use in patients with chronic lung disease.
S. pneumoniae can use a wide variety of molecules to adhere to host cells and produces an IgA
protease and a toxin, pneumolysin that can promote invasion, inflammation and tissue damage
[108]. Pneumolysin is proinflammatory and has many actions including cytolysis, inhibition of
cilial beating, and direct activation of the classical complement cascade. Although it is not a
common pathogen in CF, isolates of S. pneumoniae from CF sputum have characteristics that may
be associated with adaptation to persistence in the lung, i.e. hypermutability and the ability to
form biofilms [109, 110]. Further work is needed to clarify the role of the different virulence
factors in order to understand why S. pneumoniae may be a harmless commensal or cause noninvasive respiratory tract infection (in COPD or bronchiectasis) or produce severe invasive disease
with bacteraemia.
MICROBIOLOGY
The prevalence of antibiotic resistant S. pneumoniae has increased and in some countries very high
rates of resistance to penicillin, macrolides and tetracyclines limit the treatment options. Penicillin
resistance is due to modifications to penicillin binding proteins not by the production of a
b-lactamase and, therefore, amoxicillin–clavulanate is ineffective.
Moraxella catarrhalis
M. catarrhalis is a Gram-negative diplococcus that was previously named Branhamella or Neisseria
catarrhalis. Like NTHi it is a common commensal organism in the upper respiratory tract and can
cause otitis media or sinusitis. It was not reported in studies of non-CF bronchiectasis in the 1960s
as it was considered an oral contaminant rather than a PPM. However, it can be cultured in
significant numbers from sputum or PSB in up to 27% of patients with non-CF bronchiectasis [7].
It is also considered a significant pathogen in COPD but is only rarely isolated in CF.
A longitudinal study of M. catarrhalis in 29 patients with non-CF bronchiectasis found that patients
were colonised with a variety of strains with average colonisation duration of 2.3 months for each
strain. No association between strain acquisition and exacerbation was found and as M. catarrhalis
was often in mixed culture with other PPMs (H. influenzae or S. pneumoniae), it was difficult to
determine whether it had an independent pathogenic role [111]. In a study of 50 patients with
COPD, the average time from acquisition to clearance of a new strain of M. catarrhalis was 1 month
and re-infection with the same strain was rare, suggesting that there was an effective immune
response. Of the new acquisitions, 47% were associated with an exacerbation [112]. Acquisition of
M. catarrhalis led to an increase in airway inflammation, characterised by a rise in sputum neutrophil
elastase, interleukin (IL)-8, tumour necrosis factor (TNF)-a and a reduction in secretory leukocyte
protease inhibitor (SLPI) [113].
Putative virulence factors of M. catarrhalis include several outer membrane proteins plus LOS and
these affect cell adhesion, epithelial cell invasion, serum resistance and biofilm formation [114].
More work is needed to understand the pathogenesis of infection in both COPD and non-CF
bronchiectasis.
78
More than 90% of M. catarrhalis produce a b-lactamase (BRO-1 or BRO-2) and are resistant to
ampicillin. Acquired resistance to other antibiotics is rare with most remaining susceptible to
macrolides, tetracyclines, amoxicillin-clavulanic acid and quinolones [115].
Staphylococcus aureus
S. aureus is a Gram-positive coccus found in clusters that may be part of the normal flora in the
anterior nares, throat and on moist skin sites such as groin and axilla. Infection is characterised
by abscess formation, particularly in skin and soft tissues. It is a rare cause of respiratory tract
infection, but can cause severe pneumonia after influenza. It is a common cause of early
infection in CF but is less common in non-CF bronchiectasis where its presence may indicate
undiagnosed CF [10]. There is also an association of S. aureus with ABPA in non-CF
bronchiectasis [116].
The ability of S. aureus to rapidly adapt and persist in the lung may be a result of genomic
instability due to mobilisation of bacteriophages. Isolates from the anterior nares of CF patients
had a higher frequency of genomic alterations than those from healthy controls [120]. A higher
proportion of hypermutable strains of S. aureus were found in CF patients when compared with
isolates from bacteraemia or other respiratory infections. As with other species with high mutation
rates, many of these had defects in mutS [121].
Meticillin resistant S. aureus (MRSA) are resistant to all penicillins, cephalosporins and penems
and are often also resistant to other classes of antibiotics (macrolides, fluoroquinolones and
aminoglycosides). They can be difficult to treat, partly because oral options are limited but
also because the active parenteral options (glycopeptides) may be less effective compared
with the use of a b-lactam antibiotic to treat a susceptible isolate. It may be difficult to clear
MRSA carriage from patients with bronchiectasis, but there is data from CF that shows
that a combination of systemic treatment with skin antisepsis and inhaled antibiotics may be
effective [122].
J.E. FOWERAKER AND D. WAT
S. aureus produces a range of exotoxins that can cause tissue damage. It is also thought to form
biofilms on prosthetic devices and thereby evade the host response and resist antimicrobial
therapy [117]. Biofilm-like aggregates of S. aureus surrounded with the polysaccharide poly-Nacetyl-glucosamine have been observed in anaerobic conditions in CF mucus and can resist
nonoxidative killing [118]. Persistence of S. aureus in CF and prosthetic infections has also been
related to the presence of small colony variants. These tiny colonies are difficult to identify in vitro.
They are associated with treatment with trimethoprim/sulphamethoxazole or aminoglycosides,
are more antibiotic resistant than the typical forms in the same sputum and may survive within
host cells [119].
Burkholderia spp. and other non-fermenters
Burkholderia spp are plant pathogens and are a major cause of morbidity and mortality in CF but
are rarely encountered in other conditions. In spite of the frequent presence of these bacteria in the
environment, and their propensity for spread between CF patients, there are only two case reports
of infection in non-CF bronchiectasis, one with Burkholderia cepacia complex (not speciated) and
another with Burkholderia gladioli [123, 124].
79
A wide variety of other nonfermentative Gram-negative bacilli can occasionally act as opportunistic
pathogens in the human lung. Species of the genera Achromobacter, Stenotrophomonas, Ralstonia,
Pandoraea and Inquilinus can cause infection in the CF lung, and S. maltophilia and Achromobacter
(previously Alkaligenes) xylosoxidans have been reported in non-CF bronchiectasis (table 1). Many
are both intrinsically resistant to some antibiotics and easily acquire resistance. They can be difficult
to identify in the laboratory and molecular methods are recommended to ensure accurate
identification [101]. In particular it is important to differentiate these organisms from the
Burkholderia spp. because of the need to prevent cross infection. There is too little experience with
these microorganisms to comment on their propensity for colonisation, infection, or role in
exacerbation of non-CF bronchiectasis.
Anaerobes and other bacteria considered normal upper
respiratory tract flora
Sputum may contain microorganisms other than the PPM. These have been considered either
contaminants from the upper respiratory tract or harmless commensals colonising the sputum.
This assumption has been challenged following studies using both conventional culture methods
and culture-independent techniques.
Sputum is not routinely cultured for anaerobes partly because they are present in large numbers in
saliva and can easily contaminate expectorated sputum, but also because some are very difficult to
culture. Following the observation of a rapid drop in oxygen partial pressure just below the surface
of a CF sputum plug, investigators began to look for anaerobes in the sputum from CF and nonCF bronchiectasis [125]. Obligate anaerobes in particular Prevotella spp. were found in significant
numbers, far more than would be expected from oral contamination [126–128]. Their significance
in disease has been questioned as high numbers of bacteria were found in a stable CF patient in
one study, and the numbers of anaerobes remained constant during successful treatment of a
clinical exacerbation [129].
MICROBIOLOGY
It has been proposed that members of the Streptococcus milleri group may have a role in chronic
lung infection. One study followed the changes in the microbial flora during and between
pulmonary exacerbations of CF using both culture and culture-independent methods. The group
identified members of the S. milleri group as of potential importance in exacerbations both in CF
and in two patients with non-CF bronchiectasis [130].
Following an observation that a range of upper respiratory tract flora were seen in large numbers
in sputum from CF patients, a Staphylococcus sp. (not S. aureus) and a viridans-type Streptococcus
sp. were further studied. While not intrinsically pathogenic, they were able to enhance the
virulence of P. aeruginosa in an animal model and increase the expression of certain virulence
genes of P. aeruginosa in vitro. This could be reproduced using an inter-species QS molecule, Auto
Inducer-2 (AI-2) [131]. Of interest, the oral anaerobe Prevotella also produces AI-2 [127]. The
complex pattern of interaction between microorganisms in ecosystems other than the lung has
been described and it is known that microorganisms can enhance or inhibit growth of other cohabitants [132]. The studies in CF show that interactions may also enhance pathogenicity [133].
The CF lung, therefore, may contain a mixture of microorganisms that includes those that are
directly pathogenic, those that behave as commensals and those that are not directly pathogenic
but may increase the virulence of other organisms. Although this has not been studied in non-CF
bronchiectasis, microorganisms other than PPMs are regularly observed in sputum cultures in
combination with PPMs and further work on these potential interactions is needed.
Culture-independent studies of microbial flora in the lung
80
There have been attempts to describe the composition and diversity of the microbes in the lung
irrespective of the ability to culture individual microorganisms. One approach is to analyse the
gene encoding 16S rRNA. This is present in all true bacteria and the sequence variation is sufficient
to identify most genera and many species. Genetic material can be extracted from a clinical sample,
and the 16S rRNA gene amplified by PCR. The product may be analysed looking at terminal
restriction fragment length polymorphisms (TRFLP). This compares the size of the terminal
fragment of rRNA after cutting with a restriction enzyme; the length of the fragment being
characteristic of certain species. Alternately the PCR product can be cloned, sequenced and
compared with databases containing sequence data from a wide range of microorganisms. These
methods and other variations have been applied to patients with CF and non-CF bronchiectasis to
describe the diversity of microorganisms, and have revealed species not previously found in
respiratory samples using traditional culture methods [12, 134–136]. While the presence of nucleic
acid does not necessarily indicate the presence of viable organisms, a comparison of RT-TRFLP
with TRFLP showed that a high proportion of the bacterial species detected in CF sputum were
metabolically active [137].
There have been major technical advances facilitated by the development of next generation
sequencers plus developments in bioinformatics. These have allowed direct analysis of amplified
DNA without the cloning step, and greater depth of sequencing of 16S rRNA DNA [138, 139]. An
alternative approach is to attempt whole genome sequencing directly from the clinical sample [140].
This could include analysis of nucleic acid from eukaryotes and viruses in sputum as well as bacteria
[141]. Such techniques can provide an enormous amount of information that is a great challenge to
process. However, they offer the potential for a far more sophisticated analysis of the genetic
variability found in single species and the variety of microorganisms in chronic lung infection.
Respiratory viruses
The role of viruses in non-CF bronchiectasis is not known and remains an important area for
future research. Some data exists that viral infections in childhood may predispose to the
development of bronchiectasis in later life [142], whether it is through the development of
bronchiolitis, disruption of small airway associated innate/adaptive immunity, damage of airway
epithelia or compromise of mucociliary clearance, it is unclear.
Viral infections in asthma, COPD and CF
Viral exacerbation of asthma has been well published. In a study by JOHNSTON et al [143] using
PCR and viral culture, viruses were detected in 80% of episodes of wheeze or reduced peak
expiratory flow in children aged 9–11 years with asthma. Rhinovirus accounted for 61% of the
viruses detected, coronavirus 16%, influenza 9%, parainfluenza 9% and respiratory syncytial virus
(RSV) 5%. Similarly, NICHOLSON et al. [144] found that respiratory viruses accounted for 44% of
asthma exacerbations in adults. Respiratory viruses were also present in most patients hospitalised
for life-threatening asthma and acute not life-threatening asthma [145].
The application of molecular diagnostic methods has improved the understanding of viral
epidemiology. Respiratory viruses may induce asthma exacerbations via direct effects on the
airway epithelium as well as through a systemic immune reaction.
J.E. FOWERAKER AND D. WAT
What is also unclear is the role that viral infection plays in triggering infective exacerbations and
progressive lung damage in patients with non-CF bronchiectasis where no studies have to date
been carried out. Therefore, only cautiously can parallels be drawn from studies examining the
role of viruses in asthma, COPD and CF.
Rhinovirus is the most common respiratory virus and represents two-thirds of all upper respiratory
tract infections. It also accounts for 50% of asthma exacerbations in children [146]. Traditionally,
rhinovirus is thought to infect the upper respiratory epithelium. However, rhinovirus is also capable
of replicating in the lower airway cells during experimental infection [147]. PAPADOPOULOS et al. [148]
showed that both rhinovirus genomic material and replicative strand RNA were detectable in
bronchial biopsies using in situ hybridisation in 50% of adult volunteers subjected to an experimental
rhinovirus upper respiratory infection.
81
The mechanism by which viruses cause bronchoconstriction is not fully understood, but it is likely
to involve cytokine production in response to viral replication in the lower airways, which includes
upregulating the expression of a range of proinflammatory mediators. The proinflammatory
cytokine IL-1b is detectable in experimental infected individuals. IL-8, a key mediator in
neutrophil-mediated acute inflammation, is also detected in naturally occurring infections
correlating with neutrophilia in blood and nasal samples in children with virally precipated asthma
or experimental infection [149]. Other mediators induced by rhinovirus infections include
neutrophil-activating peptide (which induces neutrophil migration), eotaxin and RANTES
(regulated on activation, T-cell expressed and secreted), IL-16 and monocyte chemoattractant
protein (MCP-1). All of these can lead to enhance airway inflammation.
RSV was detected in only 5% of asthma episodes in the study by JOHNSTON [143]. However, it is
known to be a potent cause of wheezing, particularly in infancy. It has been shown that Gglycoprotein of RSV appears to stimulate T-helper cell (Th) type 2 immune response in the upper
airway, whether or not if the infant is atopic [150]. Th2 cytokine patterns are known to be associated
with viral immunopathology and allergic-type responses, in contrast to Th1 cytokine patterns, which
are classically associated with viral elimination. Interestingly, the nasal cytokine responses to other
viruses are of the predominant Th1 type (except RSV). This could explain the tendency for RSV to
cause wheezing, but not the association between other respiratory viruses and wheezing.
MICROBIOLOGY
Influenza A infection induces large amounts of intrapulmonary IFN-c and enhances both later
allergen specific asthma and dual Th1/Th2 responses [151]. TERAN et al. [152] also demonstarted
that the eosinophil product, major basic protein (MBP), and RANTES increased with viral
infections, and there was a correlation in the concentration of RANTES with clinical symptoms. In
addition, epithelial cells infected with influenza in vitro were associated with an increase in eotaxin
[153]. Eotaxin can in turn lead to an exaggerated inflammatory response by being an agonist for
chemokine receptor 3, which can be found on eosinophils, T-cells and basophils. These are all key
factors in asthma exacerbation.
Patients with asthma are no more susceptible to upper respiratory tract rhinovirus infections than
healthy people, but suffer from more severe consequences of the lower respiratory tract infection.
Recent epidemiological studies suggest that viruses provoke asthma attacks by additive or
synergistic interactions with allergen exposure or with air pollution. An impaired antiviral
immunity to a rhinovirus may lead to impaired viral clearance and hence prolonged symptoms.
Indirect prevention strategies focus on the reduction of overall airway inflammation to reduce the
severity of the host response to respiratory viral infections. There is a lack of specific antiviral
strategies in the prevention or reduction of viral-triggered asthma exacerbations. Recent advances
in the understanding of the epidemiology and immunopathogenesis of respiratory viral infections
in asthma may provide opportunities or the identification of specific targets for antiviral agents
and strategies for management and prevention.
COPD is the fourth leading cause of mortality worldwide and is an important cause of global
burden of disease [154]. The disease is associated with intermittent exacerbations characterised by
acute deterioration in symptoms, lung function, and quality of life [155, 156]. Exacerbations have
major effects on health status and are associated with considerable morbidity and mortality that
can lead to hospital admission with high treatment costs [157].
Infectious agents are recognised as a major pathogenic factor in exacerbations. Bacteria have a role in
the pathogenesis [158, 159] and the exacerbations of COPD. However, bacteria are absent in about
50% of exacerbations and the frequency of isolation does not increase during exacerbation [160].
82
Early studies looking at respiratory viruses and COPD have stated a 20% detection rate in COPD
exacerbations [161, 162]. However, these studies were limited by using less sensitive methods in
viral detection. SEEMUNGAL et al. [163] detected respiratory viruses from nasal samples and blood
of patients with COPD using a combination of culture, serology and PCR. They showed that 64%
of COPD exacerbations were associated with a cold occurring up to 18 days before exacerbation.
In total, there were 168 episodes of COPD exacerbation in 53 patients and 77 viruses (39 were
rhinoviruses) were detected. Viral exacerbations were associated with frequent exacerbatons,
increased symptoms, a longer median symptom recovery period (up to 13 days) and a tendency
towards higher plasma fibrinogen and serum IL-6 levels. RSV has also been shown to be an
important virus in COPD exacerbations and was detectable in 11.4% of patients admitted into
hospital [164]. Patients with stable COPD may carry respiratory viruses. Non-RSV respiratory
viruses were detected in 11 (16%), and RSV in 16 (23.5%) out of 68 stable COPD patients, with
RSV detection being associated with higher inflammatory marker levels [161, 164].
Early studies looking at respiratory viruses in CF relied on repeated serological testing, either alone
[165], or in combination with viral cultures for viral detection [166–170]. These methods are
relatively insensitive and more recent studies have utilised molecular based methodologies [171–175].
All these studies produced different results in terms of prevalence of respiratory viruses in CF, these
differences could be due to the different methodologies utilised. It is also likely that there are
differences in the populations studied, as the prognosis for CF has improved with each successive
birth cohort.
More recent studies suggest no difference in the frequency of either upper respiratory tract illness
episodes [166] or proven respiratory viral infections [168] between children with CF and healthy
controls; however, children with CF have significantly more episodes of lower airway symptoms
than controls [166, 168]. RAMSEY et al. [168] prospectively compared the incidence and effect of
viral infections on pulmonary function and clinical scores in 15 school children with CF aged
5–21 years and their unaffected siblings. Over a 2-year period, samples were taken at regular
2-month intervals and during acute respiratory illnesses for pharyngeal culture and serology for
respiratory viruses. There were a total of 68 acute respiratory illness (ARI) episodes that occurred
in the patients with CF, in 19 of these episodes an associated virus identified. A total of 49 infective
agents were identified either during ARIs or at routine testing in the patients with CF; 14 were
identified on viral isolation (rhinovirus on 11 occasions), whilst 35 were isolated on
seroconversion (parainfluenza virus on 12, RSV on nine and Mycoplasma pneumoniae on six
occasions). There was no significant difference in the rate of viral infections between the patients
with CF and their sibling controls, as measured either by culture or serology. The rate of viral
infections was higher in younger children (both CF and controls), and the rate of decline in
pulmonary function was greater in the younger children with CF with more viral infections. At the
time of an ARI, the virus isolation and seroconversion (four-fold increase in titres) rates were 8.8%
and 19.1%, respectively, in children with CF compared with 15% and 15%, respectively, for the
siblings not affected. In contrast the rates for virus isolation and seroconversion at routine
2 monthly visits were 5.6% and 16.2%, respectively, for children with CF and 7.7% and 20.2%,
respectively, for the siblings not affected.
J.E. FOWERAKER AND D. WAT
It has now been 25 years since WANG et al. [169] described the relationship between respiratory
viral infections and the deterioration in clinical status in CF patients. Viruses were identified
through repeated serology and nasal lavages for viral isolation in 49 patients with CF (mean age
13.7 years) over a 2-year period. Although the CF patients had more respiratory illnesses than the
sibling controls (3.7 per year versus 1.7 per year), there were no differences in virus identification
rates (1.7 per year). The rate of proven virus infection was significantly correlated with the decline
in forced vital capacity (FVC) and FEV1, Shwachman score, and frequency and duration of
hospitalisation.
Similarly HIATT et al. [166] assessed respiratory viral infections over three winters in 22 infants
less than 2 years of age with CF (30 patient seasons) and 27 age matched controls (28 patient
seasons). The average number of acute respiratory illness per winter was the same in the control
and the CF groups (5.0 versus 5.0). However, only four of the 28 control infants had lower
respiratory tract symptoms in association with the respiratory tract illness, compared with 13 out
of the 30 infants with CF (OR -4.6, 95% CI 1.3–16.5; p-value ,0.05). Seven of the infants with
CF cultured RSV, of whom three required hospitalisation. In contrast, none of the controls
required hospitalisation. Pulmonary function measured by rapid chest compression technique
was significantly reduced in the infants with CF after the winter months and was associated with
two interactions; RSV infection with lower respiratory tract infection and male sex with lower
respiratory tract infection.
83
From previous reports, two viral agents appear to have the greatest effect on respiratory status in
CF, namely RSV and influenza, possibly because the uses of viral culture and serology have
underestimated the effects of rhinovirus (due to the vast amount of serotypes). In younger
children, RSV is a major pathogen resulting in an increased rate of subsequent hospitalisation.
ABMAN et al. [176] prospectively followed up 48 children with CF diagnosed through newborn
screening and documented the effect of RSV infection. 18 of the infants were admitted into
hospital a total of 30 times over a mean follow-up period of 28 months (range 5–59 years). In
seven of these infants RSV was isolated, and their clinical course was severe with three requiring
mechanical ventilation and five necessitating chronic oxygen therapy. Over the next 2 years these
infants had significantly more frequent respiratory symptoms and lower chest radiograph scores
than non-RSV identified infants. In another prospective study of repeated BAL in 80 infants
identified through CF newborn screening over a 5-year period, 31 infants were hospitalised for a
respiratory exacerbation, 16 (52%) of which had a respiratory virus identified with the most
common being RSV (n57).
MICROBIOLOGY
In older children and adults with CF, influenza seems to have the greatest effect. PRIBBLE et al.
[167] assessed acute pulmonary exacerbation isolates from 54 patients with CF. Over the year of
the study 80 exacerbations were identified, of which 21 episodes were associated with an identified
viral agent (influenza A: five episodes; influenza B: four episodes; RSV: three episodes) with most
agents identified by serology. Compared with other agents, infection with influenza was associated
with a more significant drop in pulmonary function (FEV1 decreased by 26% compared with 6%,
respectively). A retrospective study in older patients with chronic P. aeruginosa infection reported
an acute deterioration in clinical status in association with influenza A virusl infection [177].
COLLINSON et al. [171] followed 48 children with CF over a 15-month period using a combination
of viral culture and PCR for picornaviruses alone [178]. 38 children completed the study and there
were 147 symptomatic upper respiratory tract infections (2.7 episodes per child per year), with
samples available for 119 episodes. Picornaviruses were identified in 51 (43%) of these episodes, of
which 21 (18%) were rhinoviruses. In those children old enough to perform spirometry there were
significant drops in both FVC and FEV1 in association with upper respiratory tract infection, with
little difference in the severity of drop whether a picornavirus was identified or not. Maximal mean
drop in FEV1 was 16.5%, at 1–4 days after onset of symptoms, but a deficit of 10.3% persisted at
21–24 days. Those with more upper respiratory tract infections appeared to have a greater change
in total Shwachman and Crispin–Norman scores over the study. Six children isolated a
P. aeruginosa for the first time during the study, five at the time of a upper respiratory tract
infection and only one was asymptomatic at the time of first isolation. The data from this study
has to be handled with care as the term ‘‘upper respiratory tract illness (URTI)’’ did not necessarily
imply a positive viral isolation.
PUNCH et al. [173] used a multiplex RT-PCR assay combined with an enzyme-linked amplicon
hybridisation assay (ELAHA) for the identification of seven common respiratory viruses in the
sputum of 38 CF patients. 53 sputum samples were collected over two seasons and 12 (23%)
samples from 12 patients were positive for a respiratory virus (influenza B n54, parainfluenza 1
n53, influenza A n53, RSV n52). There were no statistical associations between virus status and
demographics, clinical variables or isolation rates for P. aeruginosa, S. aureus or Aspergillus
fumigatus.
OLESEN et al. [174] obtained sputum/laryngeal aspirated from children with CF over a 12-month
period in outpatient clinics. They achieved a viral detection rate of 16%, with rhinovirus being the
most prevalent virus. However, this virus did not seem to have any devastating impact on lung
function. However, the other viruses detected were associated with significant reduction in lung
function. The authors failed to show a positive correlation between respiratory viruses and
bacterial infections in their studied population, as the type or frequency of bacterial infection
during or after viral infections were not altered. They also demonstrated that clinical viral
symptoms had a very poor predictive value (0.39) for a positive viral test.
84
WAT et al. [179] utilised ‘‘real-time’’ nucleic acid sequence-based amplification (NASBA) to
examine the role of respiratory viruses in CF. They achieved a rate of 46% for respiratory viruses in
their paediatric CF cohort during reported episodes of respiratory illness. The results compare
favourably with previous studies, this may be due to earlier studies relying heavily on repeated
serological testing either alone [165] or in combination with viral isolation [166–170]. These traditional
methods are relatively insensitive and once again may have underestimated the prevalence of
viruses in CF.
Detection of respiratory viruses
The principal laboratory methods utilised for the diagnosis of respiratory viruses, rely upon the
detection of the virus in respiratory secretions and therefore an important factor in respiratory
viral diagnosis is the necessity for the submission of an appropriate sample for testing. Inadequate
or improper specimen collection and transport account for the largest source of error in the
accuracy of viral detection results [180]. Nasal swabs, nasopharyngeal aspirates, nasal wash and
sputum specimens are generally considered as the specimens of choice for the detection of
respiratory viruses [173, 180–183]. A prospective study by HEIKKINEN et al. [184] showed that the
sensitivity of nasal swabs was comparable to nasopharyngeal aspirates for the detection of all major
respiratory viruses by tissue culture, with the exception of RSV.
Molecular techniques have superseded many ‘‘conventional’’ methods utilised for respiratory viral
detection, such as viral culture and serology analysis, due to the rapid turn-around time for the
results. Molecular assays have particular advantages where the starting material available is
acellular (swab) or where surveillance samples have a low copy number of the viral target. The
rapid turn-around time of results allows diagnostic virology to have an impact on patient
management, thereby avoiding prescribing the inappropriate use of antibiotics and allowing the
correct prescription for anti-virals. It may also play an important role in infection control in the
hospital setting.
There is very little known about the interaction between respiratory viruses and bacteria in non-CF
bronchiectasis but a number of publications suggest that respiratory viruses may precipitate
secondary bacterial infection in CF. In a 25-year retrospective review from the Danish CF clinic,
the most likely first isolation of P. aeruginosa was found to be occur between October and March
[185], coinciding with the peak of the RSV season. This observation implies a causal relationship
between respiratory viral and bacterial infection.
The first bacterial isolation of a given organism in CF has also been shown to often follow a viral
infection. In the 17-month prospective study reported by COLLINSON et al. [171], five of the six first
isolations of P. aeruginosa were made during the symptomatic phase of an upper respiratory tract
infection or 3 weeks thereafter. In contrast only one of the six initial infections with P. aeruginosa
was identified during the asymptomatic period. Similarly, H. influenzae was recovered for the first
time from three children within 3 weeks of an upper respiratory tract infection and the one new
S. aureus infection was identified immediately following a viral infection.
J.E. FOWERAKER AND D. WAT
Interaction between bacteria and viruses
ARMSTRONG et al. [170] have reported that 50% of CF respiratory exacerbations requiring
hospitalisation are associated with the isolation of a respiratory virus. In their prospective study of
repeated BAL in infants over a 5-year period, a respiratory virus was identified in 52% of the
infants hospitalised for a respiratory exacerbation, most commonly RSV. 11 of the 31 hospitalised
infants (35%) acquired P. aeruginosa in the subsequent 12–60-month follow-up period, compared
with three out of 49 (6%) non-hospitalised infants (relative risk 5.8).
85
Respiratory viruses can disrupt the airway epithelium and precipitate bacterial adherence. For
example influenza A infection results in epithelial shedding to basement membrane with submucosal
oedema and neutrophil infiltrate [186], while both influenza and adenovirus have a cytopathic effect
on cultured nasal epithelium leading to the destruction of the cell monolayer [187]. This epithelial
damage results in an increase in the permeability of the mucosal layer [188, 189], possibly facilitating
the bacterial adherence. Bacteria can also utilise viral glycoproteins and other virus-induced receptors
on host cell membranes as bacterial receptors, in order to adhere to virus infected cells [190, 191].
The lower respiratory tract is protected by local mucociliary mechanisms that involve the
integration of the ciliated epithelium, periciliary fluid and mucus. Mucus acts as a physical and
chemical barrier onto which particles and organisms adhere. Cilia lining the respiratory tract
propel the overlying mucus to the oropharynx where it is either swallowed or expectorated.
Influenza viral infection has been shown to lead to the loss of cilial beat, and shedding of the
columnar epithelial cells generally within 48 hours of infection [192]. PITTET et al. [193] showed
that a prior influenza infection of tracheal cells in vivo does not increase the initial number of
pneumococci found during the first hour of infection, but it does significantly reduce mucociliary
velocity, and thereby reduces pneumococcal clearance during the first 2 hours after pneumococcal
infection at both 3 and 6 days after an influenza infection. The defects in pneumococcal clearance
were greatest 6 days after an influenza infection. Changes to the tracheal epithelium induced by
influenza virus may increase susceptibility to a secondary S. pneumoniae infection by increasing
pneumococcal adherence to the tracheal epithelium and/or decreasing the clearance of
S. pneumoniae via the mucociliary escalator of the trachea, and thus increasing the risk of
secondary bacterial infection.
MICROBIOLOGY
DE VRANKRIJKER et al. [194] showed that mice that were co-infected with RSV and P. aeruginosa had
a 2,000 times higher CFU count of P. aeruginosa in the lung homogenates compared with mice
that were infected with P. aeruginosa alone. Co-infected mice also had more severe lung function
changes. These results suggest that RSV can facilitate the initiation of acute P. aeruginosa infection.
RSV has also been shown to increase adherence of NTHi and S. pneumoniae to human respiratory
epithelial cells in vitro [195]. This increase adherence could be explained by an upregulation of cell
surface receptors for bacteria, such as intercellular adhesion molecule-1 (ICAM-1), carcinoembryonic adhesion molecule 1 (CEACAM1) and platelet activating factor receptor (PAFr). Another
study also showed that NTHi and S. pneumoniae bind to both free RSV virions and epithelial cells
transfected with cell membrane-bound G protein, but not to secreted G protein. Pre-incubation with
specific anti-G antibody significantly reduce bacterial adhesion to G protein-transfected cells [196].
STARK et al. [197] showed that mice that were exposed to RSV had significantly decreased
S. pneumoniae, S. aureus or P. aeruginosa clearance between 1 to 7 days after RSV exposure. Mice
that were exposed to both RSV and bacteria had a higher production of neutrophils induced
peroxide, but less production of myeloperoxidase compared with mice that were exposed to
S. pneumoniae alone. This suggests that functional changes in the recruited neutrophils may
contribute to the decreased bacterial clearance.
More recently, CHATTORAJ et al. [198] demonstrated that acute infection of primary CF airway
epithelial cells with rhinovirus liberates planktonic bacteria from biofilm. Planktonic bacteria,
which are more proinflammatory than their biofilm counterparts, stimulate increased chemokine
responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF
exacerbations.
Collectively, these findings suggest that respiratory viruses may lead to epithelial disruption,
destruction of mucociliary escalator, increased cytokine production, neutrophil influx and
increased neutrophil induced peroxide release, indirectly facilitating bacterial infection of the
airway. Whether these are the mechanisms for infective exacerbations in the context of non-CF
bronchiectasis remains to be seen.
Prevention and treatment of infection with respiratory viruses
86
Influenza associated death is between 13,000 to 20,000 incidents per year throughout the winter
months in the UK [199], though some of the deaths may be attributed to RSV. Influenza vaccines are
the only commercially available vaccines against respiratory viruses. Recent vaccines contain
antigens of two influenza A subtypes, strains of the currently circulating H3N2 and H1N1 (Swine
flu) subtypes, and one influenza B virus. The waning of vaccine-induced immunity over time
requires annual re-immunisation even if the vaccine antigens are unchanged. Influenza vaccination
is recommended to those with chronic respiratory diseases including non-CF bronchiectasis. Despite
this recommendation, there is neither evidence for, nor against, routine annual influenza vaccination
for children and adults with non-CF bronchiectasis from a recent Cochrane review [200].
Although there is no licensed RSV vaccine to date, prophylaxis using a humanised mouse
monoclonal antibody, Palizivumab, which has been shown to reduce the rate of RSV associated
hospitalisation in premature infants [201].
Amantadine has been the conventional anti-viral against of influenza. Its mode of action involves
interfering with viral protein M2, thereby inhibiting the replication of influenza viruses by
interfering with the uncoating of the virus inside the cell. However, it is strain specific as it is only
effective against influenza A and has common side-effects such as insomnia, poor concentration
and irritability. Amantadine has now been almost completely replaced by neuraminidase
inhibitors (NI), except for some NI-resistant influenza.
NIs such as Zanamivir and Oseltamivir are licensed for the treatment of influenza A and B, avian flu
(H5N1) and Swine flu (H1N1). They work by inhibiting the function of the viral neuraminidase
protein, thus preventing the release of the progeny influenza virus from infected host cells, a process
that prevents infection of new host cells and thereby halts the spread of the infection in the
respiratory. Early initiation of these therapies within 48 hours from the onset of symptoms can
reduce the duration of common cold symptoms by 1–2 days [202, 203]. Zanamivir has a poor oral
bioavailability and intranasal application has been shown to be effective in treating experimental
influenza infection, by the reduction in symptoms caused, virus shedding and the development of
otitis media [204]. A phase III study is currently underway that looks at the efficacy of intravenous
Zanamivir preparation. However, compassionate use of i.v. Zanamivir could be considered to treat
critically ill adults and children having a life-threatening condition, due to suspected or confirmed
pandemic Influenza A (H1N1) infection or infection due to seasonal Influenza A or B virus, who are
not responding to oral or inhaled neuraminidase inhibitors. A recent systematic review metaanalysis showed that neuraminidase inhibitors only have modest effectiveness (Oseltamivir and
Zanamivir 61 and 62%, respectively) against flu-like symptoms in previously healthy subjects [205].
Ribavarin
Ribavarin, a synthetic guanosine nucleoside that has a broad spectrum of antiviral activity, is
approved treatment for lower respiratory tract disease caused by RSV [206]. It can be incorporated
into RNA as a base analog of either adenine or guanine, it pairs equally well with either uracil or
cytosine, inducing mutations in RNA-dependent replication in RNA viruses. Controlled studies
also show that the use of ribavarin is effective in reducing the clinical severity score, duration of
mechanical ventilation, supplemental oxygen use and days of hospitalisation [207].
J.E. FOWERAKER AND D. WAT
Neuraminidase inhibitors
Macrolides
Although rhinovirus is the major cause of colds, its vast amount of serotypes has made
development of anti-virals against it problematic. 90% of rhinovirus serotypes gain entry into
epithelial cells using ICAM-1 cellular receptors and blockade of these receptors in experimental
studies have shown reduced infection severity [208]. Macrolide antibiotics, bifilomycin A1 and
erythromycin, have been shown to inhibit ICAM-1 epithelial expression and hypothesis about
their potential as anti-virals have yet to be proven, more clinical proof is required [209].
Other anti-virals
87
Recently there has been a report regarding the use of an anti-rhinoviral agent known as Plecoranil.
This anti-viral binds to a hydrophobic pocket of the VP1, the major shell protein for the
rhinoviruses, thereby preventing the virus from exposing its RNA and also prevents the virus from
attaching itself to the host cell [210]. The rhinovirus 3C protease inhibitors, Ruprintrivir [211] and
soluble recombinant ICAM-1 Tremacamra [212], have shown promising results but they are
currently not widely available.
Conclusions
The role of bacteria and viruses in non-CF bronchiectasis is not presently fully understood.
Through necessity, evidence from studies in CF and COPD is used and applied to bronchiectasis.
More research using both conventional microbiological techniques as well as newer molecular
diagnostic approaches, is urgently required to address a number of important questions in non-CF
bronchiectasis. 1) What is the cause of infective exacerbations? 2) What is the role of anaerobic
bacteria and how do normal commensal bacteria interact with pathogenic bacteria? 3) How can we
clear chronic infection? 4) What proportion of exacerbations is triggered by viral infection?
5) How do viruses influence bacterial behaviour in chronically infected airways?
A greater understanding of bacterial communal behaviour and their interaction with epithelial
cells and viruses will be critical in further developments in the management of non-CF
bronchiectasis.
MICROBIOLOGY
Statement of interest
J.E. Foweraker received a consultancy fee from Novartis Pharma AG for advice on a submission to
the European Medicines Agency for licensing of Tobramycin inhaled powder and a consultancy
fee from Gilead Sciences International Ltd for advice on an application to European Medicines
Agency for licensing of Aztreonam lysine.
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