Chapter 6

Microbiology of non-
CF bronchiectasis
J.E. Foweraker* and D. Wat#

Summary
*Dept of Microbiology, and
Non-cystic fibrosis (CF) bronchiectasis is a complex disorder #
Lung Defence Unit, Papworth
Hospital, Cambridge, UK.
characterised by recurrent chest infections and poorly regulated
respiratory innate and adaptive immunity. These lead to a Correspondence: J.E. Foweraker,
Dept of Microbiology, Papworth
‘‘vicious cycle’’ of impaired mucociliary clearance, chronic Hospital, Papworth Everard,
infection, bronchial inflammation and progressive lung injury. Cambridge, CB23 3RE, UK, Email
juliet.foweraker@papworth.nhs.uk
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
MICROBIOLOGY

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 non-
CF 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.
Eur Respir Mon 2011. 52, 68–96.
Keywords: Anaerobes, Haemophilus, Moraxella, Pseudomonas, Printed in UK – all rights reserved.
Copyright ERS 2011.
Streptococcus, viruses European Respiratory Monograph;
ISSN: 1025-448x.
DOI: 10.1183/1025448x.10003610

P atients with bronchiectasis are commonly colonised with potentially pathogenic microorgan-
isms 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].
68

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.

Range of bacteria in patients with non-CF bronchiectasis
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

J.E. FOWERAKER AND D. WAT
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
H. influenzae has been reported in 14–52% of patients with non-CF bronchiectasis. It is a Gram-
negative 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.
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
69

in 1 year. GPB: Gram-positive bacilli. coli 2 (4) GPB Other the upper respiratory tract.] molecules. a com- 18 (24) RTFlora 1 (1) A. pneumoniae: Klebsiella 12 (8) S. Range of microorganisms cultured from patients with non-cystic fibrosis bronchiectasis aeruginosa tion [9]. PSB: protected specimen brush. PPM: potentially pathogenic microorganisms. A. NTHi 10 (20) 12 (16) 38 (31) 11 (12) 46 (31) 62 (43) 47 (33) 8 (9) were found in higher numbers (. influenzae was found in some patients and per- Subjects 123 150 143 92 50 75 89 sistence of the same clone in other n individuals [19]. mean¡SD or n (%). In patients with COPD the presence of NTHi in 19 (21) no PPM sputum was associated with raised 9 (18) RTFlora RTFlora/ no PPM 28 (20) inflammatory cytokines. ": bacteria were isolated on at least two occasions. maltophilia: Stenotrophomonas 2 (1) S.106 MICROBIOLOGY colony forming units (CFU)?mL-1) during exacerbation compared with when the patient was stable. This suggests 57. pneumonia Data are presented as mean (range). xylosoxidans 7 (14) K. xylosoxidans 2 (1) A. xylosoxidans 4 (3) A. They PALWATWICHAI [6] can adhere to mucus and to various MACFARLANE [11] PASTEUR [10] First author cell types in the human respiratory NICOTRA [8] ANGRILL [7] tract using pili and other adhesion KING [9] ZAID [5] [ref. influenzae and direct analysis of ND ND ND ND ND amplified DNA from sputum showed persistence of the same strain over prolonged periods [18]. suggest- aureus 14 (15) 21 (14) 39 (27) 15 (10) 2 (3) 9 (7) 3 (4) ing that even if present in the lower tract it does not have a direct patho- genic role [14. unless otherwise stated. whereas pa- Patients with organisms cultured from respiratory tract# ND ND ND tients with H. A prospective study in exacerbation COPD using molecular typing of Stable or at subgroup" Colonised Stable Stable H. GNB: Gram-negative bacilli. In COPD patients. ND: not described. coli 2 (2) E. S. Virulence factors include the endotoxin lipo-oligosaccharides 70 . pneumoniae catarrhalis There is little published data on the 30 (20) 39 (27) 9 (10) 2 (4) 3 (4) 3 (2) 7 (8) 9 (6) epidemiology of H. #: some had more than one PPM cultured. It may be cultured repeatedly from the same maltophilia. coli: Escherichia coli. and influenzae 50 (46) 24 (32) 37 (30) 42 (47) 52 (35) 75 (52) 47 (33) 7 (14) exacerbations in COPD may be as- sociated with the appearance of a new strain [16. parainfluenzae in spu- tum had similar levels of cytokines to those who had no microorganisms Haemophilus Pseudomonas Streptococcus Moraxella Staphylococcus cultured from their sputum. Haemophilus parainfluenzae. 17]. maltophilia 42 (30) Coliforms 2 (3) Anaerobes 13 (9) Coliforms mon commensal organism found in 16 (13) GNB 7 (14) GNB 1 (1) E. xylosoxidans 1 (1) A.6 in the lung or persistence in the upper respiratory tract with repeated deposition followed by clearance from Sample Sputum Sputum Sputum Sputum Sputum Sputum PSB the lung.2¡16. Country Australia Thailand Ireland NTHi have various properties that Spain USA UK UK can help explain their pathogenicity and ability to persist in the lung. influenzae in non-CF bronchiectasis. but with- 34 (37) 13 (11) 20 (13) 42 (30) 13 (9) 3 (6) 6 (8) 6 (7) out typing data it is not known if this is the persistence of a single strain or repeated episodes of infec- Table 1. may be cultured from sputum but was not found in PSB samples. 15]. RTFlora: upper respiratory tract flora. 3 months apart. sequential infection with different strains of H.7 58 (30–85) 58 (16–76) Age yrs 57¡14 (16–90) either long-term colonising infection . K.18 60. In CF. xylosoxidans: Achromobacter xylosoxidans: E. patient over several years. maltophilia pneumoniae.

P2.(LOS). direct DNA uptake- transformation. Some NTHi have a higher than usual mutation rate due to a mutation in mutS. WAT the chinchilla model of otitis media [28. 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.g. NTHi possess mechanisms to vary the structure and activity of LOS and these may explain variations in the pathogenicity of different isolates [21]. Alternatively.g. NTHi in biofilms were more resistant to antibiotics in vitro. Hypermutators are generally rare in acute infection but hypermutable H. e. 29]. influenzae grown in vitro. It may also take place in the lower respiratory tract. NTHi may be able to evade the immune response by varying its surface antigens. and altered gene content by mutation or by horizontal gene transfer. NTHi were able to enter cultured nonciliated respiratory epithelial cells and cross the respiratory epithelium [26]. 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. by phase variation. influenzae to generate phenotypic diversity include altered gene expression. Mechanisms include phase variation of LOS [24]. influenzae in non-CF bronchiectasis has yet to be assessed. Quinolone resistance is now recognised and resistance rates to trimethoprim and tetracycline are rising. which is one of the methyl-directed mismatch repair genes (MMR) that corrects errors in DNA. NTHi were identified inside cells in bronchial biopsies taken from patients with COPD [27]. has been observed in persistent infections in patients with COPD [25]. 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. or via bacteriophage [23]. 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. However. and changes to outer membrane proteins (OMP) either by horizontal gene transfer or point mutations of the immuno-dominant OMP. H. as many random mutations can reduce bacterial fitness. the hypermutable state may become beneficial to the bacterial population. and immunoglobulin (Ig)-A protease [20]. Antigenic drift. 32].e. influenzae were also found in bronchoalveolar lavage (BAL) samples from children with CF [30]. ampicillin) either due to production of b-lactamase or alteration of penicillin binding proteins. have shown that the organism is able to adapt to oxidative stress and limited nutrients [22]. Mechanisms used by H. It is an opportunistic human pathogen that can cause severe.E. influenzae have been found in patients with CF and these strains have more resistance to antibiotics compared with normo-mutators [19. The prevalence and role of hypermutable H. Structures consistent with biofilms containing H.g. aeruginosa is a versatile nonfermentative Gram-negative bacillus that is found in a range of environments. amoxicillin. Using in situ hybridisation. resistance may result from gene mutation. Pseudomonas aeruginosa P. The prevalence of antibiotic resistant NTHi increases over time in patients with non-CF bronchiectasis [9]. This hypermutability is not usually thought to be advantageous. Sub-inhibitory concentrations of azithromycin were found to reduce the size of both growing and established biofilms [31]. 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. and NTHi biofilms were seen in J. if mutations lead to antibiotic resistance. Studies comparing the proteome of H. 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. either in pooled sputum or chemically defined media. NTHi cultured from CF patients could form biofilms in vitro and on the surface of cultured airway epithelial cells. i. acute and invasive 71 . FOWERAKER AND D. NTHi from patients with COPD can form biofilms in vitro. Many are resistant to amidopenicillins (e. e. resulting from change in the P2 gene. This is thought to be because H. influenzae (along with other bacteria infecting a bronchiectatic lung) may exist in biofilms in the respiratory tract. which may be polymicrobial in non-CF bronchiectasis.

37]. of which six had CF. the mechanisms of pathogenicity and the genotypic and phenotypic changes in chronic infection have been extensively studied in CF. LES is now the most common epidemic strain in the UK affecting as many as 11% of patients in England MICROBIOLOGY and Wales [41]. There is debate over whether infection with P. More recently there have been reports in several countries of cross- infection between CF patients with what are termed ‘‘epidemic’’ strains. Chronic infection is therefore rare in COPD. aeruginosa have the typical phenotype of isolates causing acute infections and environmental strains [34]. often for many decades [36. 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]. aeruginosa can lead to an accelerated deterioration in lung function. As chronic infection with P. It is one of the most common causes of infection in non-CF bronchiectasis and other chronic lung diseases. aeruginosa is one of the most common isolates found in 12–43% of non-CF bronchiectasis patients (table 1). However. P. suggesting a common route of adaptation to chronic infection in the lung. or whether it is a marker of more damaged lungs [47. but may also be important in severe COPD. 40]. The epidemiology of P. Some siblings shared strains but it was not known whether this was cross-infection or exposure to a common environmental source. P. aeruginosa and symptoms of an exacerbation. reduced protease production and increased antibiotic resistance. aeruginosa in COPD P. aeruginosa. While there are some mixed infections. A recent study compared long-term colonisation with P. P. particularly those requiring mechanical ventilation for severe exacerbations. antibiotic treatment regimens were developed to clear early infection and delay the onset of chronic infection [35]. most CF patients carry a single genotype of P. Early studies in CF showed that individual patients were infected with distinct strains that were thought to have been acquired from the environment. aeruginosa. with fewer publications in non-CF bronchiectasis and COPD. suggesting repeated episodes of acquisition. CF patients eventually developed a persistent infection that seldom cleared despite aggressive antibiotic therapy. similar to those seen in CF [44]. Some. prior antibiotics and a low forced expiratory volume in 1 second (FEV1) [42]. aeruginosa in non-CF bronchiectasis P. aeruginosa [38]. and exacerbations do not appear to be due to the acquisition of a new strain of P. but when it does occur P. aeruginosa has a range of colony forms (morphotypes) and adaptations including increased mutability. P. infections. in particular the Liverpool epidemic strain (LES). of interest. aeruginosa from one or more sputum culture. Only 13 patients had carriage of the same clone for more than 6 months with four patients infected with mucoid strains. aeruginosa in 21 patients. two-thirds of new infections that later cleared from the sputum. aeruginosa infection was associated with steroid use. most notably CF. 10 had non-CF bronchiectasis and five had COPD. have been associated with increased morbidity [39. did so without the use of specific antibiotic treatment [43]. 48]. In a study of 126 patients with moderate-to- severe COPD over an 11-year period. 39 patients grew P. aeruginosa leads to a faster decline in lung function as is seen in CF. Stable patients with P. aeruginosa has been cultured from 4–15% of patients with COPD and was more prevalent in patients with advanced disease. The authors typed 125 sequential 72 . reduced motility. These early P. aeruginosa in CF Early infections in CF are caused by genotypically distinct isolates. There was a significant association with the culture of a new strain of P. such as necrotising ventilator-associated pneumonia and infections in immuno- compromised patients often with bacteraemia [33]. There are many similarities between the infections in these different conditions.

Imipenem induces the AmpC b-lactamase. leading to high-level resistance. but there has been one report of a patient with non- CF bronchiectasis acquiring LES from a relative with CF [50]. although their expression may differ between isolates that cause acute infection and those responsible for chronic infection. WAT by horizontal transfer [56]. can result in resistance to nearly all b-lactam antibiotics except the penems. aeruginosa possesses a range of virulence factors. More resistance can result from an increase in an efflux pump that can remove the meropenem from the cell.E. Antibiotic resistance and its regulation can be complex in P. aeruginosa from antibiotics by a variety of mechanisms [57]. Efflux mechanisms often affect more that one class of antibiotics and therefore contribute to multi-drug resistance [56]. Type I and type II secretion systems export protein toxins. Flagella. 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 J. together with other adaptations. aeruginosa P. 2) modifying the antibiotic target. pyocyanin and hydrogen cyanide [53. Both mechanisms may be present and additive. In CF the prevalence of resistant P. 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. aeruginosa can develop resistance by either: 1) producing enzymes that destroy the antibiotic. There have been no studies using genomic typing methods to investigate whether patients with non- CF bronchiectasis share strains of P. with a dominant persistent clone. aeruginosa is increasing as a result of repeated antibiotic courses. Other extra-cellular virulence factors include rhamnolipids. Pathogenicity of P. Enzymes that can destroy meropenem (penemases such as VIM) do occur but are currently rare [56]. aeruginosa. which contributes to the persistence of the organism rather than acute tissue damage and. These are called derepressed mutants and offer a survival advantage. promotes chronic infection (refer to later section). The authors found a similar pattern of colonisation in all three diseases. P. showing that the pattern of infection found in CF could also be shown in other conditions [49]. type IV pili. Antibiotic resistance P. Another pathogenicity factor is the ability to form alginate-enhanced biofilms [55]. such as alkaline protease. aeruginosa from patients with non-CF bronchiectasis. AmpC codes for an inducible cephalosporinase which. such as AmpC b-lactamase. FOWERAKER AND D. carbapenemases or aminoglycoside modifying enzymes. or 3) reducing exposure either by a decrease in permeability or increased removal of the antibiotic from the bacterial cell (efflux). exotoxin A and phospholipase C. such as gyrA for quinolone resistance. For example. elastase. Treatment with piperacillin or ceftazidime can lead to the selection of bacteria that produce the enzyme constitutively rather than just on induction. Little has been published specifically on antibiotic susceptibility of P. 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. low level resistance to meropenem may be due to reduced permeability following changes to the membrane porin OprD. lipopolysaccharide and exopolysaccharides contribute to the adherence to cells and surfaces. aeruginosa and various mechanisms that affect resistance to a single antibiotic may be present in the same organism. 52]. In addition. The regulation of ampC is exceedingly complex and 73 . Interestingly. and it also induces genes involved with alginate production [59]. when production is increased. 54]. even though it is not affected by the enzyme. the biofilm mode of growth also protects P. while type III secretion systems inject exoenzymes directly into eukaryotic cells.isolates from sputa taken at least 1 month apart.

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. resulting from various gene defects including reduced or abnormal expression of MexB and OprM [66]. ‘‘dwarf’’ forms and very slow growing ‘‘small colony variants’’. even though some of the P. is intimately linked to cell wall recycling [60–62]. mucoid forms. pyoverdin. aeruginosa would normally be intrinsically resistant. 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. The phenotypic changes found in chronic infection have been studied extensively in CF but not in non-CF bronchiectasis. AmpR does not just regulate ampC but is a global transcriptional regulator that regulates another b-lactamase PoxB. These can include colonies lacking the typical pigmentation. aeruginosa are resistant to the antibiotic used [64]. quorum sensing and other virulence factors [63]. For example. Small colony variants (SCVs) have enhanced ability to form biofilms and may also contribute to persistence [73]. Several different morphotypes may be found in the same sputum. 49. aeruginosa may be hyper-susceptible in vitro to some anti-pseudomonal antibiotics and susceptible to agents such as tetracycline and chloramphenicol to which P. MexXY uses the same exit duct OprM and can export aminoglycosides. Hyper-alginate producers were ori- ginally thought unique to CF but they are also found in non-CF bronchiectasis and COPD [45. MexXY-OprM over-expression may be due to mutation in the regulatory gene mexZ and/or to mutations in the MexXY translocase genes [66]. It is unclear why this phenomenon exists. factors. piperacillin. some that look like coliforms. 72]. P. 1). Antibiotic resistance may arise from an increase in the efflux pump activity. secretion [74]. Antibiotic resistance may therefore be associated with a change in virulence and/or fitness. Alginate may protect against phagocytosis [70] and contribute to the formation of biofilms [71. e. ceftazidime and tetracycline [65]. hydrogen cyanide) and type III found in a single sputum sample taken from a chronically infected individual. Some mutations can reduce biological competitiveness and more work is needed to assess the link between antimicrobial resistance and fitness [61]. Many virulence factors are regulated by the quorum sensing (QS) system. elastase. aztreonam. even though the isolates are clonally related. such as toxin production (e. due to mutation in the regulatory genes. One of the most easily recognised is the mucoid morphotype. This has been described in chronic infection in both CF and non-CF bronchiectasis [67. phos- logical types of Pseudomonas aeruginosa pholipase C.g. This results from over-production of the polysaccharide alginate. substrates for efflux pump MexAB-OprM include ticarcillin. Adaptations to chronic infection MICROBIOLOGY One of the characteristics of chronic infection with P. 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. They include loss of acute virulence Figure 1. These are signalling 74 . Conversely. cefepime and ciprofloxacin. but the clinical relevance of these findings has not been investigated. Different pseudomonal morpho. as well as proteases. They are thought to be an adaptation to chronic infection irrespective of the underlying cause. 68]. P. 69]. It was found that 25 out of 46 CF patients had strains hyper-susceptible to ticarcillin due to deficiencies in MexAB-OprM efflux activity.g. This could explain why some CF patients respond to treatment for acute exacerbation. SCVs have only been described so far in CF but are easily missed unless cultures are prolonged.

Las R mutants form characteristic iridescent colonies and have also been cultured from patients with non-CF bronchiectasis (J. personal communication). aeruginosa in biofilms from interferon (IFN)-c activated macrophages [79]. and only have their effect when the number of organisms reaches a critical concentration (or quorum). 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. Foweraker.molecules that act on the regulators of gene transcription. but it can block QS and alginate polymer formation in vitro [83]. such as Candida spp. Neutrophils have been observed immobilised in the extra-cellular matrix. Azithromycin is a macrolide antibiotic that does not directly inhibit or kill P. They can affect a large number of functions including pathogenicity. Multiple phenotypic variants of the underlying clonal population of P. Longitudinal studies in CF have analysed strains from patients over several years. If P. Neutrophil lysis is thought to be caused by rhamnolipid. These mutants do not produce the toxins elastase. They therefore appear to be less pathogenic but better able to adapt to the local environment. different phenotypes can co-exist so sputum may contain Las R mutants and organisms without the mutation. UK. Papworth Hospital. Biofilm fragments have been seen in CF sputum [77] and may contain a mixture of P. 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. aeruginosa with mutations in QS genes. The biofilm contains a steep oxygen gradient and is anaerobic just below the surface. aeruginosa plus proteins and DNA from other microorganisms and host cells. do not respond to QS molecules and are surprisingly common. Rhamnolipid may therefore help to protect the biofilm from disruption by neutrophils. [76]. phenazines or hydrogen cyanide. by which the bacteria possess a variety of adaptations that enable them to survive within these microniches [78]. It can disrupt early biofilms formed by nonmucoid strains but has less effect on early biofilms formed by hyper-alginate producers 75 . Cambridge. as biofilms formed in vitro in the presence of neutrophils are thicker and contain more bacteria [81]. Las R mutants can use a more diverse range of compounds as a source of carbon. metabolic adaptation and persistence [75]. Biofilms J. the neutrophils are intact and the P. Therefore. especially in the early stages of formation [82]. aeruginosa and neutrophils are combined. aeruginosa. Different concentrations of nutrients and waste products will also be found in different areas of the biofilm. the bacteria aggregate around necrotic dying neutrophils. 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]. Some QS molecules depend on population density.E. aeruginosa is thought to grow in biofilms in chronic infections in both CF and non-CF bronchiectasis. If neutrophil apoptosis is induced before the bacteria are added. aeruginosa under QS control. the biofilm contains a wide range of physiological conditions. and these may form the framework for the biofilm. unable to penetrate the biofilm [80]. they were found in 19 out of 30 CF patients in a study by SMITH et al. aeruginosa remain dispersed. FOWERAKER AND D. aeruginosa QS molecules comprise acyl-homoserine lactones and molecules of the PQS system. aeruginosa plus other bacteria and even fungi. It is thought that neutrophils may actually enhance early biofilm formation. Again. phosphorus or sulphur and have a growth advantage over the wild type when grown with phenylalanine. isoleucine or tyrosine. aeruginosa co- exist and form a complex population in the chronically infected lung. 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].E. a toxin produced by P. Neutrophils can release DNA and F-actin complexed with histones and other cations. P. most frequently las R. Alginate protects P. P. as individual organisms that have the necessary adaptation may already be present in the population. The extra- cellular matrix comprises alginate produced by P. nitrogen. WAT P.

76 . 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. 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. HM P. Other antibiotics may also influence bacterial virulence. suggesting that hyper- mutability is a general adaptation to long-term survival in the lung [93]. If the AmpC b-lactamase is over produced by some P. aeruginosa had reduced virulence and fitness both in vitro and in an animal model [97. KENNA et al. [94] suggests that hypermutability is an extremely rare finding in environmental P.g. and an anionic antibiotic. P. tobramycin) may bind to the alginate. whereas colistin can kill the non-dividing cells in the centre [88]. such as antimicrobial resistance (referred to as ‘‘hitchhiking’’). Ciprofloxacin kills organisms on the surface of the biofilm but cannot kill those deeply set within the biofilm. This was the highest prevalence that had been described for a naturally occurring population [90]. 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. These are P. such as an aminoglycoside (e. aeruginosa were found in 37% of chronically infected CF patients. 98]. most commonly mutS and mutL. P. Positively charged antibiotics such as colistin may bind to free anionic DNA and therefore not reach the bacteria. aeruginosa usually mutates at a frequency of one in 108–109. The range of metabolic conditions in the biofilm may affect antibiotic susceptibility. in changing environments or stressful conditions HM bacteria may be selected because they have adaptive mutations. 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. such as tobramycin. while mutation rates in HM bacteria can be as high as one in 100. had previously been considered high [91]. cannot act on organisms that are metabolising anaerobically. The function of these systems is to detect and repair DNA replication errors and repair oxidative damage. aeruginosa were described in 57% of chronically infected patients with COPD or non-CF bronchiectasis. However. Mutability is increased in biofilms. These affects have been shown in an in vitro model of a young biofilm in a flow chamber using live/dead staining. MMR also inhibits recombination between moderately diverged sequences and therefore reduces the acquisition of exogenous DNA through horizontal gene transfer [96]. HM P. aeruginosa with a higher than usual spontaneous mutation rate and are thought to accelerate bacterial evolution. In a longitudinal study of CF patients in Denmark. In comparison a HM prevalence of 1% in Escherichia coli and Salmonella spp. Hyper- mutability usually results from a primary mutation in genes of the MMR system. aeruginosa and in isolates from newly infected CF patients. Several mechanisms have been proposed to explain this resistance [86]. HMs are uncommon in most bacterial populations because many of the mutations are deleterious. Organisms within a biofilm may become dormant and therefore resist quinolones and b-lactam antibiotics [87]. For example ciprofloxacin can suppress alginate biosynthesis at concentration well below minimum inhibitory concentration (MIC) [85]. or defects in the GO system (mut M. Y and T). 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. aeruginosa [92]. Azithromycin also has an anti-inflammatory effect in chronic lung infection and the relative importance of its diverse actions is yet to be established. Bacteria cultured in biofilms in vitro are more resistant to most antibiotics than when they are dispersed (planktonic). Most of the information on HM P. (mucoid strains) or on established biofilms [84]. aeruginosa comes from work on isolates from CF [95]. aeruginosa it may form a high local concentration and protect bacteria that can only produce basal levels of the enzyme. aeruginosa in CF. HM P. MICROBIOLOGY 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].

It is currently unclear whether these findings can equally be applied to chronic infec- tion in non-CF bronchiectasis. pneumoniae is a Gram-positive coccus appearing in pairs and in short chains. once a chronic infection is established the range in the antibiotic susceptibility of P. Bacteria with the same morphotype may have different susceptibility and therefore resistant sub- J. WAT populations may be missed. Therefore. A variety of methods are being developed for testing biofilm susceptibility. aeruginosa in a single sputum is so diverse that susceptibility testing methods are unreliable [102. Colonies of P. 100]. aeruginosa and some of the other nonfermenting Gram-negative bacilli found in chronic infection. Two recent studies have shown that CF patients with HM had poorer lung function (FEV1 predicted). aeruginosa from non-CF bronchiectasis is that some isolates may be difficult to identify. aeruginosa infection and the clinical microbiology laboratory One practical implication of the range of phenotypic diversity of P. however. both being the result of prolonged infection [99. grow very slowly and may mimic other species. depending on which colony is picked for testing [68]. The susceptibility is proportional to the S. Work is needed on the role of HM in non-CF bronchiectasis. non-CF bronchiectasis and COPD have shown that HM are more likely to be antibiotic resistant than isolates with normal mutation rates [93. FOWERAKER AND D.25 per year in non-mutators. or lower airway infections Figure 2. aeruginosa from chronic infection may lack pigmentation. In a study of 29 CF patients over a 5-year period. The figure in patients with damaged lungs such shows the results from testing two bacteria with identical colony as non-CF bronchiectasis or COPD. Streptococcus pneumoniae S. and therefore identification methods. 103]. Chronic P. and several studies in CF. HM had more mutations leading to antibiotic resistance but also more mutations in other genes such as lasR [89]. 99]. aeruginosa in a single sputum sample (fig. but longitudinal studies are needed to determine if this was due to infection with a HM or just an association. form in sputum taken from a patient with non-cystic fibrosis but it is rare in CF. their clinical relevance still needs to be determined. 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.E. The same six different antimicrobial discs were used for both cultures. such as species-specific PCR or sequencing of the 16S ribosomal RNA gene may be required [101]. to 37% of patients with non-CF 77 . other adaptations may provide a selective advantage for HM isolates.The sequential acquisition of resistance to multiple antibiotics is seen in infection with P. In CF. It can also cause otitis media or sinusitis. Commercial identification schemes that use biochemical reactions and assimilation tests are not reliable in identifying atypical P. Variation in antimicrobial susceptibility testing. pneumoniae can be found in up diameter of the zone of diffusion around the antibiotic disc. mutations accumulated at an average mutations rate of three per year in HM P. aeruginosa compared with 0. not just antibiotic resistance. It may be a harmless commensal in the oro-pharynx but can cause severe and invasive dis- ease (pneumonia or meningitis). Another consequence of phenotypic diversity is that a range of antimicrobial susceptibility patterns can be found in a population of P. aeruginosa in CF. 2). Although bronchiectasis.

More work is needed to understand the pathogenesis of infection in both COPD and non-CF bronchiectasis. pneumoniae can use a wide variety of molecules to adhere to host cells and produces an IgA protease and a toxin. and isolates lacking the capsule are avirulent. S. inhibition of cilial beating. it can be cultured in significant numbers from sputum or PSB in up to 27% of patients with non-CF bronchiectasis [7]. catarrhalis is a Gram-negative diplococcus that was previously named Branhamella or Neisseria catarrhalis. pneumoniae may be a harmless commensal or cause non- invasive respiratory tract infection (in COPD or bronchiectasis) or produce severe invasive disease with bacteraemia. suggesting that there was an effective immune response. therefore. Acquisition of M. pneumoniae has been cultured from both stable patients and those with exacerbation [20. amoxicillin–clavulanate is ineffective. 110].e. A polyvalent vaccine containing the most common serotypes is available and recommended for use in patients with chronic lung disease. 78 . However. hypermutability and the ability to form biofilms [109. Putative virulence factors of M. amoxicillin-clavulanic acid and quinolones [115]. [106]. inflammation and tissue damage [108]. serum resistance and biofilm formation [114]. It is also considered a significant pathogen in COPD but is only rarely isolated in CF. Of the new acquisitions. catarrhalis led to an increase in airway inflammation. Pneumolysin is proinflammatory and has many actions including cytolysis. 104]. No association between strain acquisition and exacerbation was found and as M. S. characterised by a rise in sputum neutrophil elastase. i. pneumolysin that can promote invasion. bronchiectasis. 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]. and direct activation of the classical complement cascade. the average time from acquisition to clearance of a new strain of M. S. Although it is not a common pathogen in CF. tetracyclines. The prevalence of antibiotic resistant S. influenzae or S. More than 90% of M. very little has been published on its role in this condition. catarrhalis was 1 month and re-infection with the same strain was rare. pneumoniae from CF sputum have characteristics that may be associated with adaptation to persistence in the lung. In a study of 50 patients with COPD. isolates of S. pneumoniae [105]. pneumoniae has increased and in some countries very high rates of resistance to penicillin. A longitudinal study of M. macrolides and tetracyclines limit the treatment options. Like NTHi it is a common commensal organism in the upper respiratory tract and can cause otitis media or sinusitis. catarrhalis in 29 patients with non-CF bronchiectasis found that patients were colonised with a variety of strains with average colonisation duration of 2. pneumoniae). catarrhalis produce a b-lactamase (BRO-1 or BRO-2) and are resistant to ampicillin. catarrhalis was often in mixed culture with other PPMs (H. MICROBIOLOGY Moraxella catarrhalis M. The patient with non-CF bronchiectasis due to an underlying antibody deficiency may be particularly susceptible to recurrent infections with S. Acquired resistance to other antibiotics is rare with most remaining susceptible to macrolides.3 months for each strain. it was difficult to determine whether it had an independent pathogenic role [111]. epithelial cell invasion. 47% were associated with an exacerbation [112]. Further work is needed to clarify the role of the different virulence factors in order to understand why S. pneumoniae has a polysaccharide capsule that helps evade opsonisation. In COPD. interleukin (IL)-8. catarrhalis include several outer membrane proteins plus LOS and these affect cell adhesion. It was not reported in studies of non-CF bronchiectasis in the 1960s as it was considered an oral contaminant rather than a PPM. Penicillin resistance is due to modifications to penicillin binding proteins not by the production of a b-lactamase and. tumour necrosis factor (TNF)-a and a reduction in secretory leukocyte protease inhibitor (SLPI) [113]. Bronchiectasis in primary and secondary immunodeficiency patients is discussed further in the chapter by BROWN et al.

aureus produces a range of exotoxins that can cause tissue damage. As with other species with high mutation rates.E. They can be difficult to treat. 79 . because of the need to prevent cross infection.Staphylococcus aureus S. 124]. Pandoraea and Inquilinus can cause infection in the CF lung. S. 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]. aureus (MRSA) are resistant to all penicillins. but can cause severe pneumonia after influenza. They can be difficult to identify in the laboratory and molecular methods are recommended to ensure accurate identification [101]. infection. A higher proportion of hypermutable strains of S. Persistence of S. It is also thought to form biofilms on prosthetic devices and thereby evade the host response and resist antimicrobial therapy [117]. aureus in CF and prosthetic infections has also been related to the presence of small colony variants. In particular it is important to differentiate these organisms from the Burkholderia spp. and their propensity for spread between CF patients. many of these had defects in mutS [121]. but there is data from CF that shows that a combination of systemic treatment with skin antisepsis and inhaled antibiotics may be effective [122]. fluoroquinolones and aminoglycosides). They are associated with treatment with trimethoprim/sulphamethoxazole or aminoglycosides. It may be difficult to clear MRSA carriage from patients with bronchiectasis. are more antibiotic resistant than the typical forms in the same sputum and may survive within host cells [119]. It is a rare cause of respiratory tract infection. Infection is characterised by abscess formation. one with Burkholderia cepacia complex (not speciated) and another with Burkholderia gladioli [123. Ralstonia. cephalosporins and penems and are often also resistant to other classes of antibiotics (macrolides. maltophilia and Achromobacter (previously Alkaligenes) xylosoxidans have been reported in non-CF bronchiectasis (table 1). aureus surrounded with the polysaccharide poly-N- acetyl-glucosamine have been observed in anaerobic conditions in CF mucus and can resist nonoxidative killing [118]. and S. 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. there are only two case reports of infection in non-CF bronchiectasis. In spite of the frequent presence of these bacteria in the environment. aureus were found in CF patients when compared with isolates from bacteraemia or other respiratory infections. The ability of S. aureus with ABPA in non-CF bronchiectasis [116]. J. aureus to rapidly adapt and persist in the lung may be a result of genomic instability due to mobilisation of bacteriophages. 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. A wide variety of other nonfermentative Gram-negative bacilli can occasionally act as opportunistic pathogens in the human lung. Stenotrophomonas. WAT Meticillin resistant S. aureus is a Gram-positive coccus found in clusters that may be part of the normal flora in the anterior nares. particularly in skin and soft tissues. These tiny colonies are difficult to identify in vitro. There is also an association of S. or role in exacerbation of non-CF bronchiectasis. Biofilm-like aggregates of S. Species of the genera Achromobacter. Burkholderia spp. There is too little experience with these microorganisms to comment on their propensity for colonisation. FOWERAKER AND D. throat and on moist skin sites such as groin and axilla. Isolates from the anterior nares of CF patients had a higher frequency of genomic alterations than those from healthy controls [120]. Many are both intrinsically resistant to some antibiotics and easily acquire resistance.

Obligate anaerobes in particular Prevotella spp. This assumption has been challenged following studies using both conventional culture methods and culture-independent techniques. Culture-independent studies of microbial flora in the lung There have been attempts to describe the composition and diversity of the microbes in the lung irrespective of the ability to culture individual microorganisms. investigators began to look for anaerobes in the sputum from CF and non- CF bronchiectasis [125]. they were able to enhance the MICROBIOLOGY virulence of P. but also because some are very difficult to culture. 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 co- habitants [132]. the oral anaerobe Prevotella also produces AI-2 [127]. The CF lung. While not intrinsically pathogenic. 134–136]. This is present in all true bacteria and the sequence variation is sufficient to identify most genera and many species. It has been proposed that members of the Streptococcus milleri group may have a role in chronic lung infection. Their significance in disease has been questioned as high numbers of bacteria were found in a stable CF patient in one study. a Staphylococcus sp. Genetic material can be extracted from a clinical sample. those that behave as commensals and those that are not directly pathogenic but may increase the virulence of other organisms. These methods and other variations have been applied to patients with CF and non-CF bronchiectasis to describe the diversity of microorganisms. far more than would be expected from oral contamination [126–128]. were found in significant numbers. may contain a mixture of microorganisms that includes those that are directly pathogenic. This compares the size of the terminal fragment of rRNA after cutting with a restriction enzyme. While the presence of nucleic acid does not necessarily indicate the presence of viable organisms. The product may be analysed looking at terminal restriction fragment length polymorphisms (TRFLP). milleri group as of potential importance in exacerbations both in CF and in two patients with non-CF bronchiectasis [130]. microorganisms other than PPMs are regularly observed in sputum cultures in combination with PPMs and further work on these potential interactions is needed. One study followed the changes in the microbial flora during and between pulmonary exacerbations of CF using both culture and culture-independent methods. (not S. Auto Inducer-2 (AI-2) [131]. Anaerobes and other bacteria considered normal upper respiratory tract flora Sputum may contain microorganisms other than the PPM. Sputum is not routinely cultured for anaerobes partly because they are present in large numbers in saliva and can easily contaminate expectorated sputum. The studies in CF show that interactions may also enhance pathogenicity [133]. One approach is to analyse the gene encoding 16S rRNA. Alternately the PCR product can be cloned. The group identified members of the S. and the 16S rRNA gene amplified by PCR. This could be reproduced using an inter-species QS molecule. Following the observation of a rapid drop in oxygen partial pressure just below the surface of a CF sputum plug. Although this has not been studied in non-CF bronchiectasis. aureus) and a viridans-type Streptococcus sp. and the numbers of anaerobes remained constant during successful treatment of a clinical exacerbation [129]. therefore. and have revealed species not previously found in respiratory samples using traditional culture methods [12. Of interest. a comparison of RT-TRFLP 80 . aeruginosa in an animal model and increase the expression of certain virulence genes of P. sequenced and compared with databases containing sequence data from a wide range of microorganisms. the length of the fragment being characteristic of certain species. Following an observation that a range of upper respiratory tract flora were seen in large numbers in sputum from CF patients. were further studied. These have been considered either contaminants from the upper respiratory tract or harmless commensals colonising the sputum. aeruginosa in vitro.

only cautiously can parallels be drawn from studies examining the role of viruses in asthma. and greater depth of sequencing of 16S rRNA DNA [138.E. rhinovirus is also capable of replicating in the lower airway cells during experimental infection [147]. damage of airway epithelia or compromise of mucociliary clearance. COPD and CF. Viral infections in asthma. 139]. rhinovirus is thought to infect the upper respiratory epithelium. There have been major technical advances facilitated by the development of next generation sequencers plus developments in bioinformatics. Such techniques can provide an enormous amount of information that is a great challenge to process. Some data exists that viral infections in childhood may predispose to the development of bronchiectasis in later life [142]. Rhinovirus is the most common respiratory virus and represents two-thirds of all upper respiratory tract infections. An alternative approach is to attempt whole genome sequencing directly from the clinical sample [140]. FOWERAKER AND D. but it is likely to involve cytokine production in response to viral replication in the lower airways. Similarly. Traditionally. 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]. The proinflammatory cytokine IL-1b is detectable in experimental infected individuals. [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. In a study by JOHNSTON et al [143] using PCR and viral culture. Other mediators induced by rhinovirus infections include neutrophil-activating peptide (which induces neutrophil migration). viruses were detected in 80% of episodes of wheeze or reduced peak expiratory flow in children aged 9–11 years with asthma. which includes upregulating the expression of a range of proinflammatory mediators. Respiratory viruses The role of viruses in non-CF bronchiectasis is not known and remains an important area for future research. COPD and CF J. The mechanism by which viruses cause bronchoconstriction is not fully understood. However. IL-8. PAPADOPOULOS et al. parainfluenza 9% and respiratory syncytial virus (RSV) 5%. whether it is through the development of bronchiolitis. This could include analysis of nucleic acid from eukaryotes and viruses in sputum as well as bacteria [141]. The application of molecular diagnostic methods has improved the understanding of viral epidemiology. Therefore. Respiratory viruses were also present in most patients hospitalised for life-threatening asthma and acute not life-threatening asthma [145]. Respiratory viruses may induce asthma exacerbations via direct effects on the airway epithelium as well as through a systemic immune reaction. eotaxin and RANTES 81 . it is unclear. influenza 9%. disruption of small airway associated innate/adaptive immunity.with TRFLP showed that a high proportion of the bacterial species detected in CF sputum were metabolically active [137]. 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. NICHOLSON et al. Rhinovirus accounted for 61% of the viruses detected. WAT Viral exacerbation of asthma has been well published. However. These have allowed direct analysis of amplified DNA without the cloning step. [144] found that respiratory viruses accounted for 44% of asthma exacerbations in adults. 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. It also accounts for 50% of asthma exacerbations in children [146]. a key mediator in neutrophil-mediated acute inflammation. coronavirus 16%.

a longer median symptom recovery period (up to 13 days) and a tendency towards higher plasma fibrinogen and serum IL-6 levels. Infectious agents are recognised as a major pathogenic factor in exacerbations. T-cell expressed and secreted). The disease is associated with intermittent exacerbations characterised by acute deterioration in symptoms. 162]. 164]. An impaired antiviral immunity to a rhinovirus may lead to impaired viral clearance and hence prolonged symptoms. and there was a correlation in the concentration of RANTES with clinical symptoms. particularly in infancy. IL-16 and monocyte chemoattractant protein (MCP-1). Indirect prevention strategies focus on the reduction of overall airway inflammation to reduce the MICROBIOLOGY severity of the host response to respiratory viral infections. epithelial cells infected with influenza in vitro were associated with an increase in eotaxin [153]. [152] also demonstarted that the eosinophil product. 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]. and RANTES increased with viral infections. In total.4% of patients admitted into hospital [164]. lung function. Patients with stable COPD may carry respiratory viruses. Early studies looking at respiratory viruses and COPD have stated a 20% detection rate in COPD exacerbations [161.5%) out of 68 stable COPD patients. it is known to be a potent cause of wheezing. Recent epidemiological studies suggest that viruses provoke asthma attacks by additive or synergistic interactions with allergen exposure or with air pollution. Non-RSV respiratory viruses were detected in 11 (16%). the nasal cytokine responses to other viruses are of the predominant Th1 type (except RSV). RSV was detected in only 5% of asthma episodes in the study by JOHNSTON [143]. 156]. 82 . 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. T-cells and basophils. in contrast to Th1 cytokine patterns. and quality of life [155. They showed that 64% of COPD exacerbations were associated with a cold occurring up to 18 days before exacerbation. whether or not if the infant is atopic [150]. There is a lack of specific antiviral strategies in the prevention or reduction of viral-triggered asthma exacerbations. which are classically associated with viral elimination. It has been shown that G- glycoprotein of RSV appears to stimulate T-helper cell (Th) type 2 immune response in the upper airway. Influenza A infection induces large amounts of intrapulmonary IFN-c and enhances both later allergen specific asthma and dual Th1/Th2 responses [151]. COPD is the fourth leading cause of mortality worldwide and is an important cause of global burden of disease [154]. These are all key factors in asthma exacerbation. SEEMUNGAL et al. All of these can lead to enhance airway inflammation. (regulated on activation. which can be found on eosinophils. Interestingly. However. with RSV detection being associated with higher inflammatory marker levels [161. increased symptoms. but suffer from more severe consequences of the lower respiratory tract infection. and RSV in 16 (23. This could explain the tendency for RSV to cause wheezing. serology and PCR. RSV has also been shown to be an important virus in COPD exacerbations and was detectable in 11. In addition. there were 168 episodes of COPD exacerbation in 53 patients and 77 viruses (39 were rhinoviruses) were detected. bacteria are absent in about 50% of exacerbations and the frequency of isolation does not increase during exacerbation [160]. but not the association between other respiratory viruses and wheezing. [163] detected respiratory viruses from nasal samples and blood of patients with COPD using a combination of culture. these studies were limited by using less sensitive methods in viral detection. Eotaxin can in turn lead to an exaggerated inflammatory response by being an agonist for chemokine receptor 3. 159] and the exacerbations of COPD. Patients with asthma are no more susceptible to upper respiratory tract rhinovirus infections than healthy people. Viral exacerbations were associated with frequent exacerbatons. However. However. Th2 cytokine patterns are known to be associated with viral immunopathology and allergic-type responses. Bacteria have a role in the pathogenesis [158. major basic protein (MBP). TERAN et al.

Shwachman score.7 years) over a 2-year period. for the siblings not affected. respectively. respectively. compared with 13 out of the 30 infants with CF (OR -4. There was no significant difference in the rate of viral infections between the patients with CF and their sibling controls. The rate of proven virus infection was significantly correlated with the decline in forced vital capacity (FVC) and FEV1. RSV infection with lower respiratory tract infection and male sex with lower respiratory tract infection. the virus isolation and seroconversion (four-fold increase in titres) rates were 8. Seven of the infants with CF cultured RSV. and frequency and duration of hospitalisation. Viruses were identified through repeated serology and nasal lavages for viral isolation in 49 patients with CF (mean age 13. p-value . and the rate of decline in pulmonary function was greater in the younger children with CF with more viral infections. From previous reports. At the time of an ARI.7 per year). However. In contrast the rates for virus isolation and seroconversion at routine 2 monthly visits were 5. [169] described the relationship between respiratory viral infections and the deterioration in clinical status in CF patients. Over a 2-year period.6% and 16.5.0.7 per year). [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. [176] prospectively followed up 48 children with CF diagnosed through newborn 83 .7 per year versus 1. Although the CF patients had more respiratory illnesses than the sibling controls (3.2%. either alone [165]. [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). RAMSEY et al. 168]. ABMAN et al. none of the controls required hospitalisation. 95% CI 1.2%. RSV is a major pathogen resulting in an increased rate of subsequent hospitalisation. whilst 35 were isolated on seroconversion (parainfluenza virus on 12. In younger children.1%. These methods are relatively insensitive and more recent studies have utilised molecular based methodologies [171–175]. as measured either by culture or serology. there were no differences in virus identification rates (1. for children with CF and 7. It has now been 25 years since WANG et al. only four of the 28 control infants had lower respiratory tract symptoms in association with the respiratory tract illness.0).3–16. these differences could be due to the different methodologies utilised. in 19 of these episodes an associated virus identified. in children with CF compared with 15% and 15%.7% and 20. A total of 49 infective agents were identified either during ARIs or at routine testing in the patients with CF. It is also likely that there are differences in the populations studied. two viral agents appear to have the greatest effect on respiratory status in CF. samples were taken at regular 2-month intervals and during acute respiratory illnesses for pharyngeal culture and serology for respiratory viruses.05). FOWERAKER AND D. Similarly HIATT et al. 14 were J. for the siblings not affected. however. 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. 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.0 versus 5. RSV on nine and Mycoplasma pneumoniae on six occasions).E.6.Early studies looking at respiratory viruses in CF relied on repeated serological testing. In contrast. of whom three required hospitalisation. respectively. namely RSV and influenza. The average number of acute respiratory illness per winter was the same in the control and the CF groups (5. respectively. All these studies produced different results in terms of prevalence of respiratory viruses in CF.8% and 19. children with CF have significantly more episodes of lower airway symptoms than controls [166. WAT identified on viral isolation (rhinovirus on 11 occasions). There were a total of 68 acute respiratory illness (ARI) episodes that occurred in the patients with CF. possibly because the uses of viral culture and serology have underestimated the effects of rhinovirus (due to the vast amount of serotypes). as the prognosis for CF has improved with each successive birth cohort. The rate of viral infections was higher in younger children (both CF and controls). or in combination with viral cultures for viral detection [166–170].

PUNCH et al. of which 21 episodes were associated with an identified viral agent (influenza A: five episodes. They also demonstrated that clinical viral symptoms had a very poor predictive value (0. 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. In another prospective study of repeated BAL in 80 infants identified through CF newborn screening over a 5-year period. In older children and adults with CF. 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).39) for a positive viral test. Over the next 2 years these infants had significantly more frequent respiratory symptoms and lower chest radiograph scores than non-RSV identified infants. 53 sputum samples were collected over two seasons and 12 (23%) samples from 12 patients were positive for a respiratory virus (influenza B n54. However. RSV n52). of which 21 (18%) were rhinoviruses.5%. Picornaviruses were identified in 51 (43%) of these episodes. They achieved a rate of 46% for respiratory viruses in their paediatric CF cohort during reported episodes of respiratory illness. The authors failed to show a positive correlation between respiratory viruses and bacterial infections in their studied population. There were no statistical associations between virus status and demographics. They achieved a viral detection rate of 16%. 38 children completed the study and there were 147 symptomatic upper respiratory tract infections (2. In seven of these infants RSV was isolated.7 episodes per child per year). aureus or Aspergillus fumigatus. screening and documented the effect of RSV infection. influenza A n53. These traditional 84 . S. aeruginosa infection reported an acute deterioration in clinical status in association with influenza A virusl infection [177]. influenza seems to have the greatest effect. as the type or frequency of bacterial infection during or after viral infections were not altered. with rhinovirus being the most prevalent virus. Six children isolated a P. aeruginosa. Maximal mean MICROBIOLOGY drop in FEV1 was 16. at 1–4 days after onset of symptoms. but a deficit of 10. [171] followed 48 children with CF over a 15-month period using a combination of viral culture and PCR for picornaviruses alone [178]. aeruginosa for the first time during the study. [174] obtained sputum/laryngeal aspirated from children with CF over a 12-month period in outpatient clinics. Over the year of the study 80 exacerbations were identified. and their clinical course was severe with three requiring mechanical ventilation and five necessitating chronic oxygen therapy. infection with influenza was associated with a more significant drop in pulmonary function (FEV1 decreased by 26% compared with 6%. five at the time of a upper respiratory tract infection and only one was asymptomatic at the time of first isolation. A retrospective study in older patients with chronic P. [167] assessed acute pulmonary exacerbation isolates from 54 patients with CF. WAT et al. Those with more upper respiratory tract infections appeared to have a greater change in total Shwachman and Crispin–Norman scores over the study. PRIBBLE et al. OLESEN et al. this may be due to earlier studies relying heavily on repeated serological testing either alone [165] or in combination with viral isolation [166–170]. respectively). However. the other viruses detected were associated with significant reduction in lung function. [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. clinical variables or isolation rates for P. [179] utilised ‘‘real-time’’ nucleic acid sequence-based amplification (NASBA) to examine the role of respiratory viruses in CF. 16 (52%) of which had a respiratory virus identified with the most common being RSV (n57). with samples available for 119 episodes. COLLINSON et al.3% persisted at 21–24 days. parainfluenza 1 n53. The results compare favourably with previous studies. this virus did not seem to have any devastating impact on lung function. Compared with other agents. influenza B: four episodes. 31 infants were hospitalised for a respiratory exacerbation. RSV: three episodes) with most agents identified by serology. 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.

191]. It may also play an important role in infection control in the hospital setting. while both influenza and adenovirus have a cytopathic effect on cultured nasal epithelium leading to the destruction of the cell monolayer [187]. In the 17-month prospective study reported by COLLINSON et al. [170] have reported that 50% of CF respiratory exacerbations requiring hospitalisation are associated with the isolation of a respiratory virus. most commonly RSV. Interaction between bacteria and viruses J. In a 25-year retrospective review from the Danish CF clinic. nasopharyngeal aspirates. Nasal swabs. 180–183]. FOWERAKER AND D. possibly facilitating the bacterial adherence. In their prospective study of repeated BAL in infants over a 5-year period. A prospective study by HEIKKINEN et al. This epithelial damage results in an increase in the permeability of the mucosal layer [188. The rapid turn-around time of results allows diagnostic virology to have an impact on patient management. with the exception of RSV. 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]. aeruginosa in the subsequent 12–60-month follow-up period. nasal wash and sputum specimens are generally considered as the specimens of choice for the detection of respiratory viruses [173. Detection of respiratory viruses The principal laboratory methods utilised for the diagnosis of respiratory viruses. such as viral culture and serology analysis. due to the rapid turn-around time for the results. WAT 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. Molecular techniques have superseded many ‘‘conventional’’ methods utilised for respiratory viral detection. ARMSTRONG et al. the most likely first isolation of P. in order to adhere to virus infected cells [190. thereby avoiding prescribing the inappropriate use of antibiotics and allowing the correct prescription for anti-virals. compared with three out of 49 (6%) non-hospitalised infants (relative risk 5. Similarly. 85 . five of the six first isolations of P. H. a respiratory virus was identified in 52% of the infants hospitalised for a respiratory exacerbation. 189]. 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. 11 of the 31 hospitalised infants (35%) acquired P. aeruginosa was identified during the asymptomatic period. In contrast only one of the six initial infections with P. aureus infection was identified immediately following a viral infection. aeruginosa was found to be occur between October and March [185]. For example influenza A infection results in epithelial shedding to basement membrane with submucosal oedema and neutrophil infiltrate [186].methods are relatively insensitive and once again may have underestimated the prevalence of viruses in CF. influenzae was recovered for the first time from three children within 3 weeks of an upper respiratory tract infection and the one new S. Bacteria can also utilise viral glycoproteins and other virus-induced receptors on host cell membranes as bacterial receptors. aeruginosa were made during the symptomatic phase of an upper respiratory tract infection or 3 weeks thereafter. coinciding with the peak of the RSV season. [184] showed that the sensitivity of nasal swabs was comparable to nasopharyngeal aspirates for the detection of all major respiratory viruses by tissue culture. [171]. The first bacterial isolation of a given organism in CF has also been shown to often follow a viral infection. This observation implies a causal relationship between respiratory viral and bacterial infection. Respiratory viruses can disrupt the airway epithelium and precipitate bacterial adherence.E.8).

This increase adherence could be explained by an upregulation of cell surface receptors for bacteria. pneumoniae alone. Prevention and treatment of infection with respiratory viruses Influenza associated death is between 13. PITTET et al. these findings suggest that respiratory viruses may lead to epithelial disruption. Pre-incubation with MICROBIOLOGY specific anti-G antibody significantly reduce bacterial adhesion to G protein-transfected cells [196]. The waning of vaccine-induced immunity over time requires annual re-immunisation even if the vaccine antigens are unchanged. destruction of mucociliary escalator. and thus increasing the risk of secondary bacterial infection. Recent vaccines contain antigens of two influenza A subtypes. Collectively. indirectly facilitating bacterial infection of the airway. and one influenza B virus. This suggests that functional changes in the recruited neutrophils may contribute to the decreased bacterial clearance. Another study also showed that NTHi and S. aeruginosa infection. STARK et al. Cilia lining the respiratory tract propel the overlying mucus to the oropharynx where it is either swallowed or expectorated. strains of the currently circulating H3N2 and H1N1 (Swine flu) subtypes.000 to 20. Changes to the tracheal epithelium induced by influenza virus may increase susceptibility to a secondary S. but less production of myeloperoxidase compared with mice that were exposed to S. aeruginosa alone. which are more proinflammatory than their biofilm counterparts.000 times higher CFU count of P. The lower respiratory tract is protected by local mucociliary mechanisms that involve the integration of the ciliated epithelium. neutrophil influx and increased neutrophil induced peroxide release. aeruginosa clearance between 1 to 7 days after RSV exposure. pneumoniae to human respiratory epithelial cells in vitro [195]. pneumoniae bind to both free RSV virions and epithelial cells transfected with cell membrane-bound G protein. aeruginosa had a 2. aureus or P. [197] showed that mice that were exposed to RSV had significantly decreased S. increased cytokine production. periciliary fluid and mucus. [194] showed that mice that were co-infected with RSV and P. such as intercellular adhesion molecule-1 (ICAM-1). may contribute to the pathogenesis of CF exacerbations. More recently. Influenza vaccination 86 . DE VRANKRIJKER et al. but it does significantly reduce mucociliary velocity. stimulate increased chemokine responses in CF airway epithelial cells which. [198] demonstrated that acute infection of primary CF airway epithelial cells with rhinovirus liberates planktonic bacteria from biofilm. S. Whether these are the mechanisms for infective exacerbations in the context of non-CF bronchiectasis remains to be seen. Mice that were exposed to both RSV and bacteria had a higher production of neutrophils induced peroxide. These results suggest that RSV can facilitate the initiation of acute P. [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. CHATTORAJ et al. though some of the deaths may be attributed to RSV. Influenza vaccines are the only commercially available vaccines against respiratory viruses.000 incidents per year throughout the winter months in the UK [199]. carcinoem- bryonic adhesion molecule 1 (CEACAM1) and platelet activating factor receptor (PAFr). and shedding of the columnar epithelial cells generally within 48 hours of infection [192]. but not to secreted G protein. Planktonic bacteria. The defects in pneumococcal clearance were greatest 6 days after an influenza infection. RSV has also been shown to increase adherence of NTHi and S. pneumoniae infection by increasing pneumococcal adherence to the tracheal epithelium and/or decreasing the clearance of S. pneumoniae. Influenza viral infection has been shown to lead to the loss of cilial beat. pneumoniae via the mucociliary escalator of the trachea. Mucus acts as a physical and chemical barrier onto which particles and organisms adhere. Co-infected mice also had more severe lung function changes. and thereby reduces pneumococcal clearance during the first 2 hours after pneumococcal infection at both 3 and 6 days after an influenza infection. aeruginosa in the lung homogenates compared with mice that were infected with P. in turn.

However. routine annual influenza vaccination for children and adults with non-CF bronchiectasis from a recent Cochrane review [200]. have been shown to inhibit ICAM-1 epithelial expression and hypothesis about their potential as anti-virals have yet to be proven.is recommended to those with chronic respiratory diseases including non-CF bronchiectasis. A phase III study is currently underway that looks at the efficacy of intravenous Zanamivir preparation. 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]. Amantadine has now been almost completely replaced by neuraminidase inhibitors (NI). except for some NI-resistant influenza. Despite this recommendation. it is strain specific as it is only effective against influenza A and has common side-effects such as insomnia.E. nor against. virus shedding and the development of otitis media [204]. Amantadine has been the conventional anti-viral against of influenza. bifilomycin A1 and erythromycin. WAT critically ill adults and children having a life-threatening condition. duration of mechanical ventilation. its vast amount of serotypes has made development of anti-virals against it problematic. a process that prevents infection of new host cells and thereby halts the spread of the infection in the respiratory. Ribavarin Ribavarin. poor concentration and irritability. Neuraminidase inhibitors NIs such as Zanamivir and Oseltamivir are licensed for the treatment of influenza A and B. it pairs equally well with either uracil or cytosine. the major shell protein for the 87 . Controlled studies also show that the use of ribavarin is effective in reducing the clinical severity score. respectively) against flu-like symptoms in previously healthy subjects [205]. thus preventing the release of the progeny influenza virus from infected host cells. due to suspected or confirmed pandemic Influenza A (H1N1) infection or infection due to seasonal Influenza A or B virus. A recent systematic review meta- analysis showed that neuraminidase inhibitors only have modest effectiveness (Oseltamivir and Zanamivir 61 and 62%. However.v. Macrolides Although rhinovirus is the major cause of colds. thereby inhibiting the replication of influenza viruses by interfering with the uncoating of the virus inside the cell. Macrolide antibiotics. avian flu (H5N1) and Swine flu (H1N1). 203]. Other anti-virals Recently there has been a report regarding the use of an anti-rhinoviral agent known as Plecoranil. more clinical proof is required [209]. This anti-viral binds to a hydrophobic pocket of the VP1. supplemental oxygen use and days of hospitalisation [207]. a synthetic guanosine nucleoside that has a broad spectrum of antiviral activity. FOWERAKER AND D. Palizivumab. who are not responding to oral or inhaled neuraminidase inhibitors. They work by inhibiting the function of the viral neuraminidase protein. there is neither evidence for. is approved treatment for lower respiratory tract disease caused by RSV [206]. 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. Zanamivir could be considered to treat J. compassionate use of i. inducing mutations in RNA-dependent replication in RNA viruses. which has been shown to reduce the rate of RSV associated hospitalisation in premature infants [201]. Its mode of action involves interfering with viral protein M2. Zanamivir has a poor oral bioavailability and intranasal application has been shown to be effective in treating experimental influenza infection. prophylaxis using a humanised mouse monoclonal antibody. Although there is no licensed RSV vaccine to date. It can be incorporated into RNA as a base analog of either adenine or guanine. by the reduction in symptoms caused.

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