Molecular analysis of changes in Pseudomonas aeruginosa load during treatment of a pulmonary exacerbation in cystic fibrosis.

BACKGROUND: Intravenous antibiotics for pulmonary exacerbations (PEs) of cystic fibrosis (CF) usually target Pseudomonas aeruginosa. Insights into the CF lung microbiome have questioned this approach. We used RT-qPCR to determine whether intravenous antibiotics reduced P. aeruginosa numbers and whether this correlated with improved lung function. We also investigated antibiotic effects on other common respiratory pathogens in CF. METHODS: Sputa were collected from patients when stable and again during a PE. Sputa were expectorated into a RNA preservation buffer for RNA extraction and preparation of cDNA. qPCR was used to enumerate viable P. aeruginosa as well as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Burkholderia cepacia complex and Aspergillus fumigatus. RESULTS: Fifteen CF patients were followed through 21 PEs. A complete set of serial sputum samples was unavailable for two patients (three separate PEs). P. aeruginosa numbers did not increase immediately prior to a PE, but numbers during intravenous antibiotic treatment were reduced ≥4-log in 6/18 and ≥1-log in 4/18 PEs. In 7/18 PEs, P. aeruginosa numbers changed very little with intravenous antibiotics and one patient demonstrated a ≥2-log increase in P. aeruginosa load. H. influenzae and S. pneumoniae were detected in ten and five PEs respectively, but with antibiotic treatment these bacteria rapidly became undetectable in 6/10 and 4/5 PEs, respectively. There was a negative correlation between P. aeruginosa numbers and FEV1 during stable phase (r(s)=0.75, p<0.05), and reductions in P. aeruginosa load with intravenous antibiotic treatment correlated with improved FEV1 (r(s)=0.52, p<0.05). CONCLUSIONS: Exacerbations are not due to increased P. aeruginosa numbers in CF adults. However, lung function improvements correlate with reduced P. aeruginosa burden suggesting that current antibiotic treatment strategies remain appropriate in most patients. Improved understanding of PE characterised by unchanged P. aeruginosa numbers and minimal lung function improvement following treatment may allow better targeted therapies.

Antimicrobial susceptibility testing of cystic fibrosis and non-cystic fibrosis clinical isolates of Pseudomonas aeruginosa: a comparison of three methods.

Pseudomonas aeruginosa is an important pathogen in humans, particularly in the context of nosocomial infection and infections of the cystic fibrosis (CF) lung. In order to provide clinicians with information about the likely effectiveness of specific antimicrobial treatment for P. aeruginosa infections, clinical laboratories employ in vitro antimicrobial susceptibility testing. Two commonly employed methods are the CLSI disc-diffusion and Etest methods. The purpose of this study is to compare the accuracy of susceptibility results generated by these two methods against agar dilution as the reference method. Susceptible or nonsusceptible (resistant and intermediate) results of the Etest and CLSI disc-diffusion methods are compared with CLSI agar dilution results for a large cohort of clinical cystic fibrosis (n = 71) and non-cystic fibrosis (n = 83) isolates using CLSI interpretive criteria. An unacceptable number of major and very major errors were observed for various antimicrobials tested against both CF and non-CF isolates when using the Etest and CLSI disc-diffusion methods. The potential for error in standard laboratory antimicrobial susceptibility testing should be considered by clinicians when being guided by the results of such tests in the prescription of antimicrobial agents for P. aeruginosa infection.

Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa.

The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P<0.001 and P<0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P<0.01) when compared to other clinical isolates (P<0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.

Biosignificance of bacterial cyanogenesis in the CF lung.

Two recent studies have demonstrated the presence of biologically significant amounts of cyanide within the airways of cystic fibrosis (CF) patients infected with Pseudomonas aeruginosa. Whilst environmental strains of P. aeruginosa are known to synthesise cyanide, there has been a relative lack of investigation into bacterial cyanogenesis from a medical viewpoint, despite the role P. aeruginosa plays in many serious infection settings and especially in CF lung disease. This review discusses the implications of cyanogenesis in the CF airway in terms of bacterial ecology, host immune response, progression of lung disease and potential treatment options.

Increased vascular permeability precedes cellular inflammation as asthma control deteriorates.

BACKGROUND: Airway microcirculation is abnormal in asthma but the role of vascular changes in asthma deteriorations remains poorly defined. We prospectively assessed the vascular changes accompanying worsening of asthma control by using an inhaled corticosteroid (ICS) dose-reduction model. OBJECTIVES: To evaluate airway vascularity, vascular permeability and expression of vascular endothelial growth factor (VEGF) in early asthma deterioration induced by ICS back-titration. METHODS: Twenty mild-to-moderate persistent symptomatic asthmatics on low-to-moderate ICS were recruited and treated with 4 weeks of high-dose fluticasone propionate (1000 microg/day) to achieve symptom control. This was followed by dose reduction to half of the pre-study doses for 4-8 weeks until the symptoms began to return. Endobronchial biopsy and bronchoalveolar lavage (BAL) samples were obtained after both treatment periods. RESULTS: Vascularity as measured by the number and size of blood vessels, as well as VEGF expression did not change following ICS reduction. Even on high-dose ICS, perivascular albumin staining and BAL microalbumin levels in asthmatic subjects, as markers of permeability, were elevated when compared with normal subjects and both further increased significantly after ICS reduction. There was a significant association between changes in vascular leakiness and clinical deterioration. Increases in airway albumin correlated with previously reported increases in airway wall infiltration with T lymphocytes. CONCLUSIONS: Our results suggest that airway vascular leakage is a major pathophysiologic feature of early asthma deterioration, occurring before recrudescence of cellular inflammation.

Epidemiology of Pseudomonas aeruginosa in a tertiary referral teaching hospital.

A genotypically indistinguishable strain of Pseudomonas aeruginosa (Australian epidemic strain III: AES III) has previously been found in a proportion of adults with cystic fibrosis (CF) in Tasmania, Australia. The aim of this study was to identify a source of these infections within the major tertiary referral hospital for the State of Tasmania, and to determine if this strain could be isolated from settings other than the CF lung. A total of 120 isolates of P. aeruginosa were collected from clinical and environmental sources within the hospital and from environmental locations in the hospital vicinity. These isolates were genotyped by random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute method. Confirmation of similar genotypes identified by RAPD-PCR was performed using pulsed-field gel electrophoresis with restriction enzyme SpeI. AES III was not recovered from any source other than the respiratory secretions of CF patients. P. aeruginosa in the non-CF settings was found to be panmictic, and no cross-infection or acquisition of hospital environment strains by patients was observed.

Role of lung iron in determining the bacterial and host struggle in cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations. It is a multiorgan system disease that affects the lungs, gastrointestinal tract, liver, and pancreas. The majority of morbidity and mortality in CF relates to chronic airway infection with a variety of bacterial species, commencing in very early infancy, which results in lung destruction and ultimately organ failure (41, 43). This review focuses on iron homeostasis in the CF lung and its role in determining the success and chronicity of Pseudomonas aeruginosa infection. There have been previous excellent reviews regarding iron metabolism in the lower respiratory tract and mechanisms of P. aeruginosa iron acquisition, and we direct readers to these articles for further background reading (31, 53, 58, 77, 96). In this review, we have brought the "two sides of the coin" together to provide a holistic overview of the relationship between host and bacterial iron homeostasis and put this information into the context of current understanding on infection in the CF lung.

Bacterial cyanogenesis occurs in the cystic fibrosis lung.

The cystic fibrosis (CF) lung environment is poorly defined, but data suggest that bacteria may encounter reduced oxygen tensions and possibly an anaerobic environment. Pseudomonas aeruginosa produces the potent toxin cyanide under strictly microaerobic conditions. Evidence of bacterial cyanogenesis in the CF lung was investigated in the present study by measuring sputum cyanide concentrations. Sputum cyanide was measured in seven stable CF patients, as well as before and after intravenous antibiotic therapy during a hospital admission in a further eight patients experiencing acute exacerbations. All patients were chronically infected with P. aeruginosa. Comparative sputum data were obtained from nine CF patients with no documented P. aeruginosa infection and 10 healthy, nonsmoking normal controls. High levels of cyanide were detected in all the P. aeruginosa-infected stable CF patients (median (range) 0.56 (0.37-2.81) microg.mL(-1)), and in seven out of eight acute sputum samples (0.73 (0-1.43) microg.mL(-1)). In contrast, cyanide was not detectable in sputum from eight out of nine CF patients without P. aeruginosa infection or in any of the normal controls. Intravenous antibiotic treatment significantly reduced sputum cyanide levels (median 0.73 to median 0.0 microg.mL(-1)). The cyanide detected indicates that the cystic fibrosis lung provides a predominantly microaerobic environment for Pseudomonas aeruginosa. Cyanide is likely to be a potentially important virulence factor in Pseudomonas aeruginosa-infected cystic fibrosis patients.

Nonpharmacological and pharmacological interventions to prevent or reduce airway remodelling.

In the present review of airway remodelling and its response to therapies, clinical observations about airway physiological abnormalities, assumed to be caused by remodelling processes, are related to what is known about the components of structural changes from airway sampling and histopathological analysis. The review focuses on three important diseases: asthma, chronic obstructive pulmonary disease and bronchiolitis obliterans syndrome (BOS), which occurs commonly after lung transplantation as a manifestation of chronic rejection. The present authors chose to use BOS as an issue, because with routine bronchoscopic surveillance after lung transplantation there has been more opportunity to directly study airway pathology longitudinally than in more everyday conditions. In addition, the present authors have reviewed animal models of induced airway remodelling, where most information is available on the potential of therapeutic intervention. Finally, the limited information that can be gained from the literature on the effects of commonly used airway medications on remodelling components is reviewed. In conclusion, the present authors have detailed some of the gaps in knowledge surrounding the potential to improve or modulate remodelling processes in human disease. The areas where it is believed urgent research needs to be focused have also been highlighted.

Airway cell and cytokine changes in early asthma deterioration after inhaled corticosteroid reduction.

BACKGROUND: Back-titration of inhaled corticosteroid (ICS) dose in well-controlled asthma patients is emphasized in clinical guidelines, but there are few published data on the airway cell and cytokine changes in relation to ICS reduction. In our study, 20 mild-to-moderate persistent (inspite of low-moderate dose ICS treatment) asthmatic subjects prospectively rendered largely asymptomatic by high-dose ICS were assessed again by clinical, physiological, and airway inflammatory indices after 4-8 weeks of reduced ICS treatment. We aimed at assessing the underlying pathological changes in relation to clinical deterioration. METHODS: Patients recorded daily symptom scores and peak expiratory flows (PEF). Spirometry and airways hyperreactivity (AHR) were measured and bronchoscopy was performed with assessment of airway biopsies (mast cells, eosinophils, neutrophils, and T lymphocytes), bronchoalveolar lavage (BAL) IL-5 and eotaxin levels and cellular profiles at the end of high-dose ICS therapy and again after ICS dose reduction. Baseline data were compared with symptomatic steroid-free asthmatics (n=42) and non-asthmatic controls (n=28). RESULTS: After ICS reduction, subjects experienced a variable but overall significant increase in symptoms and reductions in PEF and forced expiratory volume in 1 s. There were no corresponding changes in AHR or airways eosinophilia. The most relevant pathogenic changes were increased CD4(+)/CD8(+) T cell ratio, and decreased sICAM-1 and CD18 macrophage staining (potentially indicating ligand binding). However, there was no relationship between the spectrum of clinical deterioration and the changes in cellular profiles or BAL cytokines. CONCLUSIONS: These data suggest that clinical markers remain the most sensitive measures of early deterioration in asthma during back-titration of ICS, occurring at a time when AHR and conventional indices of asthmatic airway inflammation appear unchanged. These findings have major relevance to management and to how back-titration of ICS therapy is monitored.

Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis.

Iron availability is critical to many bacteria and increased iron has been described in airway secretions in cystic fibrosis (CF). The main aim of the present study was to assess the relationship between iron in CF sputum and the quantitative bacterial burden. Iron, ferritin and total cell counts (TCC) were assessed in sputum samples obtained from 15 clinically stable CF patients chronically infected with Pseudomonas aeruginosa. Sputum samples were also obtained at the commencement of episodes of acute exacerbation in 10 subjects and analyses were repeated in six of these exacerbation cases after i.v. antibiotic treatment. The relationship between iron indices and the presence of P. aeruginosa, as well as total anaerobic bacterial load, was determined. Sputum was also obtained from 10 CF patients with no evidence of infection with P. aeruginosa and 11 normal healthy controls. Sputum iron, ferritin and TCC were significantly elevated in all CF patients, even in those not infected with P. aeruginosa, compared with healthy controls. There was a strong positive relationship between sputum iron and P. aeruginosa in clinically stable patients, but not in samples obtained during an acute exacerbation. There was no relationship between sputum iron and anaerobic bacterial load. Antibiotic treatment significantly reduced sputum TCC and anaerobic bacterial load, but not iron, ferritin or the presence of P. aeruginosa during an exacerbation. In conclusion, the present study suggests that increased airway iron may be important to Pseudomonas aeruginosa persistence in cystic fibrosis.

Effects of inhaled fluticasone on angiogenesis and vascular endothelial growth factor in asthma.

BACKGROUND: Subepithelial hypervascularity and angiogenesis in the airways are part of structural remodelling of the airway wall in asthma, but the effects of inhaled corticosteroids (ICS) on these have not been explored. Increased vascularity in asthma may contribute to a number of functional abnormalities. A study was undertaken to explore angiogenic modulation by ICS and its likely regulation via vascular endothelial growth factor (VEGF), its receptors and the angiopoietins. METHODS: A placebo-controlled intervention study with ICS in asthma was performed, examining vascularity, VEGF, its receptors (VEGFR1 and VEGFR2), and angiopoietin-1 (Ang1) to assess which of these factors were changed in the asthmatic airways after ICS treatment. Airway wall biopsy specimens, lavage fluid and cells were obtained from 35 patients with mild asthma randomised to receive ICS or placebo for 3 months, after which bronchoscopic examination and sample collection were repeated. Immunohistochemistry and image analysis were used to obtain quantitative measures of vessels, angiogenic sprouts, VEGF, VEGFR1, VEGFR2 and Ang1 staining in airway biopsy specimens. ELISA was used to assess VEGF concentrations in the lavage fluid. RESULTS: Vessel, VEGF and sprout staining were decreased after 3 months of ICS treatment. VEGF levels remained unchanged. VEGF receptors and Ang1 staining were not reduced after treatment. CONCLUSIONS: The findings of this study support an effect of ICS in downregulating angiogenic remodelling in the airways in asthma, associated with decreasing VEGF activity within the airway wall. The environment of the airways after treatment with ICS, with changes in the balance between VEGF, its receptors, Ang1 and sprouts, appears to be less angiogenic than in untreated asthma.

Inter-relationships between airway inflammation, reticular basement membrane thickening and bronchial hyper-reactivity to methacholine in asthma; a systematic bronchoalveolar lavage and airway biopsy analysis.

BACKGROUND: Asthma is accepted as a disease characterized by airway inflammation, with evidence that airway structural changes, or 'remodelling' occurs. There are few studies relating airway physiology, inflammation and remodelling, however. We have carried out a study of inter-relationships between airway inflammation, airway remodelling, reticular basement membrane (RBM) thickening, and bronchial hyper-reactivity (BHR), before and after high-dose inhaled corticosteroid (fluticasone propionate 750 microg b.d.), in a group of relatively mild but symptomatic, steroid naïve asthma patients. METHODS: Double-blind, randomized, placebo-controlled, parallel group study of inhaled corticosteroid (ICS) in 35 asthmatics, with bronchoalveolar lavage (BAL) and airway endobronchial biopsy (EBB) for inflammatory cell profiles and EBB for airway remodelling carried out at baseline, 3 and 12 months. RESULTS: At baseline RBM thickening was related to BAL mast cells and EBB eosinophil counts. In turn baseline log EBB EG2 eosinophil count, log%BAL epithelial cells and log RBM thickness explained 55% of the variability in BHR. CONCLUSION: We provide new information that airway inflammation, remodelling, and BHR in asthma are inter-related and improved by ICS therapy. Our data potentially support the need for early and long-term intervention with ICS even in relatively mild asthmatics, and the need to further assess the potential merit of longitudinal BHR testing in management of some patients, as this may reflect both airway inflammation and remodelling.