Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway

The cystic fibrosis (CF) lung is chronically inflamed and infected by Pseudomonas aeruginosa, which is a major cause of morbidity and mortality in this genetic disease. Although aerosolization of Tobramycin into the airway of CF patients improves outcomes, the lungs of CF patients, even those receiving antibiotic therapy, are persistently colonized by P. aeruginosa. Recent studies suggest that the antibiotic resistance of P. aeruginosa in the CF lung is due to the formation of drug resistant biofilms, which are defined as communities of microbes associated with surfaces or interfaces, and whose growth is facilitated by thick and dehydrated mucus in the CF lung. In this review, we discuss some of the current models used to study biofilm formation in the context of biotic surfaces, such as airway cells, as well as the contribution of host-derived factors, including DNA, actin and mucus, to the formation of these microbial communities. We suggest that better in vitro models are required, both to understand the interaction of P. aeruginosa with the host airway, and as models to validate new therapeutics, whether targeted at bacteria or host.     

Pseudomonas aeruginosa regulates flagellin expression as part of a global response to airway fluid from cystic fibrosis patients

Cystic fibrosis (CF) patients are highly susceptible to chronic lung infections by the environmental bacterium Pseudomonas aeruginosa. The overproduction and accumulation of dehydrated viscous respiratory mucus and excessive inflammation represents a defining feature of CF and constitutes the major environment encountered by P. aeruginosa during chronic infections. We applied whole-genome microarray technology to investigate the ability of P. aeruginosa to respond to signals found in muco-purulent airway liquids collected from chronically infected CF patients. Particularly notable was the activation of the Rhl-dependent quorum-sensing (QS) network and repression of fliC, which encodes flagellin. Activation of the Rhl branch of the QS network supports the observation that QS molecules are produced in the chronically infected CF lung. The shut-off of flagellin synthesis in response to CF airway liquids was rapid and independent of QS and the known regulatory networks controlling the hierarchical expression of flagellar genes. As flagellin is highly immunogenic and subject to detection by host pattern recognition receptors, its repression may represent an adaptive response that allows P. aeruginosa to avoid detection by host defense mechanisms and phagocytosis during the chronic phase of CF lung infections.     

A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an approximately 5 x 10(3) strain PAO1 P. aeruginosa. Although bacteria grew to 10(10) cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical-chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.     

Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.

Cystic fibrosis (CF) is the most common lethal hereditary disorder with autosomal recessive heredity in caucasians. The majority of CF patients suffer from chronic respiratory infection with the opportunistic bacterial pathogen Pseudomonas aeruginosa. No consensus among clinicians has been reached so far concerning antibiotic treatment against P. aeruginosa in CF patients. Consensus answers to 24 important questions in this context, based on current evidence, are presented, given by a panel of 34 European experts. Questions addressed and answered are: The diagnosis of P. aeruginosa lung colonization in CF; The impact of P. aeruginosa on the clinical state of CF patients; The assessment of P. aeruginosa susceptibility against antibiotics and the importance of these results for the clinician; The use of monotherapy versus combination therapy; The development of microbial resistance; The achievement of optimal airway concentrations; The effects of subinhibitory concentrations of antibiotics on P. aeruginosa; Statements on the pharmacokinetics of antibiotics in CF patients; Recommendations for doses and dosing intervals and length of treatment regimens; and Toxic side effects due to repeated antibiotic therapy was addressed. The expert panel answered further questions on the use of fluoroquinolones in children with CF, on the administration of nebulized antibiotics and whether prevention of P. aeruginosa lung colonization is possible in CF using antibiotic therapy. Problems of antibiotic therapy at home and in the hospital were addressed, a consensus statement on regular maintenance treatment, or treatment on demand, was given and different routes of administration of antibiotics were recommended for different clinical situations. Finally, the factors which determine the choice of the antibiotic, the dosage, and the duration of the treatment in cystic fibrosis patients were addressed and the design of future antibiotic studies in the context of Pseudomonas aeruginosa lung infection in cystic fibrosis patients were recommended.     

Pathophysiology and management of pulmonary infections in cystic fibrosis

This comprehensive State of the Art review summarizes the current published knowledge base regarding the pathophysiology and microbiology of pulmonary disease in cystic fibrosis (CF). The molecular basis of CF lung disease including the impact of defective cystic fibrosis transmembrane regulator (CFTR) protein function on airway physiology, mucociliary clearance, and establishment of Pseudomonas aeruginosa infection is described. An extensive review of the microbiology of CF lung disease with particular reference to infection with P. aeruginosa is provided. Other pathogens commonly associated with CF lung disease including Staphylococcal aureus, Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and atypical mycobacteria are also described. Clinical presentation and assessment of CF lung disease including diagnostic microbiology and other measures of pulmonary health are reviewed. Current recommendations for management of CF lung disease are provided. An extensive review of antipseudomonal therapies in the settings of treatment for early P. aeruginosa infection, maintenance for patients with chronic P. aeruginosa infection, and treatment of exacerbation in pulmonary symptoms, as well as antibiotic therapies for other CF respiratory pathogens, are included. In addition, the article discusses infection control policies, therapies to optimize airway clearance and reduce inflammation, and potential future therapies.     

Pulmonary host defense: defects that lead to chronic inflammation of the airway

Current knowledge of pulmonary host defense can help us to understand the unique relationship between CF patients and P. aeruginosa colonization of the lung. Subtle defects in CF host defense, such as those identified in mucociliary clearance and in the CF IgG opsonin, allow P. aeruginosa to persist. Not all the defects are attributable to the host. In several examples, the defects are induced by P. aeruginosa, presumably in an effort to maintain its foothold. The examples of the latter discussed here have included the effect of pseudomonas-derived products on mucociliary action, alpha 1 PI function, and the formation of ineffective IgG opsonins and immune complexes. Overall, P. aeruginosa is the cause of significant morbidity and, eventually, mortality in these patients. As we approach the identification of the genetic defect central to this disease, it is hoped that we will gain more insight into the pathogenesis of the P. aeruginosa lung lesion in CF and develop more effective ways of preventing P. aeruginosa colonization of the CF patients' lungs.     

Immunomodulatory therapies for cystic fibrosis.

Progressive pulmonary disease is the primary cause of morbidity and mortality in adult patients with cystic fibrosis (CF). The decrease in lung function associated with infection with Pseudomonas aeruginosa has been related to the severity of pulmonary inflammation. Thus therapies that reduce pulmonary inflammation may prove to be clinically efficacious. Therapeutic interventions that target pulmonary inflammation may be directed either at the infecting organism, especially P aeruginosa, or at host responses. Eradication of P aeruginosa from the airways of patients with CF has not been accomplished. However, reduction of the burden of P aeruginosa or modification of virulence factors are practical goals. Normalization of the host response ultimately depends on correction of the molecular defect. Until then, therapies are being investigated that may modulate pulmonary inflammation. These include therapies aimed at compensating for the defect in ion transport, down-regulating inflammatory cell responses, inhibiting host inflammatory products, or altering airway secretions. Preliminary data suggest that each of these approaches may have clinical efficacy. Large, multicenter trials addressing these issues are presently ongoing and hold the promise for continued improvement in the clinical course of patients with CF.     

Pseudomonas aeruginosa alginate is a potent secretagogue in the isolated ferret trachea

Airway mucus hypersecretion is in part a response to infection and inflammation. Pseudomonas aeruginosa infection is nearly universal in advanced cystic fibrosis (CF) lung disease. Mucoid strains of P. aeruginosa produce an exopolysaccharide product called alginate. The purpose of this study was to determine whether P. aeruginosa alginate stimulates secretion from mucous or serous cells in the ferret trachea exposed to alginate at concentrations reported to be present in the CF airway. We used a sandwich enzyme-linked lectin assay (ELLA) to measure mucin secretion and spectrophotometry to measure lysozyme secretion from isolated ferret tracheal segments. Purified Pseudomonas aeruginosa alginate stimulated mucin and lysozyme secretion in a dose-dependent fashion (mucin = +111%: P = 0.003; lysozyme = +20%: P = 0.024 at 200 microg/mL). This stimulated secretion was not due to proteolytic activity, and alginate exposure did not produce ultrastructural damage to the trachea. We conclude that alginate may contribute to mucus hypersecretion and respiratory morbidity associated with P. aeruginosa infection in patients with CF.     

Mucus obstruction and inflammation in early cystic fibrosis lung disease: Emerging role of the IL‐1 signaling pathway

Mucus plugging constitutes a nutrient‐rich nidus for a bacterial infection that has long been recognized as a potent stimulus for neutrophilic airway inflammation driving progressive lung damage in people with cystic fibrosis (CF). However, mucus plugging and neutrophilic inflammation are already present in many infants and young children with CF even in the absence of detectable bacterial infection. A series of observational studies in young children with CF, as well as investigations in animal models with CF‐like lung disease support the concept that mucus plugging per se can trigger inflammation before the onset of airways infection. Here we review emerging evidence suggesting that activation of the interleukin‐1 (IL‐1) signaling pathway by hypoxic epithelial cell necrosis, leading to the release of IL‐1α in mucus‐obstructed airways, may be an important mechanistic link between mucus plugging and sterile airway inflammation in early CF lung disease. Furthermore, we discuss recent data from preclinical studies demonstrating that treatment with the IL‐1 receptor (IL‐1R) antagonist anakinra has anti‐inflammatory as well as mucus modulating effects in mice with CF‐like lung disease and primary cultures of human CF airway epithelia. Collectively, these studies support an important role of the IL‐1 signaling pathway in sterile neutrophilic inflammation and mucus hypersecretion and suggest inhibition of this pathway as a promising anti‐inflammatory strategy in patients with CF and potentially other muco‐obstructive lung diseases.     

Rationale for hypertonic saline therapy for cystic fibrosis lung disease

Cystic fibrosis (CF) is caused by alterations in the CF transmembrane conductance regulator ( CFTCR) gene. More than 1400 mutations in the CFTCR gene have been described, but the most common mutation (noted in 70% of CF chromosomes) is DeltaF508. Alterations in the CFTCR gene result in deranged sodium and chloride ion transport channels. This leads to failure of airway epithelia to hydrate their surfaces normally, particularly in response to infectious or toxic insults. Additional effects include mucus adhesion to airway surface, chronic inflammation, and infections. The concept that airway surface dehydration can cause CF-like lung disease is supported by in vitro data and in vivo animal models. Rehydrating airway surfaces may reduce or prevent lung injury and damage. Short- and longer term studies have shown that inhalation of hypertonic saline is well tolerated and improves lung function, reduces exacerbations, and improves quality of life in CF patients. This review discusses the importance of airway epithelial sodium and chloride channels in the pathogenesis of CF, and strategies (particularly the use of inhaled hypertonic saline) to reverse or minimize lung inflammation and injury in this disease.     

Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways

The opportunistic bacterial pathogen Pseudomonas aeruginosa colonizes airways of individuals with cystic fibrosis (CF) with resultant chronic destructive lung disease. P. aeruginosa adaptation to the CF airway includes biofilm formation and antibiotic resistance. Isolates from asymptomatic individuals in the first 3 years of life have unique characteristics, suggesting that adaptation occurs before clinical symptoms. One defined early adaptation is expression of a specific proinflammatory lipopolysaccharide (LPS) that is associated with antimicrobial peptide resistance. This CF-specific LPS is induced when P. aeruginosa is grown in medium that is limited for magnesium. Therefore, qualitative and quantitative proteomic approaches were used to define 1,331 P. aeruginosa proteins, of which 145 were differentially expressed on limitation of magnesium. Among proteins induced by low magnesium were enzymes essential for production of 2-heptyl 3-hydroxy 4-quinolone, the Pseudomonas quinolone signal (PQS), which interacts with the homoserine lactone signaling pathway. Measurement of PQS in P. aeruginosa isolates from asymptomatic children with CF indicated that strains with increased synthesis of PQS are present during early colonization of CF patient airways.     

Early eradication therapy against Pseudomonas aeruginosa in cystic fibrosis patients.

In cystic fibrosis (CF) patients early antibiotic treatment of lung infection has been shown to lead to Pseudomonas aeruginosa eradication. The present study determined: 1) the time period from eradication to new P. aeruginosa acquisition; 2) P. aeruginosa re-growth and new acquisition; and 3) the impact of eradication therapy on lung function, antimicrobial resistance, emergence of other pathogens and treatment costs. Ciprofloxacin and colistin were used to eradicate P. aeruginosa in 47 CF patients. Bacterial pathogens, lung function decline, P. aeruginosa antimicrobial resistance and anti-pseudomonal serum antibodies were assessed quarterly and compared with an age-matched CF control group. Additionally, costs of antibiotic therapy in both groups were assessed. Early antibiotic therapy leads to a P. aeruginosa free-period of a median (range) of 18 (4-80) months. New acquisition with different P. aeruginosa genotypes occurs in 73% of episodes. It also delays the decline of lung function compared with chronically infected patients, prevents the occurrence of antibiotic resistant P. aeruginosa strains, does not lead to emergence of other pathogens, and significantly reduces treatment costs. The treatment substantially lowers P. aeruginosa prevalence in CF. In conclusion, early antibiotic therapy exerts beneficial effects on the patient's clinical status and is cost-effective compared with conventional antibiotic therapy for chronically infected cystic fibrosis patients.     

Evidence for airway surface dehydration as the initiating event in CF airway disease

Cystic fibrosis (CF) lung disease reflects persistent bacterial infection of airway lumens. Several hypotheses have been advanced to link mutations in the CFTR gene to the failure of the CF lung to defend itself against bacterial infection. Amongst the most productive hypotheses at present is the 'low airway surface liquid (ASL) volume' or 'dehydration' hypothesis. This hypothesis predicts that airway surface dehydration produces the mucus adhesion, inflammation, and bacterial biofilm formation characteristic of CF. Clinical trials of inhaled hypertonic saline have demonstrated therapeutic benefit of manoeuvres designed to rehydrate CF airway surfaces.     

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.     

Airway Biofilms

The differentiation of bacterial biofilms in the airway environment, the pathogenesis of airway biofilm, and possible therapeutic methods are discussed. Biofilm diseases that characteristically involve the respiratory system include cystic fibrosis (CF), diffuse panbronchiolitis (DPB), and bronchiectasia with Pseudomonas aeruginosa (P. aeruginosa) infection. There is evidence to suggest that almost all strains of P. aeruginosa have the genetic capacity to synthesize alginate, a main matrix of biofilms, when ecological conditions are unfavorable for their survival. The bacteria inside the mature biofilm show increased resistance to both antibacterials and phagocytic cells, express fewer virulence factors because of their stationary state of growth, and are less stimulatory to the mucosa because of the 'sandwich binding'. These factors facilitate both the colonization of bacteria and their extended survival even under unfavorable conditions. Since the biofilm limits colonization to a latent form, the clinical symptoms in this situation are unremarkable. However, the clinical progression of both CF and DPB proceeds in two characteristic directions. The first is an acute exacerbation caused by planktonic bacteria that have germinated from the biofilm. The second is a slow progression of disease that is induced by harmful immune reactions. The harmful reactions are mediated by alginate, which induces antigen antibody reactions around the airways, as well as formation of circulating immune complexes that are deposited on lung tissue. Furthermore, the highest titer of bacterial permeability increasing anti-neutrophil cytoplasmic autoantibodies (BPI-ANCA) is observed in association with highly impaired pulmonary function in patients with CF and DPB, as well as in patients with a lengthy period of colonization with P. aeruginosa. BPI-ANCA subsequently makes chronic airway infection even more intractable. The long-term use of 14- or 15-ring membered macrolides results in a favorable clinical outcome for patients with DPB and in some patients with CF. In the last 10 years, an increasing number of studies have reported secondary actions of macrolides that include effects on both airway and phagocytic cells, as well as an anti-biofilm activity. The 14- or 15-ring membered macrolides inhibit: (i) the alginate production from P. aeruginosa; (ii) the antibody reaction to alginate, which leads to a decrease in the immune complex formation; and (iii) the activation of the autoinducer 3-O-C12-homoserine lactone and subsequent expression of lasI and rhlI in quorum sensing systems in P. aeruginosa. These anti-biofilm actions of macrolides may represent their basic mechanisms of action on airway biofilm disease.     

Emergence and persistence of Pseudomonas aeruginosa in the cystic fibrosis airway.

Colonization in the respiratory tracts of cystic fibrosis (CF) patients by mucoid Pseudomonas aeruginosa correlates with the progression of bronchial airway pathology. There is a direct correlation between the incidence of Pseudomonas colonization and age, clinical score, extent of pulmonary disease, severity of radiographic changes, and level of serum immunoglobulins. The central propensity to Pseudomonas colonization in patients with CF is not freely understood, but we discuss the acquisition and persistence of P aeruginosa in the CF airway. Elucidation of pathogenetic mechanisms of CF inflammatory airways disease is the first essential step to initiating novel therapies. It has been difficult to prove that the ability of P aeruginosa to adhere to the respiratory epithelium and provide selective advantage for this gram-negative bacillus over other potential pathogens for infection in the CF airway. However, flexible filaments (pili) extending from the Pseudomonas cell wall are thought to medicate epithelial cell adherence for nonmucoid P aeruginosa, and similarly, the gelatinous exopolysaccharide alginate produced by mucoid variants of P aeruginosa seems to be the adhesive to tracheal cells. Following the signal event of adherence, this bacterial pathogen competes successfully for iron cofactor and multiplies, releasing proteases with broad substrate specificities that dramatically alter the airway antiprotease screen, and the pathogen creates defects in local antibacterial defenses. Lung inflammation in CF is characterized by massive neutrophil infiltration. Although critical to host defense, neutrophils also cause progressive airway damage by release of bioactive lipids, oxygen metabolites, and granule enzymes such as hydrolases, myeloperoxidase (MPO), lysozyme, and neutral serine proteases. The necessarily circumscribed discussion that follows will focus narrowly on the host cell-derived factors (macrophages and neutrophils) proposed as important components in this pathogenetic scheme.     

Update on pathogenesis of cystic fibrosis lung disease

PURPOSE OF REVIEW: It has been an ongoing challenge to translate knowledge pertaining to the molecular basis of cystic fibrosis (CF) into a clear understanding of the development of CF lung disease. Various hypotheses have attempted to explain the apparent breach of innate defenses in CF, although a definitive explanation has been elusive. RECENT FINDINGS: Recent data suggest that altered ion transport functions--namely sodium hyperabsorption and reduced chloride secretion--lead to a depletion of airway surface liquid. As a result, the overlying mucus layer may encroach upon cell surfaces and become adherent, thus interfering with cilia-dependent and cough clearance. These static, and ultimately anaerobic, niches provide a favorable environment for the development of bacterial biofilms and persistent infection with Pseudomonas aeruginosa. SUMMARY: With a better understanding of pathogenic steps leading to CF lung disease, we may now be able to direct the development of therapies that will substantially improve disease outcomes.     

Pseudomonas aeruginosa: role in the pathogenesis of the CF lung lesion

Lung disease is the leading cause of morbidity and mortality in individuals with cystic fibrosis (CF), with P. aeruginosa the main pulmonary infectious agent. Although CF patients can become infected with other microorganisms (such as Burkholderia cepacia complex, Staphylococcus aureus, Haemophilus influenzae, and atypical mycobacteria), P. aeruginosa predominates, eventually infecting approximately 80% of patients. Once established, P. aeruginosa infection usually persists until death. The interaction between the CF host and this opportunistic pathogen is unique and most likely directly contributes to the classical end-stage pathology of CF lung disease. However, the extent to which this constitutes success by the pathogen or failure by the host, or both, is yet to be determined. Many important questions remain regarding host susceptibility, the role of both innate and adaptive immune defenses, bacterial infectivity and transmission, and pathogen virulence factors. Here, we discuss some recent advances toward understanding this complex interaction between host and pathogen and how the interplay influences the CF lung lesion.     

Immunoglobulin-G subclasses in cystic fibrosis. IgG2 response to Pseudomonas aeruginosa lipopolysaccharide

Pulmonary macrophage phagocytosis of Pseudomonas aeruginosa is defective when this pathogen is opsonized with IgG antibodies isolated from serum samples from patients with cystic fibrosis (CF). To evaluate this defect further, IgG subclasses in the serum and lung fluids of patients with CF were quantitated. The pattern of IgG subclasses in serum specimens from patients with CF (n = 15) and in patients without CF but with chronic obstructive airway disease and recurrent P. aeruginosa infection (n = 4) was significantly altered from that found in normal subjects (n = 31). Immunoglobulin-G2 and IgG3 expressed as percentages of total IgG subclasses or in micrograms per milliliter of serum were significantly elevated in the serum specimens of these patients (p less than 0.05), and IgG1 was significantly decreased (p less than 0.01). It appears that the increase in IgG2 in the serum of patients with CF and those without CF but with chronic P. aeruginosa infection may be in response to chronic antigenic stimulation by P. aeruginosa lipopolysaccharide. Evidence presented to support this includes: (1) IgG2 is not increased in CF serum if a history of P. aeruginosa infection is absent, (2) IgG2 levels expressed as percentages of total IgG subclasses in CF lung fluids were positively correlated (r = 0.73) with the number of colony-forming units of P. aeruginosa present in CF sputum specimens, and (3) IgG antibodies specifically eluted from P. aeruginosa lipopolysaccharide ligands on affinity gels were largely restricted to IgG2. The opsonic index, ([IgG3] + [IgG1]) divided by ([IgG2] + [IgG4]), is inverted in CF lung fluids (0.73:1; normal, 2:1). Because pulmonary macrophages show surface receptors binding primarily with IgG3 and IgG1, it may be that such an alteration in IgG subclasses in the respiratory secretions of patients with CF further inhibits opsonin-mediated clearance of P. aeruginosa.