Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects.

The adherence of Pseudomonas aeruginosa PAO1 to primary cultures of cystic fibrosis nasal polyp (CFNP), normal human nasal polyp (NHNP), and immortalized CF and normal cell lines was studied. PAO1 bound significantly more to primary CFNP cells than to NHNP cells as the mean adherence +/- standard deviation of 5 x 10(7) CFU of 35S-labeled bacteria per ml per well was 15.09 x 10(6) +/- 4.25 x 10(6) CFU/ml per well and 7.62 x 10(6) +/- 2.11 x 10(6) CFU/ml per well, respectively (Mann-Whitney U test, P less than 0.0001). There was no significant difference in PAO1 adherence to the immortalized CF and normal cell lines. The primary CFNP cells had more receptors (115 per cell) than did NHNP cells (34 per cell). P. aeruginosa binding to CFNP was blocked by GlcNAc, NeuAc, L-Fuc, and D-Gal, while binding to NHNP was blocked only by GlcNAc, suggesting that receptors on the two cell types were qualitatively different. Pseudomonas supernatants containing protease, phospholipase C, and neuraminidase activity increased adherence to CFNP and NHNP cells. The Pseudomonas exoproducts modified epithelial cell glycoconjugates, as characterized by binding of fluorescein isothiocyanate-labeled lectins and the release of sialic acid. There was minimal release of fibronectin by the bacterial supernatants. The affinity of P. aeruginosa for CF epithelial cells appeared to be due to an increased number of receptors and modification of the epithelial cell surface by P. aeruginosa exoproducts that exposed asialoganglioside binding sites.     

Prevention of Pseudomonas aeruginosa infection in cystic fibrosis patients

In patients with cystic fibrosis (CF) prevention of lung infections with Pseudomonas aeruginosa is of major importance. Principles to achieve this goal include vaccination, immediate use of antibiotics in patients newly colonized with the pathogen, and hygienic measures. The purpose of this review is to discuss recent developments in this context.     

Pseudomonas aeruginosa suppresses interferon response to rhinovirus infection in cystic fibrosis but not in normal bronchial epithelial cells

Despite increased morbidity associated with secondary respiratory viral infections in cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infection, the underlying mechanisms are not well understood. Here, we investigated the effect of P. aeruginosa infection on the innate immune responses of bronchial epithelial cells to rhinovirus (RV) infection. CF cells sequentially infected with mucoid P. aeruginosa (MPA) and RV showed lower levels of interferons (IFNs) and higher viral loads than those of RV-infected cells. Unlike results for CF cells, normal bronchial epithelial cells coinfected with MPA/RV showed higher IFN expression than RV-infected cells. In both CF and normal cells, the RV-stimulated IFN response requires phosphorylation of Akt and interferon response factor 3 (IRF3). Preinfection with MPA inhibited RV-stimulated Akt phosphorylation and decreased IRF3 phosphorylation in CF cells but not in normal cells. Compared to normal, unstimulated CF cells or normal cells treated with CFTR inhibitor showed increased reactive oxygen species (ROS) production. Treatment of CF cells with antioxidants prior to MPA infection partially reversed the suppressive effect of MPA on the RV-stimulated IFN response. Together, these results suggest that MPA preinfection inhibits viral clearance by suppressing the antiviral response particularly in CF cells but not in normal cells. Further, increased oxidative stress in CF cells appears to modulate the innate immune responses to coinfection.     

Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin.

Growth and exoproduct production were examined with sputum from patients with respiratory diseases serving as the growth substrate for mucoid strains of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients. Mucoid strains are uniquely common to chronic respiratory infections of CF patients. The mucoid colonial morphology of P. aeruginosa is due to the biosynthesis of the exopolysaccharide alginate. Alginate-producing (Alg+) strains utilized CF sputum for growth and high yields of alginate; however, sputum from patients with other respiratory diseases produced comparable results. Analysis of CF sputum medium indicated that amino acids and small peptides were major substrates for P. aeruginosa in respiratory secretions. Cultures of Alg+ strains in CF sputum medium were inhibited in growth and reduced in alginate yields by a low concentration (1 mM) of D-mannose, suggesting therapeutic applications. The rates of growth of two Alg+ strains in CF sputum medium were found to be slightly lower compared with their respective spontaneous Alg- mutants, indicating that the mucoid phenotype does not enhance the ability of P. aeruginosa to utilize respiratory secretions. At all stages of growth in CF sputum medium, two Alg+ strains produced lower yields of protease than did their respective Alg- mutants. When seven Alg+ strains of CF origin were compared with their respective Alg- mutants, the Alg+ phenotype correlated with reduced yields of extracellular proteases. These data are consistent with the hypothesis that mucoid strains of P. aeruginosa are more suited to chronic rather than to acute respiratory infections in that reduced yields of proteases temper the level of damage to the lungs and result in a reduced infiltration of phagocytic cells.     

X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol 1^-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [CI], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [CI] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl^- via a Ca²⁺-dependent mechanism and inhibition of the Na⁺-K⁺-ATPase by intracellular Ca²⁺ ions causing an influx of Na⁺ and Cl^- ions.     

Serial isolates of Pseudomonas aeruginosa from a cystic fibrosis patient have identical pilin sequences.

Five isolates of Pseudomonas aeruginosa (CD2, CD3, CD4, CD5, and CD10) from a patient with cystic fibrosis were examined with regard to several genotypic and phenotypic characteristics to determine whether the patient was colonized with one or several distinct strains. Isolates CD2, CD3, and CD4 were obtained from a single sputum sample, and CD5 and CD10 were obtained 1 and 2 years later, respectively. On the basis of colonial morphology, serotyping, and antibiograms, the five isolates appeared to be different strains. However, Southern blot analysis with a 1.2-kilobase DNA probe containing the P. aeruginosa PAK pilin gene indicated that all five strains were identical at that genetic locus. The pilin genes of the five isolates were cloned and sequenced at the nucleotide level and found to be identical. Southern blot analysis with a probe from a separate region of the P. aeruginosa chromosome, a 741-base-pair PstI-NruI DNA fragment adjacent to the exotoxin A gene, also revealed genetic identity among these five clinical isolates. On this basis, it was concluded that this patient was colonized with a single strain of P. aeruginosa and that the strain had remained genetically stable over a period of 2 years. The predicted pilin sequence of the CD isolates was almost identical to that of strain PA103 (97% homology) and serologically related to PAO pilin, with which it shared 80% homology. No immunological cross-reactivity was detected between the CD and PAK pilins, which shared the least homology (62%) among the four pilins considered in this study. Although all five CD isolates contained identical pilin genes, three had acquired mutations which prevented normal expression of the pilus system. CD3 was a putative regulatory mutant which was unable to produce normal amounts of pilin, and CD4 and CD10 were putative assembly mutants which produced normal amounts of pilin but were unable to assemble the pilin subunit into intact pili.     

X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis.

X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol l-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [Cl], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [Cl] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl- via a Ca(2+)-dependent mechanism and inhibition of the Na(+)-K(+)-ATPase by intracellular Ca2+ ions causing an influx of Na+ and Cl- ions.     

Fecal isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

Fecal isolation of Pseudomonas aeruginosa was observed in 8 of 10 patients with cystic fibrosis who at the time of sampling also exhibited colonization of the respiratory tract. In contrast, P. aeruginosa cells were isolated at low frequency (9.1%) from the stools of 44 patients with cystic fibrosis with no previous history of chronic colonization. The results of this study suggest that the gastrointestinal tract is not a significant chronic reservoir of P. aeruginosa prior to pulmonary colonization.     

Protease production by Pseudomonas aeruginosa isolates from patients with cystic fibrosis

The temporal appearance of extracellular proteases produced by Pseudomonas aeruginosa was analyzed by pH 9 and pH 4 polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Ammonium sulfate precipitates of culture supernatants from various stages of growth revealed a time-dependent increase in number and amount of proteolytically active proteins. One mucoid P. aeruginosa clinical isolate and its derived nonmucoid variant, as well as two other nonmucoid variant P. aeruginosa strains (all from cystic fibrosis patients), showed similar production of five differently migrating proteases (P1 to P5, numbered according to increasing net negative charge) in pH 9 PAGE and one protease in pH 4 PAGE. P2, P3, and P5 increased to maximum concentrations at 24 to 48 h, decreasing thereafter, whereas P4 continued increasing even at 83 h, and P1 fluctuated. P3 was identified as an elastase. P2 was possibly composed of polypeptide chains bridged by disulfide bonds, since without reduction it migrated in sodium dodecyl sulfate-PAGE as a single protein, and with reduction it migrated as three protein bands. Two-dimensional PAGE revealed multiple molecular weight species within protease-positive bands in pH 9 gel strips. Isoelectric focusing gave a pattern of protein separation that correlated with two-dimensional PAGE analysis. Thus, greater heterogeneity of active proteases than previously reported has been demonstrated in all P. aeruginosa clinical isolates studied by sensitive two-dimensional PAGE analysis.     

Neutrophils enhance expression of inducible nitric oxide synthase in human normal but not cystic fibrosis bronchial epithelial cells

The bronchial epithelium in cystic fibrosis (CF) expresses very low levels of the inducible form of nitric oxide synthase (iNOS). The product of iNOS, nitric oxide (NO), mediates anti-microbial effects and can reduce neutrophil sequestration in the lung. Heavy neutrophilic infiltration of the pulmonary epithelium is a major feature of the end-stage CF lung. This study hypothesized that the system whereby the pulmonary epithelium protects itself against exaggerated neutrophilic infiltration by producing NO is compromised in CF. Human neutrophils were activated by incubation with cytokines, added to monolayers of normal (16HBE14o-) and CF (CFBE41o-) bronchial epithelial cells and co-cultured for up to 72 h. Marked up-regulation of iNOS protein expression was seen in normal bronchial epithelial cells following neutrophil co-culture but the CF cells showed a significantly smaller increase (p<0.001). To determine whether the relative lack of protein was due to a defect in translation, RT-PCR of iNOS mRNA was carried out and a pattern of mRNA expression was seen paralleling that of the protein. The reduced production of NO by CF compared with normal epithelium was shown by the presence of significantly (p<0.001) less accumulated nitrites in medium after co-culture with neutrophils. In summary, this study shows that the normal production of NO by bronchial epithelium in response to contact with neutrophils is lacking in CF. As NO has been shown to oppose neutrophil sequestration, its relative lack in CF may underlie the heavy neutrophilic infiltration that characterizes the disease.     

Induction of type I interferon signaling by Pseudomonas aeruginosa is diminished in cystic fibrosis epithelial cells

The clinical manifestations of infection in cystic fibrosis (CF) are restricted to the lung, and involve a limited number of pathogens, suggesting a specific defect in mucosal immunity. We postulated that cystic fibrosis transmembrane conductance regulator (CTFR) mutations could affect the activation of type I interferon signaling in airway epithelial cells, which function in immune surveillance and initiate the recruitment and activation of immune cells. In response to infection with Pseudomonas aeruginosa, Ifnb was induced more than 100-fold in the murine lung, and the phosphorylation of STAT1 was similarly induced by the expected TLR4/TRIF/MD2/TBK1 cascade. The stimulation by P. aeruginosa of CF (IB3) cells and control (C-38) human cell lines similarly resulted in the induction of IFN-β, but to a significantly lower extent in CF airway cells. The potential consequences of diminished type I IFN signaling were demonstrated in a murine model of P. aeruginosa pneumonia, pretreatment with polyinosinic:polycytidylic acid significantly enhanced bacterial clearance and correlated with increased numbers of mature CD11c(+)/CD86(+) dendritic cells (DCs) in the lung. Using culture supernatants from CF or control cell lines stimulated with P. aeruginosa, we similarly demonstrated the diminished activation of human monocyte-derived DCs by incubation with CF compared with normal epithelial cell culture supernatants, which was dependent on IFN-β. These observations suggest that dysfunction of the CFTR in airway epithelial cells may contribute to impaired immune surveillance in the CF airway and resultant colonization by P. aeruginosa.     

Ecology of Pseudomonas aeruginosa in patients with cystic fibrosis

The occurrence of various Pseudomonas aeruginosa strains in the sputum of 15 patients with cystic fibrosis (CF) was monitored over periods ranging from 2 to 60 months. Isolates of P. aeruginosa were typed by four different techniques, namely serotyping, active and passive pyocin typing, and phage typing. The maximum number of different serotypes found in the patients was three (one serotype in nine patients; two serotypes in five patients; three serotypes in one patient). Pyocin and phage typing showed no marked differences between strains of the same serotype in individual patients. Exacerbations of chronic respiratory infection were not associated with changes in the sputum flora, the composition of P. aeruginosa strains in which remains constant over long periods in patients with CF.     

Pyocin typing of Pseudomonas aeruginosa isolates from children with cystic fibrosis.

Pyocin typing and serotyping of 433 strains of Pseudomonas aeruginosa from children with cystic fibrosis (CF) showed that pyocin type 9 was predominant, particularly in association with polyagglutinating serotype. The common pyocin groups, 1, 5 and 10, made up only 20% of these isolates in contrast to reported rates of up to 89% in other studies using non-CF strains. No strains of pyocin type 3 were found. Polyagglutinating strains made up 72% of strains from patients colonized with P. aeruginosa for more than 12 mths. Pyocin type 9 was associated with 93% of polyagglutinating strains. The parallel between pyocin type 9 and polyagglutinating serotype suggests that these may both be characteristics acquired by P. aeruginosa colonizing patients with CF. Because of confounding between duration of colonization and exposure to cross-infection, this study does not allow definition of the role of cross-infection in determining the characteristics of these strains in most patients. In siblings, however, evidence supports a role for cross-infection either between siblings or from a common source. In 6 pairs of siblings studied, each pair had at least 1 pyocin group in common concurrently, either at entry to the study or after an interval of several months. Identical and unusual pyocin groups were recognized in samples obtained on the same day from pairs of siblings. More studies are needed to compare results of pyocin typing with methods such as genome fingerprinting to characterize these strains and determine whether the observed distribution of pyocin groups in CF isolates is related to cross-infection or whether the combination of pyocin type 9 with polyagglutinating serotype is a characteristic of CF strains.     

Protease phenotypes of Pseudomonas aeruginosa isolated from patients with cystic fibrosis.

The protease phenotypes expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients were evaluated. The majority of isolates tested produced elastase (65%) or alkaline protease (64%) or both. The mucoid phenotype expressed by many CF isolates of P. aeruginosa did not absolutely restrict the expression of protease activity, although a higher percentage of nonmucoid isolates was proteolytic. When isolates from CF patients chronically infected with P. aeruginosa were compared to isolates from CF patients colonized with this organism, both groups were found to contain comparable percentages of elastase-producing strains and mucoid strains. However, the group of isolates from colonized patients contained a higher percentage of strains producing alkaline protease and expressing general protease activity. In addition, the group of isolates from chronically infected patients contained more weakly proteolytic isolates than either the group from colonized CF patients or a group of isolates from pediatric patients without CF. These data suggest that protease production may be important in the initial colonization of the respiratory tract of CF patients by P. aeruginosa.     

Typing of polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients

The study assesses the reproducibility, typability and discriminatory power of several typing methods for Pseudomonas aeruginosa isolated from cystic fibrosis patients. 178 polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients were serotyped using polyclonal sera and monoclonal antibodies, phage typed, pyocin typed and reverse phage typed. 31 of these polyagglutinable isolates, six monoagglutinable isolates and three nontypable isolates were also typed by means of hybridization using a DNA probe. In a comparison of the methods used, on polyagglutinable isolates only, typability was 0% with polyclonal sera, 90% with monoclonal sera, 94% with phage typing, 85% with pyocin typing, 36% with reverse phage typing and 100% with DNA-prope typing. Using monoclonal antibodies, the reproducibility was 75%, while that of phage typing was 88%, pyocin typing 53% and reverse phage typing 62%. Typing with the DNA probe was not repeated. using polyclonal sera, repeated typing showed that 94% of the isolates were polyagglutinable. Using phage typing, 40% of the isolates belonged to phage type 31, while 60% were distributed amongst 32 phage types. Using monoclonal antibodies, 71% of the isolates belonged to 0-group 3, and these isolates showed 16 different phage types. Subdivision of the phage types was further achieved by both pyocin typing and reverse phage typing. The DNA probe typing made it possible in some cases to discriminate between isolates which were otherwise found identical with the conventional typing methods, while in other cases typing with the DNA probe recorded as identical isolates which conventional methods had typed as being different. These differences may be due to a high mutation rate caused by the selection pressure of antibiotics, and by the host immune response. According to our results, investigations of reproducibility and typability of old and new typing methods are essential when they are used in clinical situations. The low reproducibility of some of the typing methods in the present study affects the reliability of epidemiological investigations in cystic fibrosis patients. Usage of only one method may not be sufficient in cases of polyagglutinable strains from cystic fibrosis patients.     

Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.     

Pseudomonas aeruginosa strains from the chronically infected cystic fibrosis lung display increased invasiveness of A549 epithelial cells over time

The invasive properties of Pseudomonas aeruginosa pose a serious threat to the wellbeing of cystic fibrosis (CF) patients; however the specific factors affecting invasiveness are not well understood, especially in chronic infection. This study characterises the invasive profiles of sequential isolates of the same P. aeruginosa strain collected five to eight years apart from five chronically infected adult CF patients. Strains from three patients were characterised as unique isolates and from two patients as the Australian Epidemic strain (AES-1) by pulsed field gel electrophoresis. The capacity of these strains to invade the human alveolar A549 cell line was examined. Later isolates were significantly more invasive than earlier counterparts from the same patient. Quantitative real-time PCR and Western blotting showed that the increase in invasiveness over time was independent of ExoS expression and secretion. A link between clonality and invasiveness was also identified, with AES-1 isolates more invasive than unique isolates. These results suggest that despite a reduction in some virulence factors such as the Type-3 Secretion System (T3SS) during chronic infection, a particular strain can become more invasive over time. Defining mechanisms behind the increased invasiveness during chronic infection may help identify new therapeutic targets for CF patients.     

Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells.

The ability of pili from Pseudomonas aeruginosa K (PAK) to act as an adhesin to human respiratory epithelial cells was examined using an in vitro adhesion assay. Equilibrium analysis of PAK binding to human buccal epithelial cells (BECs) and tracheal epithelial cells (TECs) by means of a Langmuir adsorption isotherm revealed that the maximum numbers of binding sites per epithelial cell (N) were 255 for BECs and 236 for TECs, with apparent association constants (Ka) of 2.8 x 10(-9) and 5.8 x 10(-9) ml/CFU, respectively. Trypsinization of the BECs before the binding assay increased N to 605 and decreased the Ka to 1.7 x 10(-9) ml/CFU. Addition of homologous pili to the binding assay with BECs or TECs or the addition of anti-pilus Fab fragments inhibited PAK adherence. Binding of purified pili to BECs was shown to reach saturation. Purified pili and PAK competed for the same receptor on the BEC surface. Further, by using peptide fragments of PAK pilin (derived from the native pili or produced synthetically) in the binding assay for PAK to BECs, we have presumptively identified the pilus binding domain in the C-terminal region of the pilin and shown that the C-terminal disulfide bridge is important in maintaining the functionality of the binding domain.