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.     

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.     

Mutators in cystic fibrosis chronic lung infection: Prevalence,mechanisms, and consequences for antimicrobial therapy

Hypermutable (or mutator) microorganisms are those that have an increased spontaneous mutation rate due to defects in DNA repair or error avoidance systems. Over last decade, several studies have provided strong evidence of a relevant role of mutators in the evolution of natural bacterial populations, with a special emphasis in the field of infectious diseases. Among them, chronic respiratory infection by Pseudomonas aeruginosa in cystic fibrosis (CF) patients was the first natural environment revealing a high prevalence and important role of mutators. High frequencies of mutators have also been noted for other pathogens in the CF setting, particularly Staphylococcus aureus and Haemophilus influenzae. In addition to a strong statistical link between hypermutation and antibiotic resistance (i.e., mutators are more frequently resistant against antibiotics) obtained from the analysis of collections of clinical isolates from CF patients, several in vitro and in vivo experiments have further highlighted the dramatic consequences of hypermutation, especially when acting on microorganisms that are genetically equipped to acquire efficient resistance to most antibiotics by mutations in chromosomal genes such as P. aeruginosa. Recent studies additionally showed that mutators accelerate genetic adaptation of P. aeruginosa to the airways of CF patients and that patients infected by mutator strains have a poorer lung function. Further prospective clinical studies are nevertheless needed for a deep evaluation of the impact of mutators in disease progression and outcome. Future research should particularly focus on the investigation of the optimal therapeutical strategies to prevent the emergence of mutator strains in the lungs of CF patients and to avoid multiple-antimicrobial resistance development if they have already been selected.     

Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.     

Lipopolysaccharide is present in immune complexes isolated from sputum in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection.

Sputum samples from seven patients with cystic fibrosis and chronic P. aeruginosa lung infection were investigated for immune complexes by PEG precipitation and in two different complement binding assays. All seven patients were immune complex positive. The components involved in immune complex formation were identified by SDS-PAGE and immunoblotting. We found P. aeruginosa lipopolysaccharide as a major antigen. Both core and O-specific saccharide antigens could be demonstrated. IgG and IgA were the immunoglobulins involved, with IgG2 as the dominating IgG subclass. Lipopolysaccharide has a number of biological activities and its presence in sputum may have consequences for the pathogenesis of lung disease in cystic fibrosis.     

Cytokines and inflammatory mediators do not indicate acute infection in cystic fibrosis

Various treatment regimens and difficulties with research design are encountered with cystic fibrosis (CF) because no standard diagnostic criteria exist for defining acute respiratory exacerbations. This study evaluated the role of serial monitoring of concentrations of selected cytokines and inflammatory mediators in serum and sputum as predictors of respiratory exacerbation, as useful outcome measures for CF, and to guide therapy. Interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), neutrophil elastase-alpha-1-protease inhibitor complex (NE complex), protein, and alpha-1-protease inhibitor (alpha-1-PI) were measured in serum and sputum collected from CF patients during respiratory exacerbations and periods of well-being. Levels of NE complex, protein, and alpha-1-PI in sputum rose during respiratory exacerbations and fell after institution of antibiotic therapy (P = 0.078, 0.001, and 0.002, respectively). Mean (+/- standard error of the mean) levels of IL-8 and TNF-alpha were extremely high in sputum (13,780 +/- 916 and 249.4 +/- 23.5 ng/liter, respectively) but did not change significantly with clinical deterioration of the patient (P > 0.23). IL-8 and TNF-alpha were generally undetectable in serum, and therefore these measures were unhelpful. Drop in forced expiratory volume in 1 s was the only clinical or laboratory parameter that was close to being a determinant of respiratory exacerbation (P = 0.055). This study provides evidence of intense immunological activity occurring continually within the lungs of adult CF patients. Measurement of cytokines and inflammatory mediators in CF sputum is not helpful for identifying acute respiratory exacerbations.     

Use of immunoblot detection of serum antibodies in the diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

Antibodies to Pseudomonas aeruginosa were investigated in serum from cystic fibrosis (CF) patients by immunoblotting (Western blotting). The results were compared with determinations of precipitating antibodies in serum by crossed immune electrophoresis (CIE). The number of CIE precipitins is a sensitive and specific indication of infection and is used, with sputum bacteriology, to distinguish between colonisation and invasive lung infection. Immunoblotting was considerably more sensitive than CIE for detecting antibodies to P. aeruginosa. Paired serum samples from 64 CF patients, taken before a diagnosis of P. aeruginosa lung infection and immediately afterwards, showed a marked increase in the number of serum antibodies with the onset of infection. The intensity of the reaction, as shown by the density of blotted bands, was also increased. Laser scanning densitometry of immunoblots, and of photographic negatives taken from them, was used to quantify the increases. Differences in the number and intensity of blotted bands were highly significant between the two groups. The reproducibility of the method was good. An immunoblot assay may be a sensitive and useful method for routine diagnosis of early P. aeruginosa lung infection in CF.     

Serum antibodies to Pseudomonas aeruginosa outer-membrane proteins and iron-regulated membrane proteins at different stages of chronic cystic fibrosis lung infection

Serum samples collected over periods up to 15 years from nine patients with cystic fibrosis (CF) were investigated by immunoblotting and crossed immuno-electrophoresis (CIE) for antibodies to Pseudomonas aeruginosa outer-membrane proteins (OMPs). The earliest antibody response to OMPs was directed against proteins G, H1 and I. Detection by immunoblotting sometimes preceded the CIE response; the appearance of antibodies to the other major OMPs was co-incident with an increase in CIE precipitins. Isolation of the mucoid form of P. aeruginosa was associated with a rapid increase in both precipitin numbers and antibodies detected by immunoblotting. Antibodies to iron-regulated OMPs could be detected in all the serum samples that showed eight or more CIE precipitins but their presence became pronounced only in the advanced stages of disease. The clinical strain used in this study and other isolates from CF patients showed several atypical OMPs, perhaps as a consequence of antibiotic therapy or related to the serum sensitivity of mucoid P. aeruginosa. Their expression in vivo was confirmed by detecting antibodies to them in patients' serum.     

True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis

Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the microorganisms present in transplanted CF lungs (n = 5) by standard culturing and 16S rRNA gene analysis. A correspondence between culturing and the molecular methods was observed. In conclusion, standard culturing seems reliable for the identification of the dominating pathogens.     

Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens.     

Heart-lung and lung transplantation for cystic fibrosis

End-stage lung disease in Cystic Fibrosis (CF) now is considered to be one of the indications for heart-lung or double lung transplantation. Results of this surgery for 50 or so CF patients in the US and Europe are about the same as for other diseases, although there are some postoperative problems specific for this diagnosis. These include: need for higher oral dosages of cyclosporine, likelihood of precipitation of diabetes mellitus with high dosage corticosterbid therapy for acute lung rejection, constant threat of pathogens remaining in the sinuses, increased likelihood of drug toxicity to the liver and kidneys, and need to make a psychological transition from a patient with a fatal disease to one with optimism about the future. Although improved postoperative management likely will improve postoperative mortality and morbidity, scarcity of donor organs and the high cost of the procedure will limit the impact of this procedure on the general CF population.     

Impact of Staphylococcus aureus on the pathogenesis of chronic cystic fibrosis lung disease

One of the first pathogens which can be isolated from the airways of cystic fibrosis (CF) patients is Staphylococcus aureus, which often persists in this hostile environment for many months or even years. The increase in infections due to the methicillin-resistant S. aureus (MRSA) worldwide and even more the emergence of community-acquired MRSA, which differ from nosocomial MRSA by lack of multiresistance and carriage of a phage-encoded toxin, Panton-Valentine leukocidin, attracts new attention to the epidemiology, pathogenesis, and impact of S. aureus in the background of CF. In this review, recent data and studies will be reported and discussed to give an overview of the latest research.     

Genotypic and phenotypic variation in Pseudomonas aeruginosa reveals signatures of secondary infection and mutator activity in certain cystic fibrosis patients with chronic lung infections

Evolutionary adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung is limited by genetic variation, which depends on rates of horizontal gene transfer and mutation supply. Because each may increase following secondary infection or mutator emergence, we sought to ascertain the incidence of secondary infection and genetic variability in populations containing or lacking mutators. Forty-nine strains collected over 3 years from 16 patients were phenotyped for antibiotic resistance and mutator status and were genotyped by repetitive-sequence PCR (rep-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Though phenotypic and genetic polymorphisms were widespread and clustered more strongly within than between longitudinal series, their distribution revealed instances of secondary infection. Sequence data, however, indicated that interlineage recombination predated initial strain isolation. Mutator series were more likely to be multiply antibiotic resistant, but not necessarily more variable in their nucleotide sequences, than nonmutators. One mutator and one nonmutator series were sequenced at mismatch repair loci and analyzed for gene content using DNA microarrays. Both were wild type with respect to mutL, but mutators carried an 8-bp mutS deletion causing a frameshift mutation. Both series lacked 126 genes encoding pilins, siderophores, and virulence factors whose inactivation has been linked to adaptation during chronic infection. Mutators exhibited loss of severalfold more genes having functions related to mobile elements, motility, and attachment. A 105-kb, 86-gene deletion was observed in one nonmutator that resulted in loss of virulence factors related to pyoverdine synthesis and elements of the multidrug efflux regulon. Diminished DNA repair activity may facilitate but not be absolutely required for rapid evolutionary change.     

The immune response to chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is predominantly of the Th2 type

Most cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection have a persistent acute type lung inflammation dominated by polymorphonuclear neutrophils (PMN) and a pronounced antibody response against P. aeruginosa. We speculated whether this immune response in CF is of the Th2 type and whether a change to a Th1 type immune response could improve the prognosis. Therefore, we studied 14 CF patients with (CF +P) and 14 CF patients without (CF -P) chronic P. aeruginosa lung infection. The specific production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by peripheral blood mononuclear cells was determined. Cells from CF +P patients had lower IFN-gamma (p<0.05) and higher IL-4 (p<0.005) production as compared to cells from CF -P patients. Furthermore, a positive correlation between IFN-gamma production and lung function was found (FVC: Rho = 0.637; p<0.03; FEV1: Rho=0.524; p<0.07). We conclude that a Th2 type immune response is most frequent in CF patients with chronic P. aeruginosa lung infection, and the patients with a Th1-dominated immune response had the best lung function. The clinical implication is that a change to a Th1 type immune response might improve the prognosis in these patients.     

Burkholderia cepacia complex infection in patients with cystic fibrosis

The word 'complex' has several meanings and synonyms such as composite, obsession, heterogeneous, mixed and network, can all be used in its place. Our obsession with bacteria from the Burkholderia cepacia complex started in the early 1990s. In less than 10 years, we have seen the status of this bacterium move from: (i) a lesser known pseudomonad opportunist pathogen, (ii) to devastating infections transmitted between patients with cystic fibrosis (CF), (iii) through divisions into several new species, and (iv) now on towards one of the largest gram-negative genome sequencing projects. For microbiologists, hospital infection control officers, caregivers, and most of all the CF community, the changes in our understanding of the taxonomy, epidemiology and pathogenesis of the bacterium 'B. cepacia' are complex.     

Fatal Burkholderia gladioli infection misidentified as Empedobacter brevis in a lung transplant recipient with cystic fibrosis

Data describing the risk of lung transplantation (LT), clinical features, and outcomes of patients with cystic fibrosis (CF) infected with Burkholderia gladioli are limited. Herein, we report a case of disseminated B. gladioli infection characterized by bacteremia, necrotizing pneumonia, lung abscess, and empyema in a lung transplant recipient with CF, highlight the importance of accurate microbiological identification, and review published outcomes of LT in CF patients infected with B. gladioli, which include cases of pneumonia, tracheobronchitis, bacteremia, and abscesses, and demonstrate an all-cause 1-year mortality of approximately 23%, often after combined medical and surgical treatment.