Respiratory Tract Of Cystic Fibrosis Patients Research Articles

Association Between Respiratory Tract Methicillin-Resistant S<emph type="ital">taphylococcus aureus</emph> and Survival in Cystic Fibrosis

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the respiratory tract of individuals with cystic fibrosis (CF) has increased dramatically; however, its impact on outcomes in CF is unclear. Because the time between infection with bacteria in CF and death can be decades, observational studies with long periods of follow-up are well suited to address the current gap in knowledge. To determine whether isolation of MRSA from the respiratory tract of CF patients is associated with worse survival compared with patients who never have a culture positive for MRSA. Cohort study of 19,833 CF patients aged 6 to 45 years seen at centers accredited by the Cystic Fibrosis Foundation in the United States. Patients entered between January 1996 and December 2006 and were followed up through December 2008. Cox regression models with time-varying covariates were used to compare survival between CF patients with and without respiratory tract MRSA. Time from age at entry until age at death from any cause. In 137,819 patient-years of observation (median, 7.3 years/patient), 2537 CF patients died and 5759 patients had MRSA detected. The mortality rate was 18.3 deaths (95% confidence interval [CI], 17.5-19.1) per 1000 patient-years in patients without MRSA and 27.7 deaths (95% CI, 25.3-30.4) per 1000 patient-years in those with MRSA. Among those with MRSA, the attributable risk percentage of death associated with MRSA was 34.0% (95% CI, 26.7%-40.4%). The unadjusted hazard ratio associated with MRSA was 1.47 (95% CI, 1.32-1.62). After adjustment for time-varying covariates associated with severity of illness, MRSA remained associated with a higher risk of death (1.27; 95% CI, 1.11-1.45). Detection of MRSA in the respiratory tract of CF patients was associated with worse survival.

Emergence of Respiratory Streptococcus agalactiae Isolates in Cystic Fibrosis Patients

Streptococcus agalactiae is a well-known pathogen for neonates and immunocompromized adults. Beyond the neonatal period, S. agalactiae is rarely found in the respiratory tract. During 2002–2008 we noticed S. agalactiae in respiratory secretions of 30/185 (16%) of cystic fibrosis (CF) patients. The median age of these patients was 3–6 years older than the median age CF patients not harboring S. agalactiae. To analyze, if the S. agalactiae isolates from CF patients were clonal, further characterization of the strains was achieved by capsular serotyping, surface protein determination and multilocus sequence typing (MLST). We found a variety of sequence types (ST) among the isolates, which did not substantially differ from the MLST patterns of colonizing strains from Germany. However serotype III, which is often seen in colonizing strains and invasive infections was rare among CF patients. The emergence of S. agalactiae in the respiratory tract of CF patients may represent the adaptation to a novel host environment, supported by the altered surfactant composition in older CF patients.

Persistence ofCandidaspecies in the respiratory tract of cystic fibrosis patients

It is still controversial as to whether Candida spp. are transient or persistent colonizers of the respiratory tract of cystic fibrosis (CF) patients. We conducted a prospective study of 56 CF patients over a 30 month period to assess the distribution and persistence of different Candida spp. In vitro antifungal susceptibility testing was performed and the C. albicans isolates were typed with CARE-2 hybridization and other Candida spp. by RAPD-PCR for persistence and transmission. We found that the mean persistence of the most frequent Candida spp. was >or= 9 months. In patients from whom more than 10 isolates were recovered, we noted that at least 30% were genetically related and transmission of C. albicans in siblings was observed. The majority of all isolates were susceptible to all antifungals tested. We concluded that there was long-term persistence of Candida in the respiratory tract of CF patients and that transmission between siblings may be one possible means of acquisition. Whether long-term colonization with Candida strains can contribute to the chronic infection and inflammation in the CF lung requires further investigation.

Aerosol Antibiotics in Cystic Fibrosis

Chronic airways infection and inflammation is the greatest source of morbidity and mortality in cystic fibrosis (CF) patients. Many organisms can be found in the lower respiratory tract of CF patients, but infection with mucoid Pseudomonas aeruginosa is common, is associated with poorer outcomes, and is the main target for antimicrobial strategies in CF. Aerosol antibiotics achieve high local concentrations in the airways, reduce systemic toxicity, and have been used successfully for chronic suppressive treatment for established P. aeruginosa infections. Eradication of early P. aeruginosa airway infection has also been tried with aerosol antibiotics, though the ideal treatment strategy is still being investigated. There are several variables to consider when choosing an antibiotic formulation to develop for topical inhalation. Tobramycin solution for inhalation (TSI) is currently the only approved inhaled antibiotic in the United States. The time burden for patients to administer TSI by jet nebulizer is substantial, so efforts have focused on more efficient, faster delivery methods. Novel formulations of aerosol antibiotics are being studied for CF, including beta-lactams, fluoroquinolones and aminoglycosides. Phase-3 studies of aztreonam lysinate for inhalation delivered via a proprietary eFlow nebulizer showed improved outcomes and a short (< 3 min) delivery time. Liposome formulations are being studied as a way to penetrate mucoid biofilms and prolong the residence time of the antibiotic in the lungs. Light, porous, dry-powder formulations are also in clinical trials to reduce delivery time. These new formulations and delivery systems promise to expand our armamentarium against microbes while reducing the time burden for patients.

Selective Isolation of Pseudallescheria and Scedosporium Species from Respiratory Tract Specimens of Cystic Fibrosis Patients

Background: Fungi of the Pseudallescheria/Scedosporium complex are known to be colonizers and infectious agents of the respiratory tract of cystic fibrosis (CF) patients. Colonized CF patients are at high risk for the development of disseminated scedosporiosis after lung transplantation. The detection of these fungi may be difficult, because they grow slowly and so will be overgrown by faster-developing microorganisms on the media routinely used in diagnostic laboratories. Objectives: To examine the usefulness of the isolation medium SceSel+ agar as a diagnostic tool for clinical samples from CF patients. Methods: 150 respiratory tract samples from 42 CF patients were inoculated on SceSel+ agar. During the incubation phase, which lasted up to 30 days at 36 ± 1°C, the cultures were inspected every 2 or 3 days. Results: The isolation of Scedosporium species was successful in 3 samples (2%) with standard microbiological media and procedures, while the isolation rate on SceSel+ agar was 5.3% (8 of 150 specimens). Conclusions: Our results suggest that standard microbiological media and procedures are not sufficient to detect colonization of the respiratory tract by Pseudallescheria/Scedosporium in CF patients. By use of SceSel+ agar, fungi belonging to this complex were isolated more frequently. Therefore, this semiselective mycological isolation medium should be used for the detection of these fungi in the respiratory tract of CF patients, especially in patients in whom a fungal infection is assumed or who are scheduled for lung transplantation.

P1541 Characteristics of Staphylococcus aureus isolated from the respiratory tract of cysticfibrosis patients

P1541 Characteristics of Staphylococcus aureus isolated from the respiratory tract of cysticfibrosis patients

Sequence variability and functional analysis of MutS of hypermutable Pseudomonas aeruginosa cystic fibrosis isolates

In this study, we investigated the variability of MutS among Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. Sequencing of the mutS gene of 15 hypermutable P. aeruginosa isolates obtained from different patients revealed high rates of nucleotide substitutions as compared to that of strain PAO1. Significantly more synonymous than non-synonymous nucleotide substitutions have been found, indicating that generally MutS is highly conserved. The functional analysis of MutS variants by complementation of a PAO1 mutS mutant revealed 5 isolates with a defective MutS due to frameshift mutations or amino acid substitutions. This work supports the hypothesis that the respiratory tract of CF patients represents an environment that favors the selection of highly adaptive mutator phenotypes.

Eradication of early Pseudomonas aeruginosa infection

Chronic pulmonary infection with Pseudomonas aeruginosa is responsible for most of the morbidity and mortality in cystic fibrosis (CF). Once established as a biofilm, chronic P. aeruginosa infection caused by the mucoid phenotype cannot be eradicated. However, a period of intermittent colonization with P. aeruginosa precedes the establishment of the chronic infection. This window of opportunity can be utilized to eradicate P. aeruginosa from the respiratory tract of CF patients by means of oral ciprofloxacin in combination with nebulized colistin for 3 weeks or, even better, for 3 months or by means of inhaled tobramycin as monotherapy for 4 weeks or longer. This early, aggressive eradication therapy has now been used for 15 years without giving rise to resistance to the antibiotics and without serious side effects. The therapeutic results have been very successful and have completely changed the epidemiology in the Danish Cystic Fibrosis Center and a few other centers which have used this strategy for several years. The chronic P. aeruginosa lung infection is not seen in CF infants and children anymore due to the aggressive therapy, and no other bacteria have replaced P. aeruginosa in these young patients. The aggressive therapy has been shown to very cost-effective, and a European Consensus report recommends this approach.

Identification of genomic groups in the genus Stenotrophomonas using gyrB RFLP analysis

Stenotrophomonas maltophilia isolates have been recovered from various clinical samples, including the respiratory tract of cystic fibrosis (CF) patients, but this organism is also widespread in nature. Previously it has been shown that there is a considerable genomic diversity within S. maltophilia. The aims of our study were to determine the taxonomic resolution of restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction-amplified gyrB gene for the genus Stenotrophomonas. Subsequently, we wanted to use this technique to screen a set of S. maltophilia isolates (with emphasis on a specific subset, isolates recovered from CF patients), to assess the genomic diversity within this group. In this study we investigated 191 Stenotrophomonas sp. isolates (including 40 isolates recovered from CF patients) by means of gyrB RFLP. The taxonomic resolution of gyrB RFLP, and hence its potential as an identification tool, was confirmed by comparison with results from published and novel DNA–DNA hybridisation experiments. Our data also indicate that the majority of CF isolates grouped in two clusters. This may indicate that isolates from specific genomic groups have an increased potential for colonisation of the respiratory tract of CF patients.

Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov.

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii, Burkholderia fungorum, Comamonas testosteroni, Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis, Pandoraea genomospecies 4, Ralstonia gilardii, Ralstonia mannitolilytica, Rhizobium radiobacter, and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the alpha-PROTEOBACTERIA: Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family ENTEROBACTERIACEAE: The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.

Elevation of nitrotyrosine and nitrate concentrations in cystic fibrosis sputum.

Nitric oxide (NO) is increased in the exhaled air of some patients with inflammatory lung disorders, but not in others. NO may combine with superoxide to form peroxynitrite, which lowers NO gas concentrations, increases formation of nitrate, and increases nitration of tyrosine residues on proteins. We hypothesized that superoxide released from neutrophils in the lower respiratory tract of cystic fibrosis (CF) results in increased nitrate and nitrotyrosine levels in sputum. In order to test this hypothesis, exhaled NO was collected from 5 stable adult CF subjects and from 5 nonsmoking normal controls. Consistent with previous reports, exhaled NO concentrations were not increased in CF exhaled air (22.6 +/- 1.5 ppb vs. 28.6 +/- 1.5 ppb in normals, P > 0.05). Sputum was collected from 9 adult CF subjects and the same 5 normal controls and evaluated for nitrite, nitrate, and nitrotyrosine. Nitrate and nitrotyrosine levels, but not nitrite, were significantly elevated in CF. Recently, myeloperoxidase has also been implicated as a mechanism of nitrotyrosine formation. Therefore, myeloperoxidase was measured and found to be elevated in the CF sputum (64.2 +/- 35.9 vs. 0.73 +/- 0.16 U/mL, P < 0.001), and was found to correlate with concentrations of nitrotyrosine (r = 0.87, P < 0.05). However, in vitro studies with myeloperoxidase and murine lung epithelial cells did not demonstrate a reduction of NO gas with nitrotyrosine or an increase in nitrate formation. These data demonstrate that nitrate and nitrotyrosine are elevated in the sputa of CF subjects and suggest increased production of NO in the lower respiratory tract of CF patients, despite the relatively low exhaled NO levels. Pediatr Pulmonol. 2000; 30:79-85. Published 2000 Wiley-Liss, Inc.

Antimicrobial susceptibility of Haemophilus influenzae in the respiratory tracts of patients with cystic fibrosis.

We analyzed the antimicrobial susceptibilities of Haemophilus influenzae isolates from 157 sputum specimens prospectively collected from 39 cystic fibrosis (CF) patients during a 2-year study. These isolates were characterized by random amplified polymorphic DNA analysis and major outer membrane protein (MOMP) analysis to identify H. influenzae strains and MOMP variants and to assess their persistence in the respiratory tract. Among the 247 H. influenzae isolates, 16 (6.5%) produced beta-lactamase. The 231 beta-lactamase-negative isolates represented 85 H. influenzae strains, 61 MOMP variants derived from 27 of these strains, and 85 persistent isolates identical to strains or MOMP variants. All beta-lactamase-negative isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, cefuroxime, cefotaxime, cefaclor, imipenem, tetracycline, and trimethoprim-sulfamethoxazole by disk diffusion testing. Eleven (13%) H. influenzae strains, 18 (30%) MOMP variants, and 30 (35%) persistent isolates were resistant to one or more of the antibiotics tested. Antimicrobial susceptibility was decreased among MOMP variants and persistent isolates compared to nonpersistent H. influenzae strains, and changes in susceptibility occurred irrespective of MOMP variation. We conclude that the decreased antimicrobial susceptibility of H. influenzae during persistence contributes to the poor eradication of H. influenzae from the respiratory tracts of CF patients.

Chloride channels of intracellular organelles and their potential role in cystic fibrosis.

Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels. A 64 x 10(3) M(r) protein, p64, identified as a component of this chloride channel, was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, also depleted solubilized kidney cortex from all chloride channel activity. The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinases A and C. There was no significant homology to other protein (or DNA) sequences in the data base including other anion channels or the cystic fibrosis transmembrane conductance regulator. The protein is expressed in all cells tested and probably represents the chloride channel of intracellular organelles. Cystic fibrosis (CF) is associated with a defect in a cyclic-AMP-activated chloride channel in secretory epithelia which leads to decreased fluid secretion. In addition, many mucus glycoproteins show decreased sialylation but increased sulfation. We have recently shown that the pH of intracellular organelles is more alkaline in CF cells, an abnormality that is due to defective chloride conductance in the vesicle membranes. We postulate that the defect in the intracellular chloride channel, and hence the alkalization, could explain the glycosylation abnormalities since the pH optimum of Golgi sialyltransferase is acid while that of focusyl- and sulfotransferases is alkaline. Defects in sialyation of glycolipids might also generate receptors for Pseudomonas, which is known to colonize the respiratory tract of CF patients.

Chloride channels, Golgi pH and cystic fibrosis

Cystic fibrosis (CF) is associated with a defect in a cAMP-activated chloride channel in secretory epithelia, which leads to decreased fluid secretion. In addition, many mucus glycoproteins show decreased sialylation but increased sulphation. We have recently shown that the pH of intracellular organelles is elevated in CF cells, due to defective chloride conductance in the vesicle membranes. We postulate that this may affect the activity of sialyl-, fucosyl- and sulphotransferases, and thus explain the abnormal glycosylation. Defects in sialylation of glycolipids might also generate receptors for Pseudomonas, which infects the respiratory tract of CF patients.

Cryopreservation of cystic fibrosis nasal epithelium

Cystic fibrosis (CF) is the most common lethal genetic disease which affects Caucasians. The respiratory system is almost universally affected in CF patients, and the basic defect in this organ system as well as in others appears to be an abnormality of the regulation of chloride ion transport; the transport of other ions may also be either primarily or secondarily affected. Both the nasal mucosa and the lower respiratory tracts of CF patients exhibit this defect, but the nasal mucosa is free of the infectious complications common in the lower tract regions. Electron probe x ray microanalysis (EPXMA) promises to be a useful tool in the investigation, at the cellular and subcellular levels, of this ion transport defect as well as possible defects in the transport of other ions. This report documents the development of techniques for obtaining well-preserved cryosections of human nasal mucosa which are suitable for subsequent EPXMA.

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.

Role of adherence in the pathogenesis of Pseudomonas aeruginosa lung infection in cystic fibrosis patients.

A correlation has been demonstrated between the in vitro adherence of Pseudomonas aeruginosa to upper respiratory tract epithelium and colonization of the respiratory tract by this organism. Twenty patients with cystic fibrosis (CF) and 20 age-matched controls were examined in this study. All of the CF patients but none of the controls were colonized with P. aeruginosa at the time of study. P. aeruginosa adherence to isolated epithelial cells, as determined by an in vitro assay, was 19.1 +/- 1.1 bacteria per buccal epithelial cell in the CF patients and 2.3 +/- 0.3 bacteria per cell in the controls (P < 0.01). P. aeruginosa strains of the mucoid colony type adhered in significantly lower numbers to buccal epithelial cells than did strains of the rough colony type (1.8 + 0.1 versus 24.8 +/- 0.9, P < 0.001). This difference might explain the common observation that the initial pseudomonas colonization of the respiratory tract of CF patients is due to organisms of the rough colony type. We have further demonstrated that increased P. aeruginosa adherence in vitro varies directly with the loss of a protease-sensitive glycoprotein, fibronectin, from the cell surface, as well as increased levels of salivary proteases in CF patients. When examined by a direct radioimmune binding assay, buccal cells from CF patients possessed only 17% of the total cell surface fibronectin present on similar cells obtained from controls. Salivary protease levels, as measured by (125)I release from an (125)I-labeled insoluble fibrin matrix, were increased about threefold in CF patients versus controls. Thus, colonization of the respiratory tract by P. aeruginosa in CF patients correlates well with buccal cell adherence of this organism; increased adherence is associated with decreased amounts of fibronectin on respiratory epithelial cell surfaces and increased levels of salivary proteases.