C.R. Hansen

Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients

The present study was undertaken to investigate the appearance and location of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung and in sputum. Samples include preserved tissues of CF patients who died due to chronic P. aeruginosa lung infection prior to the advent of intensive antibiotic therapy, explanted lungs from 3 intensively treated chronically P. aeruginosa infected CF patients and routine sputum from 77 chronically P. aeruginosa infected CF patients. All samples were investigated microscopically using hematoxylin–eosin (HE), Gram and alcian‐blue stain, PNA FISH and immunofluorescence for alginate.

Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonization in cystic fibrosis patients: 15 years experience.

BACKGROUND Since 1989, CF-patients intermittently colonized with Pseudomonas aeruginosa have been treated with inhaled colistin and oral ciprofloxacin in the Copenhagen CF-centre. The study evaluates 15 years results of this treatment. METHODS All isolates of P. aeruginosa from CF-patients intermittently colonized with P. aeruginosa from 1989 to 2003 were identified All anti-P. aeruginosa treatments were evaluated for antibiotics used, treatment duration, pseudomonas-free interval and development of chronic infection. All P. aeruginosa isolates were assessed for resistance and for non-mucoid or mucoid phenotype. RESULTS 146 CF-patients were included in the study (1106 patient-years). 99 patients had first ever isolate during the study period. Median observation time 7 years (0.1-14.9). 12 patients developed chronic infection. A Kaplan Meyer plot showed protection from chronic infection in up to 80% of patients for up to 15 years. 613 colistin/ciprofloxacin treatments were given. There was no difference in pseudomonas-free interval comparing 3 weeks (5 months) and 3 months (10.4 months) of colistin and ciprofloxacin, but a significant difference compared to no treatment (1.9 months). Patients developing chronic infection had significantly shorter pseudomonas-free interval after treatment of first ever isolate compared to patients remaining intermittently colonized (p<0.003). Treatment failure (P. aeruginosa-positive culture immediately after ended treatment of first ever isolate) was a strong risk factor for development of chronic infection after 3-4 years, OR 5.8. 1093 pseudomonas-isolates were evaluated (86.6% non-mucoid). No colistin-resistance was found. Ciprofloxacin-resistance was found in 4% of isolates. CONCLUSION Treatment of intermittent P. aeruginosa colonization in CF-patients using colistin and ciprofloxacin can protect up to 80% of patients from development of chronic infection for up to 15 years. A positive culture immediately after treatment of first ever isolate is a strong risk factor for development of chronic infection. We found no colistin-resistance and minimal ciprofloxacin-resistance.

Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis

Background: Chronic lung infection with Pseudomonas aeruginosa is the most severe complication for patients with cystic fibrosis (CF). This infection is characterised by endobronchial mucoid biofilms surrounded by numerous polymorphonuclear leucocytes (PMNs). The mucoid phenotype offers protection against the PMNs, which are in general assumed to mount an active respiratory burst leading to lung tissue deterioration. An ongoing respiratory burst by the PMNs has, however, not been demonstrated previously in endobronchial secretions from chronically infected patients with CF. Objective: Based on the accumulating evidence for depletion of molecular oxygen (O2) in the mucus in infected CF bronchi, it was hypothesised that the O2 depletion in the mucus in infected CF bronchi may be accelerated by the respiratory burst of the PMNs due to the reduction of O2 to the superoxide anion (O-2) by the phagocyte NADPH oxidase (Phox). Methods: Methods were established to isolate the O2 consumption by the respiratory burst from aerobic respiration in freshly expectorated sputum from chronically infected patients with CF. Results: Inhibition of the Phox with diphenylene iodonium (DPI) delayed O2 depletion, nearly abolished staining of O-2-producing PMNs with hydroethidine and inhibited the rapid luminol-enhanced chemiluminescence in sputum. Furthermore, the total O2 consumption was correlated to the concentration of PMNs in the sputum samples. Conclusion: The results demonstrate that CF sputum contains PMNs with an active consumption of O2 for O-2 production and suggest that the respiratory burst is ongoing and causes accelerated O2 depletion due to formation of O-2 in the lungs of chronically infected patients with CF.

Respiratory bacterial infections in cystic fibrosis.

Purpose of review Bacterial respiratory infections are the main cause of morbidity and mortality in patients with cystic fibrosis (CF). Pseudomonas aeruginosa remains the main pathogen in adults, but other Gram-negative bacteria such as Achromobacter xylosoxidans and Stenotrophomonas maltophilia as well as nontuberculous mycobacteria have been shown to play an important role in the lung disease. The purpose of this review is to summarize the knowledge on disease and treatment of infection with CF-related pathogens. Recent findings The role of the paranasal sinuses as a source of infection and site of bacterial adaptation has been recognized. This review will focus on the different conditions encountered by the bacteria in sinuses and lung, as well as the principles of treatment in the different infection sites. Summary Chronic, pulmonary infections remain the single most prominent cause of the increased morbidity and mortality in CF. After the increasing efficiency of anti-P. aeruginosa treatment, newer pathogens have been identified, with individual clinical characteristics. Microbiological surveillance is very important in keeping the patients stable. Samples from both the lower and upper respiratory tract (nasal sampling) should be investigated and both infection sites should be treated.

Inflammation in Achromobacter xylosoxidans infected cystic fibrosis patients

BACKGROUND Achromobacter xylosoxidans infection may cause conspicuous chronic pulmonary inflammation in cystic fibrosis (CF) patients similar to Pseudomonas aeruginosa and the Burkholderia cepacia complex (Bcc). Evolution in lung function was compared in chronically infected patients. Cytokine concentrations in CF patients with and without chronic infection were compared to healthy controls. METHODS Cytokines in serum and sputum were measured using multiplex bead based immunoassay. RESULTS Sixty CF patients, 11 with A. xylosoxidans, 11 with Bcc, 21 with P. aeruginosa and 17 non-infected CF patients were compared to 11 healthy controls. A. xylosoxidans patients were younger, but had a FEV(1) decline similar to P. aeruginosa patients. Bcc patients had the steepest decline in FEV(1). Serum levels of G-CSF, IL-6 and TNF-alpha were significantly higher in CF patients compared to healthy controls. Chronically infected CF patients had significantly higher serum levels of IFN-gamma and IL-6 compared to non-infected CF patients. Bcc patients had significantly lower serum G-CSF and A. xylosoxidans patients had significantly higher sputum TNF-alpha compared to the other groups of chronically infected patients. CONCLUSION A. xylosoxidans can cause a level of inflammation similar to P. aeruginosa in chronically infected CF patients. A. xylosoxidans is a clinically important pathogen in CF and should be treated accordingly.

Epidemiology of nontuberculous mycobacteria among patients with cystic fibrosis in Scandinavia

Background Nontuberculous mycobacteria (NTM) are an emerging threat to cystic fibrosis (CF) patients but their epidemiology is not well described. Methods In this retrospective observational study we identified all Scandinavian CF patients with a positive NTM culture from airway secretions from 2000 to the end of 2012 and used national CF databases to describe microbiological and clinical characteristics. Results During the 13-year period 157 (11%) CF patients were culture positive for NTM at least once. Mycobacterium abscessus complex (MABSC) (45%) and Mycobacterium avium complex (MAC) (32%) were the predominant species with geographical differences in distribution. Younger patients were more prone to MABSC (p < 0.01). Despite treatment, less than one-third of MABSC patients with repeated positive cultures cleared their infection and a quarter had a lung transplant or died. Conclusion NTM are significant CF pathogens and are becoming more prevalent in Scandinavia. MABSC and MAC appear to target distinct patient groups. Having multiple positive cultures despite treatment conveys a poor outcome.

Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO3 − and NO2 − in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N2O (range 1.4–157.9 µM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3 − decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.

Complete Genome Sequence of the Cystic Fibrosis Pathogen Achromobacter xylosoxidans NH44784-1996 Complies with Important Pathogenic Phenotypes

Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen.

Long-term, low-dose azithromycin treatment reduces the incidence but increases macrolide resistance in Staphylococcus aureus in Danish CF patients

BACKGROUND Since 2001, long-term, low-dose azithromycin treatment has been used for CF patients chronically infected with Pseudomonas aeruginosa in the Copenhagen CF centre. Our study investigates changes in incidence of colonization with Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis and changes in macrolide sensitivity in these microorganisms during azithromycin treatment. METHODS CF patients treated continuously with azithromycin for at least 3 months were included. Results of microbiological examination, including phage typing results of S. aureus, obtained during treatment were compared to results obtained 2 years before treatment. RESULTS 70 patients (median age 29.1 years) treated for a median of 4 years (range 0.7-5.1) were included. Before treatment, 44 patients had at least one culture positive for S. aureus compared to 25 patients during treatment (p<0.01). Mean percentage of sputum samples with growth of S. aureus decreased from 12.1% (range 0-82.6%) before treatment to 6.1% (range 0-93.2) during treatment (p<0.0006). Prevalences of H. influenzae and S. pneumoniae also decreased significantly. Fifteen of 214 isolates (7%) of S. aureus were macrolide resistant before treatment, increasing to 95 of 181 isolates (52.5%) during treatment (p<0.001). Macrolide resistant strains were found in 3 of 44 S. aureus colonized patients before treatment and in 11 of 25 patients at some time during treatment (p<0.03), all belonging to different phage types. First resistant S. aureus isolate was isolated after a median treatment duration of 1.5 years (range 0.3-2.9). No MRSA were isolated. Only 1 macrolide resistant isolate of M. catarrhalis was found during treatment. No macrolide resistance was found in H. influenzae or S. pneumoniae. CONCLUSION Long-term, low-dose treatment with azithromycin in CF patients leads to reduced prevalence of S. aureus, S. pneumoniae, and H. influenzae, but increased macrolide resistance in S. aureus. Reduction in the prevalence of S. aureus will make increasing macrolide resistance clinically insignificant in these patients.

Chronic pulmonary infection with Stenotrophomonas maltophilia and lung function in patients with cystic fibrosis

BACKGROUND The clinical consequences of chronic Stenotrophomonas maltophilia infection in cystic fibrosis (CF) patient are still unclear. METHOD All patients treated in the Copenhagen CF centre (N=278) from 1 January 2008 to 31 December 2009 were included. Each patient chronically infected with S. maltophilia for at least 2 years without any other chronic Gram-negative infection were matched to two non-infected CF controls. RESULTS Twenty-one patients were chronically infected with S. maltophilia during the 2-year study period. Fifteen were infected for at least 2 years. The patients in the S. maltophilia group had a steeper decline (-3.2%/year vs. -0.3%/year) in FEV(1) compared to the non-infected CF controls (P=0.03). The rate of decline was the same as observed 3 years before the patients became chronically infected. DISCUSSIONS Chronic infection with S. maltophilia does not lead to a steeper decline in lung function when compared to the period before chronic infection.