Kasper Aanaes

Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection

The opportunistic pathogen Pseudomonas aeruginosa is a frequent colonizer of the airways of patients suffering from cystic fibrosis (CF). Depending on early treatment regimens, the colonization will, with high probability, develop into chronic infections sooner or later, and it is important to establish under which conditions the switch to chronic infection takes place. In association with a recently established sinus surgery treatment program for CF patients at the Copenhagen CF Center, colonization of the paranasal sinuses with P. aeruginosa has been investigated, paralleled by sampling of sputum from the same patients. On the basis of genotyping and phenotypic characterization including transcription profiling, the diversity of the P. aeruginosa populations in the sinuses and the lower airways was investigated and compared. The observations made from several children show that the paranasal sinuses constitute an important niche for the colonizing bacteria in many patients. The paranasal sinuses often harbor distinct bacterial subpopulations, and in the early colonization phases there seems to be a migration from the sinuses to the lower airways, suggesting that independent adaptation and evolution take place in the sinuses. Importantly, before the onset of chronic lung infection, lineages with mutations conferring a large fitness benefit in CF airways such as mucA and lasR as well as small colony variants and antibiotic-resistant clones are part of the sinus populations. Thus, the paranasal sinuses potentially constitute a protected niche of adapted clones of P. aeruginosa, which can intermittently seed the lungs and pave the way for subsequent chronic lung infections.

Environmental Heterogeneity Drives Within-Host Diversification and Evolution of Pseudomonas aeruginosa

ABSTRACT Microbial population polymorphisms are commonly observed in natural environments, including long-term infected hosts. However, the underlying processes promoting and stabilizing diversity are difficult to unravel and are not well understood. Here, we use chronic infection of cystic fibrosis airways by the opportunistic pathogen Pseudomonas aeruginosa as a system for investigating bacterial diversification processes during the course of infection. We analyze clonal bacterial isolates sampled during a 32-year period and map temporal and spatial variations in population diversity to different infection sites within the infected host. We show that the ancestral infecting strain diverged into distinct sublineages, each with their own functional and genomic signatures and rates of adaptation, immediately after initial colonization. The sublineages coexisted in the host for decades, suggesting rapid evolution of stable population polymorphisms. Critically, the observed generation and maintenance of population diversity was the result of partitioning of the sublineages into physically separated niches in the CF airway. The results reveal a complex within-host population structure not previously realized and provide evidence that the heterogeneity of the highly structured and complex host environment promotes the evolution and long-term stability of pathogen population diversity during infection. IMPORTANCE Within-host pathogen evolution and diversification during the course of chronic infections is of importance in relation to therapeutic intervention strategies, yet our understanding of these processes is limited. Here, we investigate intraclonal population diversity in P. aeruginosa during chronic airway infections in cystic fibrosis patients. We show the evolution of a diverse population structure immediately after initial colonization, with divergence into multiple distinct sublineages that coexisted for decades and occupied distinct niches. Our results suggest that the spatial heterogeneity in CF airways plays a major role in relation to the generation and maintenance of population diversity and emphasize that a single isolate in sputum may not represent the entire pathogen population in the infected individual. A more complete understanding of the evolution of distinct clonal variants and their distribution in different niches could have positive implications for efficient therapy. Within-host pathogen evolution and diversification during the course of chronic infections is of importance in relation to therapeutic intervention strategies, yet our understanding of these processes is limited. Here, we investigate intraclonal population diversity in P. aeruginosa during chronic airway infections in cystic fibrosis patients. We show the evolution of a diverse population structure immediately after initial colonization, with divergence into multiple distinct sublineages that coexisted for decades and occupied distinct niches. Our results suggest that the spatial heterogeneity in CF airways plays a major role in relation to the generation and maintenance of population diversity and emphasize that a single isolate in sputum may not represent the entire pathogen population in the infected individual. A more complete understanding of the evolution of distinct clonal variants and their distribution in different niches could have positive implications for efficient therapy.

Colonisation and infection of the paranasal sinuses in cystic fibrosis patients is accompanied by a reduced PMN response

BACKGROUND We studied whether the sinuses might be foci for Pseudomonas aeruginosa lung infection. METHODS Endoscopic Sinus Surgery was performed in 78 CF patients; PFGE was used for bacterial genotyping. Material from sinuses and lungs were Gram-stained to detect biofilms. Immunoglobulins were measured in serum and saliva. RESULTS When P. aeruginosa was cultured simultaneously from the sinuses and the lungs they were genetically identical in 38 of the 40 patients (95%). In the sinuses, P. aeruginosa formed biofilms with minimal cellular inflammation, probably because of a significantly higher local production of secretory IgA compared with IgG (p<0.001). CONCLUSIONS We have shown that P. aeruginosa form biofilm in the sinuses, which constitute an important bacterial reservoir for subsequent lung infection. The high amount of IgA in the upper airways probably protects P. aeruginosa from the inflammatory immune system, and they can proceed unnoticed into a permanent infectious focus that cannot be eradicated with antibiotics.

P. aeruginosa in the paranasal sinuses and transplanted lungs have similar adaptive mutations as isolates from chronically infected CF lungs

BACKGROUND Pseudomonas aeruginosa cells are present as biofilms in the paranasal sinuses and the lungs of chronically infected cystic fibrosis (CF) patients. Since different inflammatory responses and selective antibiotic pressures are acting in the sinuses compared with the lungs, we compared the adaptive profiles of mucoid and non-mucoid isolates from the two locations. METHODS We studied the genetic basis of phenotypic diversification and gene expression profiles in sequential lung and sinus P. aeruginosa isolates from four chronically infected CF patients, including pre- and post-lung transplantation isolates. RESULTS The same phenotypes caused by similar mutations and similar gene expression profiles were found in mucoid and non-mucoid isolates from the paranasal sinuses and from the lungs before and after transplantation. CONCLUSION Bilateral exchange of P. aeruginosa isolates between the paranasal sinuses and the lungs occurs in chronically infected patients and extensive sinus surgery before the lung transplantation might prevent infection of the new lung.

Decreased mucosal oxygen tension in the maxillary sinuses in patients with cystic fibrosis

BACKGROUND Pseudomonas aeruginosa in the sinuses plays a role in the lungs in cystic fibrosis (CF) patients, but little is known about the sinus environment where the bacteria adapt. Anoxic areas are found in the lower respiratory airways but it is unknown if the same conditions exist in the sinuses. METHODS The oxygen tension (pO(2)) was measured, using a novel in vivo method, in the maxillary sinus in a group of 20 CF patients. RESULTS The CF patients had a significant lower pO(2) on the mucosa but not in the sinus lumen as compared with a control group of non-CF patients. Anoxic conditions were found in 7/39 (18%) of the sinuses from where we cultured P. aeruginosa, Stenotrophomonas maltophilia and/or coagulase negative staphylococci. CONCLUSION These findings support our hypothesis that P. aeruginosa can adapt or acclimate to the environment in the lungs, during growth in anoxic parts of the paranasal sinuses.

Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization

BACKGROUND Pseudomonas aeruginosa sinusitis may be the focus for intermittent lung colonization in patients with cystic fibrosis (CF). The sinusitis may induce elevated IgA levels in nasal secretion and saliva against P. aeruginosa. METHODS 120 CF patients chronically infected, intermittently colonized or without P. aeruginosa in the lungs participated in this cross-sectional study. IgA and IgG against P. aeruginosa sonicate and alginate were measured in nasal secretions, saliva, and in serum by ELISA. RESULTS The intermittently colonized patients had significantly higher IgA levels in nasal secretions and saliva than those without P. aeruginosa in the lungs, indicating that P. aeruginosa sinusitis may precede intermittent colonization and chronic infection of the lungs. CONCLUSIONS Specific IgA against P. aeruginosa in nasal secretions and saliva can contribute to differentiation between patients chronically infected, intermittently colonized, and without P. aeruginosa in the lungs. The diagnostic value of the IgA ELISA awaits a prospective study.

CT of the paranasal sinuses is not a valid indicator for sinus surgery in CF patients

BACKGROUND No guidelines comprise when or to what extent sinus surgery should be done in patients with cystic fibrosis (CF) or how a CT scan of the paranasal sinuses should influence the decision. Symptoms of rhinosinusitis and/or eradication of pathogenic bacteria from the sinuses are reasons for sinus surgery. METHODS In this observational cross sectional study, 55 CF cases had their preoperative CT scans scored according to the Lund Mackay- and the Nair-system. Correlations between the CT scans, symptoms, surgical findings and cultures obtained during sinus surgery were made. RESULTS There was no significant correlation between the CT score and detection of pus, pathogenic bacteria or symptoms. Pus and pathogenic bacteria were found in several cases without sinus opacification on the CT scan. Non pathogenic and sterile cultures were also found in sinuses with opacification. CONCLUSIONS A CT scan is not a valid criterion for sinus surgery in CF patients.

Simultaneous sinus and lung infections in patients with primary ciliary dyskinesia.

Abstract Conclusion: The sinuses should be considered as a bacterial reservoir and a target for surgery and antibiotic treatment in patients with primary ciliary dyskinesia (PCD). The observed decrease in serum precipitating antibodies (precipitins) against Pseudomonas aeruginosa may indicate a beneficial effect of combined endoscopic sinus surgery (ESS) and concomitant medical treatment. Objectives: The purpose of this research, which is the first study addressing bacteriology in the sinuses of patients with PCD, was to examine the association between sinus and lung infections. Methods: We reviewed findings of bacterial pathogens from the sinuses obtained during ESS and the lung infection status in eight PCD patients over a 6 year period. Precipitins against P. aeruginosa were used as a marker of severity of chronic infection and effect of treatment. Results: Preoperatively, seven of the eight patients (88%) exhibited intermittent or chronic pulmonary infection with P. aeruginosa. Sinus cultures were obtained during ESS in seven patients. The sinuses were colonized with P. aeruginosa in four of seven patients (57%). Bacterial sinusitis was found in five of seven patients (71%) and the same bacterium was found in the sinuses and lungs in all cases. Decreasing precipitins against P. aeruginosa were observed postoperatively in three of four evaluable patients.

Sinus surgery can improve quality of life, lung infections, and lung function in patients with primary ciliary dyskinesia.

Chronic rhinosinusitis (CRS) and bacterial sinusitis are ubiquitous in patients with primary ciliary dyskinesia (PCD). From the sinuses, Pseudomonas aeruginosa can infect the lungs.

National long-lasting effect of endonasal endoscopic sphenopalatine artery clipping for epistaxis

Abstract Conclusion: We consider sphenopalatine artery ligation to be a safe and effective treatment of posterior epistaxis as the long-term need for revision surgery and the complication rates are low. Surgery should be considered earlier in the treatment of posterior epistaxis. Objectives: Posterior epistaxis is common and surgical endoscopic ligation of the sphenopalatine arteries is indicated in severe cases. Knowledge about long-term effects and complications is sparse. Methods: Within 2001–2006, 78 patients underwent endonasal endoscopic-guided surgery for posterior epistaxis in one of the eight ENT clinics in Denmark treating these patients. In 2011, 45 patients were still alive and eligible for the study. Patients were contacted by telephone and invited to complete an interview questionnaire on late adverse affects and recurrence. Results: In all, 42 of 45 patients participated in the mean follow-up. The mean follow-up was 6.7 years: 90% of patients (n = 38) obtained an effect of the treatment during follow-up; 78% (n = 33) had no recurrent epistaxis, 12% (n = 5) had recurrent epistaxis but only needed non-surgical specialized treatment; 10% (n = 4) required revision surgery due to recurrent epistaxis within the 6.7 mean years of follow-up; and 26% of the patients had minor postoperative complications, permanent nasal crusting being most persistent and frequent.