Metagenomic Analysis of Respiratory Tract DNA Viral Communities in Cystic Fibrosis and Non-Cystic Fibrosis Individuals

The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5′-triphosphate,3′-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota.     

Psychological Implications of Cystic Fibrosis

A psychological and psychiatric study of 11 children with cystic fibrosis revealed major psychological problems in all of them. Among the parents of the majority of these children, marked psychopathology and gross marital discord were noted. Popular literature concerning cystic fibrosis had a negative effect on the child''s attitude toward the disease. Virtually all of these patients showed a preoccupation with death. In this study, the necessity of psychiatric consultation as an integral part of current intensive treatment programs in cystic fibrosis clinics was demonstrated.     

Proteomic Analysis of Nasal Epithelial Cells from Cystic Fibrosis Patients

The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.     

Immune Complexes in Cystic Fibrosis

Circulating immune complexes were detected in serum and sputum of patients with cystic fibrosis (C.F.). There were extensive deposits of immunoglobulins and complement immune complexes in several of the C.F. organs, especially the respiratory and gastrointestinal tracts, but not in the kidneys. Significant concentrations of IgG and of complement complexes could be eluted from the lungs of the C.F. patients but not from those of controls. Studies involving immunoabsorption, autoradiography, and molecular sieving through Sephadex G-200 columns identified both bovine serum albumin and staphylococcal α-haemolysin as two of the antigens present in the immune complexes. The sedimentation constant of the immune complexes was about 8S to 11S. The clinical significance of these immune complexes and the wide variety of antibodies detected in C.F. patients are discussed.     

Evidence for a Cystic Fibrosis Enteropathy

BackgroundPrevious studies have suggested the existence of enteropathy in cystic fibrosis (CF), which may contribute to intestinal function impairment, a poor nutritional status and decline in lung function. This study evaluated enterocyte damage and intestinal inflammation in CF and studied its associations with nutritional status, CF-related morbidities such as impaired lung function and diabetes, and medication use.MethodsSixty-eight CF patients and 107 controls were studied. Levels of serum intestinal-fatty acid binding protein (I-FABP), a specific marker for enterocyte damage, were retrospectively determined. The faecal intestinal inflammation marker calprotectin was prospectively studied. Nutritional status, lung function (FEV1), exocrine pancreatic insufficiency (EPI), CF-related diabetes (CFRD) and use of proton pump inhibitors (PPI) were obtained from the medical charts.ResultsSerum I-FABP levels were elevated in CF patients as compared with controls (p<0.001), and correlated negatively with FEV1 predicted value in children (r-.734, p<0.05). Faecal calprotectin level was elevated in 93% of CF patients, and correlated negatively with FEV1 predicted value in adults (r-.484, p<0.05). No correlation was found between calprotectin levels in faeces and sputum. Faecal calprotectin level was significantly associated with the presence of CFRD, EPI, and PPI use.ConclusionThis study demonstrated enterocyte damage and intestinal inflammation in CF patients, and provides evidence for an inverse correlation between enteropathy and lung function. The presented associations of enteropathy with important CF-related morbidities further emphasize the clinical relevance.     

Assessment of the Microbial Constituents of the Home Environment of Individuals with Cystic Fibrosis (CF) and Their Association with Lower Airways Infections

Introduction

Cystic fibrosis (CF) airways are colonized by a polymicrobial community of organisms, termed the CF microbiota. We sought to define the microbial constituents of the home environment of individuals with CF and determine if it may serve as a latent reservoir for infection.

Methods

Six patients with newly identified CF pathogens were included. An investigator collected repeat sputum and multiple environmental samples from their homes. Bacteria were cultured under both aerobic and anaerobic conditions. Morphologically distinct colonies were selected, purified and identified to the genus and species level through 16S rRNA gene sequencing. When concordant organisms were identified in sputum and environment, pulsed-field gel electrophoresis (PFGE) was performed to determine relatedness. Culture-independent bacterial profiling of each sample was carried out by Illumina sequencing of the V3 region of the 16s RNA gene.

Results

New respiratory pathogens prompting investigation included: Mycobacterium abscessus(2), Stenotrophomonas maltophilia(3), Pseudomonas aeruginosa(3), Pseudomonas fluorescens(1), Nocardia spp.(1), and Achromobacter xylosoxidans(1). A median 25 organisms/patient were cultured from sputum. A median 125 organisms/home were cultured from environmental sites. Several organisms commonly found in the CF lung microbiome were identified within the home environments of these patients. Concordant species included members of the following genera: Brevibacterium(1), Microbacterium(1), Staphylococcus(3), Stenotrophomonas(2), Streptococcus(2), Sphingomonas(1), and Pseudomonas(4). PFGE confirmed related strains (one episode each of Sphinogomonas and P. aeruginosa) from the environment and airways were identified in two patients. Culture-independent assessment confirmed that many organisms were not identified using culture-dependent techniques.

Conclusions

Members of the CF microbiota can be found as constituents of the home environment in individuals with CF. While the majority of isolates from the home environment were not genetically related to those isolated from the lower airways of individuals with CF suggesting alternate sources of infection were more common, a few genetically related isolates were indeed identified. As such, the home environment may rarely serve as either the source of infection or a persistent reservoir for re-infection after clearance.     

Genetic Adaptation of Achromobacter sp. during Persistence in the Lungs of Cystic Fibrosis Patients

Achromobacter species are increasingly isolated from the respiratory tract of cystic fibrosis patients and often a chronic infection is established. How Achromobacter sp. adapts to the human host remains uncharacterised. By comparing longitudinally collected isolates of Achromobacter sp. isolated from five CF patients, we have investigated the within-host evolution of clonal lineages. The majority of identified mutations were isolate-specific suggesting co-evolution of several subpopulations from the original infecting isolate. The largest proportion of mutated genes were involved in the general metabolism of the bacterium, but genes involved in virulence and antimicrobial resistance were also affected. A number of virulence genes required for initiation of acute infection were selected against, e.g. genes of the type I and type III secretion systems and genes related to pilus and flagellum formation or function. Six antimicrobial resistance genes or their regulatory genes were mutated, including large deletions affecting the repressor genes of an RND-family efflux pump and a beta-lactamase. Convergent evolution was observed for five genes that were all implicated in bacterial virulence. Characterisation of genes involved in adaptation of Achromobacter to the human host is required for understanding the pathogen-host interaction and facilitate design of future therapeutic interventions.     

Cystic Fibrosis Complicated by Heart Failure

Survival studies were done on 36 children with cystic fibrosis and heart failure. Thirty percent did not survive the first four weeks, and the median survival for the group was between two and three months. By the end of the first year from the onset of failure, 74 percent had died and at 30 months, 87 percent had died.     

Voice Disorder in Cystic Fibrosis Patients

Cystic fibrosis is a common autosomal recessive disorder with drastic respiratory symptoms, including shortness of breath and chronic cough. While most of cystic fibrosis treatment is dedicated to mitigating the effects of respiratory dysfunction, the potential effects of this disease on vocal parameters have not been systematically studied. We hypothesized that cystic fibrosis patients, given their characteristic respiratory disorders, would also present dysphonic symptoms. Given that voice disorders can severely impair quality of life, the identification of a potential cystic fibrosis-related dysphonia could be of great value for the clinical evaluation and treatment of this disease. We tested our hypothesis by measuring vocal parameters, using both objective physical measures and the GRBAS subjective evaluation method, in male and female cystic fibrosis patients undergoing conventional treatment and compared them to age and sex matched controls. We found that cystic fibrosis patients had a significantly lower vocal intensity and harmonic to noise ratio, as well as increased levels of jitter and shimmer. In addition, cystic fibrosis patients also showed higher scores of roughness, breathiness and asthenia, as well as a significantly altered general grade of dysphonia. When we segregated the results according to sex, we observed that, as a group, only female cystic fibrosis patients had significantly lower values of harmonic to noise ratio and an abnormal general grade of dysphonia in relation to matched controls, suggesting that cystic fibrosis exerts a more pronounced effect on vocal parameters of women in relation to men. Overall, the dysphonic characteristics of CF patients can be explained by dysfunctions in vocal fold movement and partial upper airway obstruction, potentially caused by the accumulation of mucus and chronic cough characteristic of CF symptomatology. Our results show that CF patients exhibit significant dysphonia and suggest they may potentially benefit from voice therapy as a parallel treatment strategy.     

Pathogenesis of Fungal Infections in Cystic Fibrosis

For a long time, the microbiology of cystic fibrosis has been focussed on Pseudomonas aeruginosa and associated Gram-negative pathogens. An increasing body of evidence has been compiled demonstrating an important role for moulds and yeasts within this complex patient group. Whether or not fungi are active participants, spectators or transient passersby remain to be elucidated. However, functionally, they do appear to play a contributory role in pathogenesis, albeit we do not know if this is a direct or indirect effect. The following review examines some of the key evidence for the role of fungi in CF pathogenesis.