Gerdien A. Tramper-Stranders

Maintenance azithromycin treatment in pediatric patients with cystic fibrosis: long-term outcomes related to macrolide resistance and pulmonary function.

Background: Maintenance azithromycin therapy may improve pulmonary function in patients with cystic fibrosis (CF) with Pseudomonas aeruginosa infection because of its antiinflammatory properties. However, azithromycin therapy might increase macrolide resistance in Staphylococcus aureus cultured from respiratory secretions. We studied the emergence of macrolide resistance in S. aureus and correlated this to pulmonary function decline in pediatric patients with CF on daily azithromycin therapy. Methods: Respiratory cultures of 100 patients with CF were analyzed for S. aureus colonization and its resistance pattern before and during 3 years after initiation of azithromycin maintenance therapy. Mean annual change in forced expiratory volume as percent of predicted (FEV1 %) was calculated to compare pulmonary function before and after azithromycin therapy. Results: Staphylococcal colonization did not significantly decrease after initiation of azithromycin (50% versus 48%). Before start of therapy, 10% of patients with staphylococcal colonization had macrolide-resistant strains. Staphylococcal resistance increased to 83% in the first year; 97% in the second and 100% in the third year after initiation of azithromycin therapy (P < 0.001). Half of macrolide-resistant S. aureus comprised the macrolide–lincosamide–streptogramin phenotype. Percent forced expiratory volume in 1 second improved in the first year after initiation of azithromycin (mean annual change: −4.75% before versus +3.09% after initiation; P < 0.01) but decreased during the second and third years after initiation (−5.15% and −3.65%, respectively). Emergence of macrolide-resistant S. aureus was not related to pulmonary function decline. Conclusion: Maintenance azithromycin therapy in patients with CF leads to macrolide resistance in nearly all S. aureus carriers. Pulmonary function improvement after initiation of azithromycin therapy seems to be temporary and appears not to be related to macrolide resistance of S. aureus.

Development of the Nasopharyngeal Microbiota in Infants with Cystic Fibrosis.

RATIONALEnCystic fibrosis (CF) is characterized by early structural lung disease caused by pulmonary infections. The nasopharynx of infants is a major ecological reservoir of potential respiratory pathogens.nnnOBJECTIVESnTo investigate the development of nasopharyngeal microbiota profiles in infants with CF compared with those of healthy control subjects during the first 6 months of life.nnnMETHODSnWe conducted a prospective cohort study, from the time of diagnosis onward, in which we collected questionnaires and 324 nasopharynx samples from 20 infants with CF and 45 age-matched healthy control subjects. Microbiota profiles were characterized by 16S ribosomal RNA-based sequencing.nnnMEASUREMENTS AND MAIN RESULTSnWe observed significant differences in microbial community composition (P < 0.0002 by permutational multivariate analysis of variance) and development between groups. In infants with CF, early Staphylococcus aureus and, to a lesser extent, Corynebacterium spp. and Moraxella spp. dominance were followed by a switch to Streptococcus mitis predominance after 3 months of age. In control subjects, Moraxella spp. enrichment occurred throughout the first 6 months of life. In a multivariate analysis, S. aureus, S. mitis, Corynebacterium accolens, and bacilli were significantly more abundant in infants with CF, whereas Moraxella spp., Corynebacterium pseudodiphtericum and Corynebacterium propinquum and Haemophilus influenzae were significantly more abundant in control subjects, after correction for age, antibiotic use, and respiratory symptoms. Antibiotic use was independently associated with increased colonization of gram-negative bacteria such as Burkholderia spp. and members of the Enterobacteriaceae bacteria family and reduced colonization of potential beneficial commensals.nnnCONCLUSIONSnFrom diagnosis onward, we observed distinct patterns of nasopharyngeal microbiota development in infants with CF under 6 months of age compared with control subjects and a marked effect of antibiotic therapy leading toward a gram-negative microbial composition.

Initial Pseudomonas aeruginosa infection in patients with cystic fibrosis: characteristics of eradicated and persistent isolates.

Despite intensive eradication therapy, some CF patients with early Pseudomonas aeruginosa infection rapidly develop a chronic infection. To elucidate factors associated with this persistence, bacterial characteristics of early P. aeruginosa isolates were analysed that were either eradicated rapidly or persisted despite multiple antimicrobial treatments. Eighty-six early infection episodes were studied. First P. aeruginosa isolates from patients with eradication (36) or persistent infection (16) were included; isolates from patients with intermittent infection (34) were omitted from the study. Virulence assays, antimicrobial resistance, cytotoxicity and mutation frequencies were analysed in vitro. P. aeruginosa was genotyped by SNP-array. Transcriptomic profiles of two eradicated and two persistent strains were compared. Nineteen per cent of patients developed persistent infection; 42% achieved eradication. Secretion of virulence factors and mutation frequencies were highly variable among both eradicated and persistent isolates and were not different between the groups. Cytotoxicity was present in 57% of eradicated vs. 100% of persistent isolates (p <0.01). None of the isolates were resistant to antibiotics. The isolates were genotypically highly diverse. Multivariate analysis showed that in vitro determined bacterial characteristics could not predict persistence after first P. aeruginosa infection. Preliminary transcriptomic data showed increased expression of some genes related to a metabolic pathway. The early onset of chronic infection was not associated with (in vitro determined) bacterial characteristics only. Although the persistent isolates were more often cytotoxic, for the individual patient it was not possible to predict the risk of persistence based on bacterial characteristics. Unknown factors such as host-pathogen and pathogen-pathogen interactions should be further explored.

Outcome of assisted ventilation for acute respiratory failure in cystic fibrosis

ObjectivesTo assess outcome of assisted ventilation in cystic fibrosis (CF) patients with acute respiratory failure (ARF), to identify risk factors associated with poor outcome and to compare long-term outcome of CF children who were mechanically ventilated for ARF with unventilated CF controls.DesignRetrospective cohort study.SettingTwo large CF centres in the Netherlands.PatientsCF patients who required assisted ventilation for ARF and unventilated CF controls.InterventionsNone.Measurements and resultsThirty-one CF patients required assisted ventilation for ARF between January 1990 and March 2005. All five children (under 2xa0years of age) and seven adults (27%) survived. In the total population, age was axa0statistically significant risk factor for poor outcome (pu202f=u202f0.02). In adult CF patients who required invasive mechanical ventilation, acute on chronic respiratory failure was associated with poor outcome. In children who required mechanical ventilation for ARF, lung function and CF related complications 5xa0years later were not significantly different compared with controls matched for age, gender and genotype.ConclusionsCF patients younger than 2xa0years old, who are ventilated because of ARF, have axa0good prognosis and their long-term outcome seems identical to unventilated CF controls. ARF in adult CF patients still is associated with high mortality, especially among patients with acute on chronic respiratory failure.

Controlled trial of cycled antibiotic prophylaxis to prevent initial Pseudomonas aeruginosa infection in children with cystic fibrosis

Background Initial pulmonary Pseudomonas aeruginosa infection in patients with cystic fibrosis (CF) is currently treated with intensive antibiotic therapy. At this stage, inflammation and tissue injury might have already occurred. Moreover, bacterial eradication is not always achieved. Prophylactic treatment against P aeruginosa seemed to have a preventive effect in retrospective studies. A study was undertaken to establish prospectively the effect of cycled prophylactic treatment on prevention of initial P aeruginosa infection in children with CF. Methods This 3-year triple-blind randomised controlled trial included 65 children with CF without P aeruginosa infection. Intervention existed of 3-monthly 3-week treatments with oral ciprofloxacin and inhaled colistin or both placebo controls. The primary outcome was P aeruginosa infection. Secondary outcomes were serum anti-Pseudomonas antibodies, pulmonary function, exacerbations, chest x-ray scores, inflammation parameters, respiratory pathogens and antimicrobial resistance. Results There was no difference in acquisition of P aeruginosa infection between the control and treatment groups (annual incidence 14% vs 11%; HR 0.738, 95% CI 0.299 to 1.822). Anti-Pseudomonas antibodies emerged earlier in the control group, but this difference had disappeared after 3u2005years. Chronic infection was observed in 19% of controls and 12% of treated patients. Decline in pulmonary function and other clinical outcomes did not differ between the two groups. In the treatment group, significantly fewer Gram-positive bacteria and Enterobacteriaceae were observed but there were more non-P aeruginosa non-fermentative Gram-negative bacteria. Conclusions Three-monthly cycled anti-P aeruginosa prophylaxis does not reduce the risk of initial and chronic infection in P aeruginosa-negative children with CF of all ages. Shifts in bacterial colonisation demand caution. Trial Registration Number ISRCTN 11604593.

Pseudomonas aeruginosa diversity in distinct paediatric patient groups

Pseudomonas aeruginosa is a pathogen that often infects patients who are either immunocompromised or have local defects in host defences. It is known that cystic fibrosis (CF) patients are sometimes infected with certain clonal isolates. It is not clear whether these clonal isolates also infect non-CF patients and whether clonality of isolates occurs in other patient groups. The aim of this study was to investigate P. aeruginosa diversity and the occurrence of clones within five distinct paediatric patient groups susceptible to P. aeruginosa infection. P. aeruginosa isolates were cultured from 157 patients (CF first infection (CF-1 group) (29); CF chronic infection (CF-chronic group) (27); urinary tract infection (34); chronic suppurative otitis media (43); and intensive-care hospitalization/immunodeficiency (24)). All 202 phenotypically different isolates were tested for antimicrobial resistance and further typed by pulsed-field gel electrophoresis. Simpsons diversity index was calculated for the five groups. CF-chronic patients carried the highest number of distinct P. aeruginosa phenotypes and genotypes per culture. Isolates from the CF-chronic group were significantly less diverse than those from the other groups. A group of clonal isolates was observed among patients from the CF-chronic and CF-1 groups. These or different clonal isolates were not encountered among the three other patient groups. No characteristic resistance pattern could be identified among isolates from the distinct patient groups and among the clonal isolates. In conclusion, isolates of the CF-chronic group were less diverse than those in the other patient groups with P. aeruginosa infection; clonal isolates were not encountered in non-CF patients. Transmission of clonal CF isolates to other patient groups was not observed.

Concordance between upper and lower airway microbiota in infants with cystic fibrosis

Nasopharyngeal and oropharyngeal samples are commonly used to direct therapy for lower respiratory tract infections in non-expectorating infants with cystic fibrosis (CF). We aimed to investigate the concordance between the bacterial community compositions of 25 sets of nasopharyngeal, oropharyngeal and bronchoalveolar lavage (BAL) samples from 17 infants with CF aged ∼5u2005months (n=13) and ∼12u2005months (n=12) using conventional culturing and 16S-rRNA sequencing. Clustering analyses demonstrated that BAL microbiota profiles were in general characterised by a mixture of oral and nasopharyngeal bacteria, including commensals like Streptococcus, Neisseria, Veillonella and Rothia spp. and potential pathogens like Staphylococcus aureus, Haemophilus influenzae and Moraxella spp. Within each individual, however, the degree of concordance differed between microbiota of both upper respiratory tract niches and the corresponding BAL. The inconsistent intra-individual concordance between microbiota of the upper and lower respiratory niches suggests that the lungs of infants with CF may have their own microbiome that seems seeded by, but is not identical to, the upper respiratory tract microbiome. Lungs of CF infants have a microbiome that seems seeded by, but is not identical to, the URT microbiome http://ow.ly/1NlA306DuPv

DYNAMIC DEVELOPMENT OF THE GUT MICROBIOTA IN CHILDREN WITH CYSTIC FIBROSIS AND HEALTHY CHILDREN DURING THE FIRST 18 MONTHS OF LIFE

Newly synthesized domains of the cystic fibrosis transmembrane conductance regulator (CFTR) fold cotranslationally and then undergo cooperative domain-domain assembly. Folding process during synthesis by the ribosome has biologically evolved to reduce misfolding and maximize native folding outcome. During this process, the rate of translation is controlled by several factors including, codon usage, tRNA availability, mRNA structure, RNA binding proteins, and ribosome cofactors. In this study, we monitored the process of translation using ribosome profiling to obtain a high-resolution and quantitative profile of the location of actively translating ribosomes across the entire cellular transcriptome. Ribosome-protected fragments (“footprints”) were isolated and sequenced by next-generation sequencing from HEK293 cells expressing exogenous CFTR proteins. Footprints were predominantly recovered on protein coding region within mRNA, and showed a three-nucleotide periodicity, indicating that they originated from actively engaged ribosomes. Footprint density, which inversely correlates with the rate of translation, varied across CFTR mRNA. Transmembrane domain 1 (TMD1) was among CFTR domains with the highest ribosome occupancy, whereas nucleotide binding domain 2 (NBD2) had the lowest (TMD1>>TMD2~R~NBD1>NBD2). Assuming that all engaged ribosomes generate a full length CFTR protein, this suggests that TMD1 is most slowly translated, while NBD2 is translated at the fastest rate. Particularly high footprint occupancy was observed at the start codon region, which is mainly caused by bias of cycloheximide used for stabilizing ribosomes. Within TMD1 and TMD2, slower translation was observed for extracellular loop 2 (EL2) and EL5 regions respectively. Considering that the ribosome occludes about 40 amino acid residues within the exit tunnel, the result suggests that newly synthesized intracellular loop 1 (IL1) and IL3 emerge slowly from the ribosome during TMD folding. Few footprints were observed on the 3UTR, implying efficient termination at the stop codon. Overall ribosome occupancy across CFTR coding region, however, did not correspond well to a predicted relative rate of CFTR translation using an algorithm devised by Spencer et al. (J Mol Biol 2012;422:328-35) that is based on the rate of tRNA abundance and mRNA codon-tRNA anticodon pairings, suggesting that additional factors may be involved. ΔF508 mutation impairs not only NBD1 folding but also interdomain assembly of CFTR. Recent studies on the mechanism of action of VX-809 have shown that it improves NBD1-TMD2 interdomain assembly by acting primarily on cotranslational folding. Ribosome profiling revealed no significant difference in the translation rate between wildtype and ΔF508 CFTR. Furthermore, C18, a VX-809 analog, also had no significant effects on CFTR translation as evidenced by overall ribosome occupancy across CFTR mRNA or location of ribosomes across the entire transcriptome. These results suggest that cotranslational effects of C18 on CFTR folding are not mediated by changes in translation per se, but rather operate at the level of domain folding and/or interdomain assembly. Supported by NIH and CFF. 2w NOVEL MODULATORS OF PROTEIN UBIQUITINATION IMPROVE VX-809-DEPENDENT RESCUE OF F508DEL-CFTR Goeckeler-Fried, J.; Chiang, A.; Larsen, M.; Chung, W.; Denny, R.; Weissman, A.; Watkins, S.C.; Sorscher, E.J.; Frizzell, R.A.; Brodsky, J.L. 1. Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA; 2. Center for Biologic Imaging, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA; 3. Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; 4. Pfizer Worldwide Medicinal Chemistry, Cambridge, MA, USA; 5. Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD, USA; 6. Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USAThe current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified a novel potentiator GLPG1837 by compound screening on mutant CFTR. YFP-halide efflux assays and single channel measurements showed ∼2.5-fold improvement in channel activity by GLPG1837 compared to VX-770 (ivacaftor/Kalydeco) on G551D CFTR (1, 2). GLPG1837 successfully passed the Phase-2 clinical trials and proved to be the first potentiator after VX-770 to show competitive results on G551D patients. To identify potential differences in the mode of actions of these potentiators we studied their effects on CFTR folding and function. Biochemical radiolabeling experiments showed that mutations in the intracellular loop 2 (ICL2) disrupt domain assembly between TMD1 and NBD2, a late folding event in CFTR, but in most cases do not impair CFTR trafficking towards the cell surface. Protease-susceptibility assays showed that VX-770 improved late TMD1 folding of many ICL2 mutations, but GLPG1837 did not. YFP-halide efflux assays showed that these ICL2 mutants had varying effect on channel function, ranging from wild-type-like to function-defective mutants. GLPG1837 restored function of non-CF gating mutant E267K much better than VX-770. Residue E267 in ICL2 electrostatically interacts with K1060 in ICL4 to promote channel opening (3). This indicates that GLPG1837 is more efficient in compensating for this lost interaction. Altogether, our biochemical and functional data suggests that potentiators VX-770 and GLPG1837 have a different mode of action.

Interhospital transfer of premature neonates from intensive to lower care settings: impact on the clinical condition

Limited data arexa0available on the outcomes of interhospital neonatal transports of premature infants from intensivexa0to lowerxa0care settings by ambulance. Available data come from studies that only covered the outcomes of the transportation of critically ill premature neonates to neonatal intensive care unitsxa0(NICU); undesirable outcomes have been reported.1 2 Transport of clinically stable premature infants from NICUs to lowerxa0care settings allows further neonatal treatment in the vicinity of the family home. The clinical impact of this interhospital transport is unknown and not quantified before. Therefore, we investigated the impact of these transfers from an NICU to regional hospitals in the Netherlands.nnAll premature neonates who were transferred between a tertiary centre NICU …

WS07.5 Gut microbiome in healthy children and children with cystic fibrosis during the first 18 months of life

Introduction: Progressive lung damage, associated with bacterial infection and neutrophil mediated inflammation, is the major cause of morbidity and death in CF. Ivacaftor improves lung function in CF patients with the G551D mutation and this may be associated with changes in airway bacterial community composition. Objectives: To determine the effect of ivacaftor on CF airway bacterial community composition and to examine the relationship with lung function and measures of systemic inflammation. Methods: Sputum and blood samples from CF patients (n = 20) were collected prior to and every three months (for 12 months) after initiation of ivacaftor therapy. Molecular based analysis (Illumina MiSeq) was used to define airway bacterial community composition. ELISA’s were used to quantify measures of systemic inflammation in blood (IL-8, IL-6 & CRP). Results: Mean relative abundance of Pseudomonas decreased after ivacaftor therapy whilst relative abundance of Streptococcus, Haemophilus, Rothia and Prevotella increased. There was no correlation between relative abundance of individual taxa and lung function. However, there was a significant inverse correlation between the relative abundance of both Streptococcus and Prevotella and IL-8 (p < 0.05). There was no association between relative abundance of individual taxa and IL-6 or CRP. Conclusion: Alterations in CF airway bacterial community composition post-ivacaftor therapy are associated with reduced levels of the potent neutrophil chemoattractant IL-8. This may result in decreased neutrophil influx into the airways and improve lung function by limiting neutrophil mediated inflammation. Acknowledgement: Funding: DEL NI Studentship & CFMATTERS (603038)