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80 The microbiome in early cystic fibrosis lung disease: A longitudinal analysis
Posters
4. Microbiology
S77
77 Whole genome sequencing of multiple isolates of the Pseudomonas aeruginosa Liverpool epidemic strain reveals transmission and geographical clustering
79 Development of the upper respiratory tract microbiome was investigated longitudinally in cystic fibrosis infants and controls 0−6 months of age
M. Moore1 , D. Williams2 , S. Paterson2 , N. Tucker3 , D. Kenna4 , J. Turton4 , N. Loman5 , D. Taylor-Robinson6 , K. Southern7 , M.J. Walshaw6 , J.L. Fothergill1 , C. Winstanley1 . 1 Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom; 2 Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom; 3 University of Strathclyde, Liverpool, United Kingdom; 4 Public Health England, London, United Kingdom; 5 University of Birmingham, Birmingham, United Kingdom; 6 Liverpool Heart and Chest Hospital, Liverpool, United Kingdom; 7 Alder Hey Children’s Hospital, Liverpool, United Kingdom
S.M.P.J. Prevaes1 , K.M. de Winter-de Groot1 , H.M. Janssens2 , X. Wang1 , G.A. Tramper-Stranders1 , R. Hasrat1 , H.A.W.M. Tiddens2 , M. van Westreenen2 , C.K. van der Ent1 , E.A.M. Sanders1 , D. Bogaert1 . 1 University Medical Center Utrecht, Utrecht, Netherlands; 2 ErasmusMC-Sophia Children’s Hospital, Rotterdam, Netherlands
Objectives: Pseudomonas aeruginosa is the primary cause of chronic lung infection in cystic fibrosis (CF) and is associated with a decrease in morbidity and mortality. Discovery of the Liverpool Epidemic Strain (LES) provided the first molecular evidence for a transmissible strain of P. aeruginosa. The LES has since been discovered in Canada, remains the most common clone of P. aeruginosa in UK CF clinics and is associated with poorer prognoses. In 2009, the earliest archived isolate, LESB58 from 1988, was whole genome sequenced. The aims of this study were to utilise whole genome sequencing to provide insight into the population structure and epidemiology of the strain and characterise genomic variations between isolates. Methods: Using the Illumina HiSeq platform, 73 LES isolates, chosen to represent a historically and geographically diverse panel, were sequenced and compared to 50 LES genomes already available. Results: Phylogenetic analysis of the LES core genome revealed 3533 polymorphic sites. Isolates clustered geographically, with Canadian and UK isolates forming distinct separate clusters. Conclusion: By closer analysis of the distribution of specific mutations we can gain a better understanding of the transmission and evolution of this important strain. We would like to acknowledge the Cystic Fibrosis Trust for funding this research.
Background: Cystic Fibrosis (CF) is characterized by early damage of the lungs by infections. The responsible pathogens find their ecological niche in the upper respiratory tract (URT). Information on microbial colonization dynamics in the URT of CF infants is lacking. Objective: To examine colonization dynamics of URT microbiota in CF infants over time. Methods: Case-control study. Questionnaires and nasopharyngeal (NP) samples were collected monthly from 20 CF patients and 45 controls. We studied the dynamics of URT microbiota from 0−6 months of life by 16SrRNA-based sequencing. Results: The bacterial density of URT microbiota is lower in CF infants compared to controls, and inversely correlated with antibiotic use. Also, the microbial community composition is significantly different between groups over the first 6 months of life. Where in CF infants the URT microbiota is initially predominated by Corynebacterium and Staphylococcus followed by Streptococcus and Moraxella, in controls Moraxella, Haemophilus and another Corynebacterium sp. predominate throughout the first 6 months. Multivariate analyses show significantly more Staphylococcus, Pseudomonas, Enterobacteriaceae and Bacillus spp. and less Moraxella, Haemophilus, Dolosigranulum and specific Corynebacterium spp. in CF infants vs. Controls. Antibiotic use is independently associated with increased carriage of Gram-negative bacteria and reduced carriage of non-pathogenic commensals. Conclusion: CF infants have a significantly different development of URT microbiota compared to controls from birth on. This information may help to improve targeted therapy, and allows surveillance of ecological side effects of antibiotics.
78 Differences in infection, inflammation and structural abnormalities between the right and left lung
80 The microbiome in early cystic fibrosis lung disease: A longitudinal analysis
R. Carzino1 , L. King1 , S. Ranganathan1 . 1 Murdoch Children’s Research Institute, Respiratory Medicine, Parkville, Australia
K.B. Frayman1,2,3 , D. Armstrong2,4 , R. Carzino1,2 , T. Ferkol5 , K. Grimwood6,7 , G. Storch5 , K. Wylie5 , S. Ranganathan1,2,3 . 1 Royal Children’s Hospital, Department of Respiratory Medicine, Melbourne, Australia; 2 Murdoch Children’s Research Institute, Infection and Immunity Theme, Melbourne, Australia; 3 University of Melbourne, Department of Paediatrics, Melbourne, Australia; 4 Monash Children’s Hospital, Respiratory Medicine, Melbourne, Australia; 5 Washington University, Department of Pediatrics, St Louis, United States; 6 Griffith University, Gold Coast, Australia; 7 Gold Coast University Hospital, Gold Coast, Australia
Background: Previous studies, albeit small sample sizes have shown early infection in young children with cystic fibrosis (CF) can be detected in bronchoalveolar lavage (BAL) samples on one side despite inflammation being present on both sides of the lung. Aim: To investigate differences between the right and left lung in terms of infection, inflammation and lung structure using microbiological culture taken from the right and left lobes of BAL samples of children. Methods: Microbiological culture data were collected from BAL samples of the right and left lobes from children with CF. Samples were analysed using routine microbiological processing procedures at The Royal Children’s Hospital, Melbourne. Respective Chest Computed Tomography (CT) images were analysed using the Brody scoring system and all lung lobes were assessed. Results: Data from 527 BAL samples from 124 children were analysed. Neutrophil elastase (ne) was detected and reported in 163 (31%) of samples and interleukin-8 (IL-8) measured in 519 (98%). Mean (SD) ne reported was 2654.08 (7249.51) mg/ml, and mean (SD) IL8 measured 3.24 (7.65) ng/ml. A difference between presence of infection in the right and left lung existed (p < 0.001) for each type of infection analysed. Bronchiectasis was more apparent in the left lung [c2 (1) = 188.72; p < 0.001] for volumetric scans as was the case for limited scans [c2 (1)=56.23; p < 0.001]. Bronchiectasis of the left lung was greater when Pseudomonas aeruginosa infection was not present on the left side [c2 (1) = 4.72; p < 0.05]. Conclusion: Lower respiratory infection and early lung structural changes appeared to be more common in the left lung in infants and young children with CF.
Objectives: The evolution of the respiratory microbiome in CF and its contribution to the development of lung disease is yet to be characterised. The few longitudinal studies in older patients suggest a decrease in microbial diversity over time, possibly occurring in conjunction with declining pulmonary function. This study describes the lower airway microbiome in CF and its evolution in early childhood. We aim to assess the correlation between the microbiome, traditional culture-based microbiology and inflammatory status, and to examine the impact of the early lung microbiome on pulmonary function at six years of age. Methods: Serial surveillance bronchoscopies were performed on newly diagnosed infants with CF in 1992–2001. Quantitative bacterial culture and inflammatory markers (cell count, IL-8 and neutrophil elastase) were performed contemporaneously. 16S rDNA analysis was performed on stored samples. Results: 16S rDNA analysis was successfully performed on serial BAL samples from 18 infants (77% P.Phe508del homozygous, 50% male). Samples were obtained at median ages of 2.8 months (range 1.2–27.3), 15.2 months (range 12.3–39.7), 27.5 months (range 20.4–78.3) and 46.7 months (range 28.4–63.6). Microbial diversity decreased over time, with increasing dominance of traditional CF pathogens, particularly staphylococcal and Pseudomonas species. There was often concordance between culture results and the dominant species on 16S analysis. Conclusion: 16S rDNA analysis of these historical BAL samples provides a unique insight into the early lung microbiome in CF, its evolution over time and its correlation with long term clinical outcomes.
4. Microbiology
S77
77 Whole genome sequencing of multiple isolates of the Pseudomonas aeruginosa Liverpool epidemic strain reveals transmission and geographical clustering
79 Development of the upper respiratory tract microbiome was investigated longitudinally in cystic fibrosis infants and controls 0−6 months of age
M. Moore1 , D. Williams2 , S. Paterson2 , N. Tucker3 , D. Kenna4 , J. Turton4 , N. Loman5 , D. Taylor-Robinson6 , K. Southern7 , M.J. Walshaw6 , J.L. Fothergill1 , C. Winstanley1 . 1 Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom; 2 Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom; 3 University of Strathclyde, Liverpool, United Kingdom; 4 Public Health England, London, United Kingdom; 5 University of Birmingham, Birmingham, United Kingdom; 6 Liverpool Heart and Chest Hospital, Liverpool, United Kingdom; 7 Alder Hey Children’s Hospital, Liverpool, United Kingdom
S.M.P.J. Prevaes1 , K.M. de Winter-de Groot1 , H.M. Janssens2 , X. Wang1 , G.A. Tramper-Stranders1 , R. Hasrat1 , H.A.W.M. Tiddens2 , M. van Westreenen2 , C.K. van der Ent1 , E.A.M. Sanders1 , D. Bogaert1 . 1 University Medical Center Utrecht, Utrecht, Netherlands; 2 ErasmusMC-Sophia Children’s Hospital, Rotterdam, Netherlands
Objectives: Pseudomonas aeruginosa is the primary cause of chronic lung infection in cystic fibrosis (CF) and is associated with a decrease in morbidity and mortality. Discovery of the Liverpool Epidemic Strain (LES) provided the first molecular evidence for a transmissible strain of P. aeruginosa. The LES has since been discovered in Canada, remains the most common clone of P. aeruginosa in UK CF clinics and is associated with poorer prognoses. In 2009, the earliest archived isolate, LESB58 from 1988, was whole genome sequenced. The aims of this study were to utilise whole genome sequencing to provide insight into the population structure and epidemiology of the strain and characterise genomic variations between isolates. Methods: Using the Illumina HiSeq platform, 73 LES isolates, chosen to represent a historically and geographically diverse panel, were sequenced and compared to 50 LES genomes already available. Results: Phylogenetic analysis of the LES core genome revealed 3533 polymorphic sites. Isolates clustered geographically, with Canadian and UK isolates forming distinct separate clusters. Conclusion: By closer analysis of the distribution of specific mutations we can gain a better understanding of the transmission and evolution of this important strain. We would like to acknowledge the Cystic Fibrosis Trust for funding this research.
Background: Cystic Fibrosis (CF) is characterized by early damage of the lungs by infections. The responsible pathogens find their ecological niche in the upper respiratory tract (URT). Information on microbial colonization dynamics in the URT of CF infants is lacking. Objective: To examine colonization dynamics of URT microbiota in CF infants over time. Methods: Case-control study. Questionnaires and nasopharyngeal (NP) samples were collected monthly from 20 CF patients and 45 controls. We studied the dynamics of URT microbiota from 0−6 months of life by 16SrRNA-based sequencing. Results: The bacterial density of URT microbiota is lower in CF infants compared to controls, and inversely correlated with antibiotic use. Also, the microbial community composition is significantly different between groups over the first 6 months of life. Where in CF infants the URT microbiota is initially predominated by Corynebacterium and Staphylococcus followed by Streptococcus and Moraxella, in controls Moraxella, Haemophilus and another Corynebacterium sp. predominate throughout the first 6 months. Multivariate analyses show significantly more Staphylococcus, Pseudomonas, Enterobacteriaceae and Bacillus spp. and less Moraxella, Haemophilus, Dolosigranulum and specific Corynebacterium spp. in CF infants vs. Controls. Antibiotic use is independently associated with increased carriage of Gram-negative bacteria and reduced carriage of non-pathogenic commensals. Conclusion: CF infants have a significantly different development of URT microbiota compared to controls from birth on. This information may help to improve targeted therapy, and allows surveillance of ecological side effects of antibiotics.
78 Differences in infection, inflammation and structural abnormalities between the right and left lung
80 The microbiome in early cystic fibrosis lung disease: A longitudinal analysis
R. Carzino1 , L. King1 , S. Ranganathan1 . 1 Murdoch Children’s Research Institute, Respiratory Medicine, Parkville, Australia
K.B. Frayman1,2,3 , D. Armstrong2,4 , R. Carzino1,2 , T. Ferkol5 , K. Grimwood6,7 , G. Storch5 , K. Wylie5 , S. Ranganathan1,2,3 . 1 Royal Children’s Hospital, Department of Respiratory Medicine, Melbourne, Australia; 2 Murdoch Children’s Research Institute, Infection and Immunity Theme, Melbourne, Australia; 3 University of Melbourne, Department of Paediatrics, Melbourne, Australia; 4 Monash Children’s Hospital, Respiratory Medicine, Melbourne, Australia; 5 Washington University, Department of Pediatrics, St Louis, United States; 6 Griffith University, Gold Coast, Australia; 7 Gold Coast University Hospital, Gold Coast, Australia
Background: Previous studies, albeit small sample sizes have shown early infection in young children with cystic fibrosis (CF) can be detected in bronchoalveolar lavage (BAL) samples on one side despite inflammation being present on both sides of the lung. Aim: To investigate differences between the right and left lung in terms of infection, inflammation and lung structure using microbiological culture taken from the right and left lobes of BAL samples of children. Methods: Microbiological culture data were collected from BAL samples of the right and left lobes from children with CF. Samples were analysed using routine microbiological processing procedures at The Royal Children’s Hospital, Melbourne. Respective Chest Computed Tomography (CT) images were analysed using the Brody scoring system and all lung lobes were assessed. Results: Data from 527 BAL samples from 124 children were analysed. Neutrophil elastase (ne) was detected and reported in 163 (31%) of samples and interleukin-8 (IL-8) measured in 519 (98%). Mean (SD) ne reported was 2654.08 (7249.51) mg/ml, and mean (SD) IL8 measured 3.24 (7.65) ng/ml. A difference between presence of infection in the right and left lung existed (p < 0.001) for each type of infection analysed. Bronchiectasis was more apparent in the left lung [c2 (1) = 188.72; p < 0.001] for volumetric scans as was the case for limited scans [c2 (1)=56.23; p < 0.001]. Bronchiectasis of the left lung was greater when Pseudomonas aeruginosa infection was not present on the left side [c2 (1) = 4.72; p < 0.05]. Conclusion: Lower respiratory infection and early lung structural changes appeared to be more common in the left lung in infants and young children with CF.
Objectives: The evolution of the respiratory microbiome in CF and its contribution to the development of lung disease is yet to be characterised. The few longitudinal studies in older patients suggest a decrease in microbial diversity over time, possibly occurring in conjunction with declining pulmonary function. This study describes the lower airway microbiome in CF and its evolution in early childhood. We aim to assess the correlation between the microbiome, traditional culture-based microbiology and inflammatory status, and to examine the impact of the early lung microbiome on pulmonary function at six years of age. Methods: Serial surveillance bronchoscopies were performed on newly diagnosed infants with CF in 1992–2001. Quantitative bacterial culture and inflammatory markers (cell count, IL-8 and neutrophil elastase) were performed contemporaneously. 16S rDNA analysis was performed on stored samples. Results: 16S rDNA analysis was successfully performed on serial BAL samples from 18 infants (77% P.Phe508del homozygous, 50% male). Samples were obtained at median ages of 2.8 months (range 1.2–27.3), 15.2 months (range 12.3–39.7), 27.5 months (range 20.4–78.3) and 46.7 months (range 28.4–63.6). Microbial diversity decreased over time, with increasing dominance of traditional CF pathogens, particularly staphylococcal and Pseudomonas species. There was often concordance between culture results and the dominant species on 16S analysis. Conclusion: 16S rDNA analysis of these historical BAL samples provides a unique insight into the early lung microbiome in CF, its evolution over time and its correlation with long term clinical outcomes.