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98 The cystic fibrosis lower airways microbial metagenome
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Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
97 Nocardia and cystic fibrosis: the impact of Gram staining O. Tuncer1 , S. Olmez1 , B. Sancak1 , G.D. Tugcu2 , N. Emiralioglu2 , B. Otlu3 , 2 1 1 ¨ celik B. Er4 , E. Yalcın ¸ 2 , D. Dogru2 , U. Oz ¸ , N. Kiper2 , B. Sener ¸ . Hacettepe University Medical Faculty, Clinical Microbiology, Ankara, Turkey; 2 Hacettepe University Ihsan Dogramaci Children’s Hospital, Pediatric Pulmonology, Ankara, Turkey; 3 In¨ on¨ u University Medical Faculty, Clinical Microbiology, Malatya, Turkey; 4 Hacettepe University Medical Faculty, Pulmonology, Ankara, Turkey Objectives: We describe Nocardia isolation from multiple respiratory specimens of two CF patients with chronic Pseudomonas colonization. We aimed to investigate the genetic relatedness of the sequential Nocardia isolates in each patient. Methods: When branching Gram-positive bacilli were observed in Gram stained smears, buffered charcoal yeast extract agar was also included to the culture panel and incubation period was extended to minimum 7 days. The plates were incubated at 37o C in 5% CO2 /air environment. Smears were prepared from suspected colonies and stained by Gram and modified Ziehl-Neelsen to confirm the presence of Gram positive, variable staining, branching bacilli that were positive by modified ZiehlNeelsen. 16S rRNA gene sequence analysis was used to identify the Nocardia isolates to species level. The genetic relatedness between the isolates were analysed by pulsed field gel electrophoresis and AP-PCR. Results: In patient A N. transvalensis/N. wallacei and in patient B N. asteroides were identified. After the first isolation of N. transvalensis/ N. wallacei in sputum culture of patient A, she received TMP/SMX for 4 weeks, no new Nocardia-positive culture was obtained. Patient B received piperacillin–tazobactam for two weeks, TMP/SMX treatment still continues now. Last cultures are negative for Nocardia. Conclusion: The presence of Nocardia in sputum cultures does not always imply disease but rather simple colonization. In order to understand the impact of Nocardia in CF lungs, more reports are needed describing the clinical and microbiological features of CF patients who have Nocardia spp.-positive sputum culture. 98 The cystic fibrosis lower airways microbial metagenome P. Moran Losada1 , P. Chouvarine1 , M. Dorda1 , S. Hedtfeld1 , S. Mielke1 , 1 1 A. Schulz1 , L. Wiehlmann1 , B. Tummler ¨ . Medizinische Hochschule Hannover, Clinic for Paediatric Pneumology, Allergology and Neonatology, Hannover, Germany Objectives: Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Here we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, molds and fungi in CF lower airways. Methods: The microbial contents of induced sputa collected on several occasions from 10 PS and 15 Phe508del homozygous CF children, adolescents and adults was resolved by high-throughput whole genome sequencing. Results: Deep sputum metagenome sequencing identified on average about ten DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple lowly abundant species superimposed by few disease-associated pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus as major components. The host-associated signatures ranged from inconspicuous poly-microbial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource which could rapidly expand as a response to habitat alterations such as antimicrobial chemotherapy or invasion of novel microbes.
99 Investigating the airway microbiome in cystic fibrosis patients with normal and severe pulmonary function decline: an opportunity for a personalized microbiome-based therapy G. Bacci1 , P. Paganin2 , N. Segata3 , F. Armanini3 , G. Taccetti4 , D. Dolce4 , A. De Alessandri5 , P. Morelli5 , V. Tuccio6 , E.V. Fiscarelli6 , V. Lucidi6 , A. Mengoni1 , A. Bevivino2 . 1 University of Florence, Department of Biology, Florence, Italy; 2 ENEA Casaccia Research Center, Sustainable Territorial and Production Systems Department, Rome, Italy; 3 University of Trento, Centre for Integrative Biology, Trento, Italy; 4 Cystic Fibrosis Center, Anna Meyer Children’s University Hospital, Department of Pediatrics Medicine, Florence, Italy; 5 Cystic Fibrosis Center, G. Gaslini Institute, Department of Pediatrics, Genoa, Italy; 6 Children’s Hospital and Research Institute Bambino Ges` u, CF Microbiology and CF Center, Rome, Italy Objectives: To gain new insights into CF microbiome composition, metabolism and function, trying to unravel the underlying causes of the severe lung disease and identify potential determinants of serious decline in lung function. Methods: Twelve CF subjects attending three Italian CF centres were enrolled. Patients were stratified according to lung function decline rates and disease severity. Shotgun sequencing was performed following standard pipelines in Illumina Hiseq 2000 platform. To infer functional and taxonomic patterns among patients, extrinsic and intrinsic metagenomic data analysis was used. Results: We identified a core set of bacterial metabolic pathways and functions. In particular, the Kegg pathway One carbon pool by folate related to cysteine/methionine metabolism was found more abundant in patients with normal/mild disease. Moreover, antibiotic resistance genes, mainly belonging to efflux and transporter systems, were found to be more present in patients with severe lung disease. A longitudinal analysis on twenty CF patients followed over one-year is ongoing. Conclusion: The different pulmonary function in CF patients co-occurs with potentially different microbiome capabilities, mainly related to an increasing number of genes for multiple antibiotic resistance (efflux systems) and a potential differential presence of metabolic pathways. The ongoing longitudinal analysis will permit us to identify the microbial markers possible related to severe lung function decline in CF patients, and allow a risk assessment and a better focused and personalized therapy of CF patients. Acknowledgement: Supported by grants from the Italian CF Foundation (FFC #10/2014 and FFC #14/2015). 100 (More) viable cough aerosols from individuals with cystic fibrosis M.E. Wood1,2,3 , G.R. Johnson4 , R.E. Stockwell1 , L.J. Sherrard1,5 , N. Jabbour4 , K.A. Ramsay1,2 , L.D. Knibbs2 , T.J. Kidd2,5 , C.E. Wainwright2,6 , L. Morawska4 , S.C. Bell1,3 . 1 QIMR Berghofer Medical Research Institute, Brisbane, Australia; 2 The University of Queensland, Brisbane, Australia; 3 The Prince Charles Hospital, Brisbane, Australia; 4 Queensland University of Technology, Brisbane, Australia; 5 Queen’s University Belfast, Belfast, United Kingdom; 6 Lady Cilento Children’s Hospital, Brisbane, Australia Objectives: Cough-generated airborne P. aeruginosa droplet nuclei can remain viable for up 45 minutes. This prompted changes to infection control guidelines for patients with cystic fibrosis (CF). This study investigated if other CF respiratory pathogens are detectable in cough aerosols and assessed their survival over distance and time. Methods: 27 subjects with CF, mean age 28.6 years (SD 9.7); mean FEV1 63.1% (26.2) predicted were enrolled into 2 groups: 1. non-P. aeruginosa Gram-negative bacteria (GNB), 2. Gram-positive bacteria (GP), Staphylococcus aureus. Subjects performed a series of voluntary coughing into 2 validated aerosol-sampling devices to measure viability at 2 and 4 m and after 5, 15 and 45 min following generation. An Anderson Impactor collected and sized the aerosols, and total CFU (stages 1–6) was measured. Quantitative sputum and cough aerosol cultures were performed. Results: 11/16 (69%) subjects with mean sputum GNB concentration of 1.9×108 CFU/mL (8.0×103 –2.2×109 ) produced viable aerosol containing the target pathogen. The mean (range) CFU identified in cough aerosol at 4 m and at 45 min was 21.6 (0–164) and 15.6 (0–123),
Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
97 Nocardia and cystic fibrosis: the impact of Gram staining O. Tuncer1 , S. Olmez1 , B. Sancak1 , G.D. Tugcu2 , N. Emiralioglu2 , B. Otlu3 , 2 1 1 ¨ celik B. Er4 , E. Yalcın ¸ 2 , D. Dogru2 , U. Oz ¸ , N. Kiper2 , B. Sener ¸ . Hacettepe University Medical Faculty, Clinical Microbiology, Ankara, Turkey; 2 Hacettepe University Ihsan Dogramaci Children’s Hospital, Pediatric Pulmonology, Ankara, Turkey; 3 In¨ on¨ u University Medical Faculty, Clinical Microbiology, Malatya, Turkey; 4 Hacettepe University Medical Faculty, Pulmonology, Ankara, Turkey Objectives: We describe Nocardia isolation from multiple respiratory specimens of two CF patients with chronic Pseudomonas colonization. We aimed to investigate the genetic relatedness of the sequential Nocardia isolates in each patient. Methods: When branching Gram-positive bacilli were observed in Gram stained smears, buffered charcoal yeast extract agar was also included to the culture panel and incubation period was extended to minimum 7 days. The plates were incubated at 37o C in 5% CO2 /air environment. Smears were prepared from suspected colonies and stained by Gram and modified Ziehl-Neelsen to confirm the presence of Gram positive, variable staining, branching bacilli that were positive by modified ZiehlNeelsen. 16S rRNA gene sequence analysis was used to identify the Nocardia isolates to species level. The genetic relatedness between the isolates were analysed by pulsed field gel electrophoresis and AP-PCR. Results: In patient A N. transvalensis/N. wallacei and in patient B N. asteroides were identified. After the first isolation of N. transvalensis/ N. wallacei in sputum culture of patient A, she received TMP/SMX for 4 weeks, no new Nocardia-positive culture was obtained. Patient B received piperacillin–tazobactam for two weeks, TMP/SMX treatment still continues now. Last cultures are negative for Nocardia. Conclusion: The presence of Nocardia in sputum cultures does not always imply disease but rather simple colonization. In order to understand the impact of Nocardia in CF lungs, more reports are needed describing the clinical and microbiological features of CF patients who have Nocardia spp.-positive sputum culture. 98 The cystic fibrosis lower airways microbial metagenome P. Moran Losada1 , P. Chouvarine1 , M. Dorda1 , S. Hedtfeld1 , S. Mielke1 , 1 1 A. Schulz1 , L. Wiehlmann1 , B. Tummler ¨ . Medizinische Hochschule Hannover, Clinic for Paediatric Pneumology, Allergology and Neonatology, Hannover, Germany Objectives: Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Here we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, molds and fungi in CF lower airways. Methods: The microbial contents of induced sputa collected on several occasions from 10 PS and 15 Phe508del homozygous CF children, adolescents and adults was resolved by high-throughput whole genome sequencing. Results: Deep sputum metagenome sequencing identified on average about ten DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple lowly abundant species superimposed by few disease-associated pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus as major components. The host-associated signatures ranged from inconspicuous poly-microbial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource which could rapidly expand as a response to habitat alterations such as antimicrobial chemotherapy or invasion of novel microbes.
99 Investigating the airway microbiome in cystic fibrosis patients with normal and severe pulmonary function decline: an opportunity for a personalized microbiome-based therapy G. Bacci1 , P. Paganin2 , N. Segata3 , F. Armanini3 , G. Taccetti4 , D. Dolce4 , A. De Alessandri5 , P. Morelli5 , V. Tuccio6 , E.V. Fiscarelli6 , V. Lucidi6 , A. Mengoni1 , A. Bevivino2 . 1 University of Florence, Department of Biology, Florence, Italy; 2 ENEA Casaccia Research Center, Sustainable Territorial and Production Systems Department, Rome, Italy; 3 University of Trento, Centre for Integrative Biology, Trento, Italy; 4 Cystic Fibrosis Center, Anna Meyer Children’s University Hospital, Department of Pediatrics Medicine, Florence, Italy; 5 Cystic Fibrosis Center, G. Gaslini Institute, Department of Pediatrics, Genoa, Italy; 6 Children’s Hospital and Research Institute Bambino Ges` u, CF Microbiology and CF Center, Rome, Italy Objectives: To gain new insights into CF microbiome composition, metabolism and function, trying to unravel the underlying causes of the severe lung disease and identify potential determinants of serious decline in lung function. Methods: Twelve CF subjects attending three Italian CF centres were enrolled. Patients were stratified according to lung function decline rates and disease severity. Shotgun sequencing was performed following standard pipelines in Illumina Hiseq 2000 platform. To infer functional and taxonomic patterns among patients, extrinsic and intrinsic metagenomic data analysis was used. Results: We identified a core set of bacterial metabolic pathways and functions. In particular, the Kegg pathway One carbon pool by folate related to cysteine/methionine metabolism was found more abundant in patients with normal/mild disease. Moreover, antibiotic resistance genes, mainly belonging to efflux and transporter systems, were found to be more present in patients with severe lung disease. A longitudinal analysis on twenty CF patients followed over one-year is ongoing. Conclusion: The different pulmonary function in CF patients co-occurs with potentially different microbiome capabilities, mainly related to an increasing number of genes for multiple antibiotic resistance (efflux systems) and a potential differential presence of metabolic pathways. The ongoing longitudinal analysis will permit us to identify the microbial markers possible related to severe lung function decline in CF patients, and allow a risk assessment and a better focused and personalized therapy of CF patients. Acknowledgement: Supported by grants from the Italian CF Foundation (FFC #10/2014 and FFC #14/2015). 100 (More) viable cough aerosols from individuals with cystic fibrosis M.E. Wood1,2,3 , G.R. Johnson4 , R.E. Stockwell1 , L.J. Sherrard1,5 , N. Jabbour4 , K.A. Ramsay1,2 , L.D. Knibbs2 , T.J. Kidd2,5 , C.E. Wainwright2,6 , L. Morawska4 , S.C. Bell1,3 . 1 QIMR Berghofer Medical Research Institute, Brisbane, Australia; 2 The University of Queensland, Brisbane, Australia; 3 The Prince Charles Hospital, Brisbane, Australia; 4 Queensland University of Technology, Brisbane, Australia; 5 Queen’s University Belfast, Belfast, United Kingdom; 6 Lady Cilento Children’s Hospital, Brisbane, Australia Objectives: Cough-generated airborne P. aeruginosa droplet nuclei can remain viable for up 45 minutes. This prompted changes to infection control guidelines for patients with cystic fibrosis (CF). This study investigated if other CF respiratory pathogens are detectable in cough aerosols and assessed their survival over distance and time. Methods: 27 subjects with CF, mean age 28.6 years (SD 9.7); mean FEV1 63.1% (26.2) predicted were enrolled into 2 groups: 1. non-P. aeruginosa Gram-negative bacteria (GNB), 2. Gram-positive bacteria (GP), Staphylococcus aureus. Subjects performed a series of voluntary coughing into 2 validated aerosol-sampling devices to measure viability at 2 and 4 m and after 5, 15 and 45 min following generation. An Anderson Impactor collected and sized the aerosols, and total CFU (stages 1–6) was measured. Quantitative sputum and cough aerosol cultures were performed. Results: 11/16 (69%) subjects with mean sputum GNB concentration of 1.9×108 CFU/mL (8.0×103 –2.2×109 ) produced viable aerosol containing the target pathogen. The mean (range) CFU identified in cough aerosol at 4 m and at 45 min was 21.6 (0–164) and 15.6 (0–123),