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WS19.5 Alginate phenotypes of Pseudomonas aeruginosa from a Brazilian CF center compared to Scandinavian CF centers
S36
Workshop 19. Influence of host environment on bacterial diversity
Oral Presentations
WS19.5 Alginate phenotypes of Pseudomonas aeruginosa from a Brazilian CF center compared to Scandinavian CF centers
WS19.7 Pseudomonas aeruginosa genotyping: Predicting transition to chronic colonization in cystic fibrosis patients
N.C. Ca¸cador1,2 , A.L.C. Darini1 , L.A.G.M.M. Torres3 , O. Ciofu2 , N. Høiby2,4 . 1 School of Pharmaceutical Sciences of Ribeir˜ ao Preto, University of S˜ao Paulo, Ribeir˜ao Preto, Brazil; 2 Institute of International Health, Immunology and Microbiology, University of Copenhagen, Rigshospitalet, Copenhagen, Denmark; 3 Department of Pediatrics, Ribeir˜ ao Preto Medical School, University of S˜ao Paulo, Hospital das Cl´ınicas, Ribeir˜ao Preto, Brazil; 4 Department of Clinical Microbiology, University Hospital, Rigshospitalet, Copenhagen, Denmark
L. Jonckheere1 , M. Vaneechoutte2 , F. De Baets3 , S. Van Daele3 , F. Haerynck3 , L. Van Simaey2 , P. Schelstraete3 . 1 Ghent University, Ghent, Belgium; 2 Ghent University, Laboratory for Microbiology Research, Ghent, Belgium; 3 Ghent University Hospital, Pediatric Pulmonology, Ghent, Belgium
Objectives: Alginate production (mucoid phenotype) of P. aeruginosa is a marker of chronic biofilm infection in the lungs of CF patients. Little is known about the occurrence of alginate-producing P. aeruginosa in CF patients outside Europe and North America. We therefore decided to study the occurrence of the alginate phenotype in P. aeruginosa from chronically infected CF patients in a CF center in Brazil and CF centers in Scandinavia (Ciofu et al., 2008). Methods: The phenotype was classified according to Schurr et al. (1994): Type I: mucoid (M) on PIA (Pseudomonas isolation agar) and LB agar, type II: mucoid on PIA but non-mucoid (NM) on LB, type III: non-mucoid on both, type IV: very slight but detectable mucoidy on both media after 4 days incubation. Results: See the table. Table: Distribution of different alginate phenotypes Type I Type II Type III Type IV Both Both Only Only
M & NM isolates from 20 Brazilian patients 95% M & NM isolates from 83 Scandinavian patients 89% NM isolates from 12 Brazilian patients NM isolates from 8 Scandinavian patients
17%
40% 87% 92% 75%
65% 6% 8% 25%
Objectives: Chronic respiratory infection with Pseudomonas aeruginosa (Pa) is known to increase morbidity in cystic fibrosis (CF) patients. Following a first ever Pa isolation, patients may go through subsequent episodes of intermittent colonization, preceding chronic colonization by a variable time span, before chronic infection eventually is established. Currently, definitions of chronic colonization are based on the frequency of Pa isolation. This study evaluates whether genotyping of subsequent Pa isolates can be predictive towards persistence of Pa, and therefore towards transition from intermittent to chronic colonization. Methods: Between January 2002 and December 2014 respiratory samples were prospectively collected and cultured from 80 CF patients with a first ever Pa isolation and all available isolates were compared using McRAPD genotyping (Deschaght et al.). Results: Sixty-eight patients, 22 of which became chronically colonized − according to the European consensus criteria − during the study period, had at least one subsequent Pa isolate. Isolates were genotyped for a sample group of 25 patients. For 10 out of the 11 patients becoming chronically colonized during the study period. the first and second isolate were identical, as compared to for only 7 out of the 14 patients remaining non-chronically colonized (p < 0.0001). Conclusion: Our results indicate that the presence of a genotypically identical subsequent Pa isolate can predict impending chronic colonization. As such, genotyping and comparison of the subsequent Pa isolates could − for many patients − speed up the diagnosis of chronic colonization, compared to the currently used criteria.
Conclusion: Generally, the results show rather similar distributions of the Brazilian and Scandinavian isolates on the different phenotypes (Table). This probably reflects common selective environment in the lungs of CF patients in Brazil and Scandinavia, since the countries are widely separated and have completely different climate and geography, whereas the public medical systems are tax-based in all 4 countries. We are now sequencing the mucA and algT genes of the isolates which are responsible for the alginate phenotypes. WS19.6 Diversity of Pseudomonas aeruginosa in cystic fibrosis airways L.M. Sommer1 , R.L. Marvig2 , A. Luj´an3 , A. Koza1 , T. Pressler4 , S. Molin1,5 , H.K. Johansen2 . 1 Technical University of Denmark, Novo Nordic Foundation, Center for Biosustainability, Hørsholm, Denmark; 2 Rigshospitalet, Department of Clinical Microbiology, Copenhagen, Denmark; 3 Exeter University, Cornwall, United Kingdom; 4 Rigshospitalet, CF Centre, Copenhagen, Denmark; 5 Technical University of Denmark, Systems Biology, Lyngby, Denmark Objectives: Previous investigations of the population dynamics of P. aeruginosa in the CF airways have been based on longitudinally collected single isolates, but recently this approach has been questioned. The aim of this study is to determine whether genotypic characterization of single isolates can represent the adaptation of P. aeruginosa populations in CF airways. Methods: Five metagenomes from sputum samples from four CF patients were sequenced without pre-culturing. The metagenomes were analysed and compared with single isolates from the same patients. Results: For all the investigated patients the most recent clone type identified among the single isolates was compared with the metagenome of the specific patient. In all cases the metagenomes were linked to a significantly higher degree of the mutations found in the single isolates sampled from the same patient, than to single isolates sampled from other patients(p < 0.05). Conclusion: Our results document that single isolates represent the dominant clone type in the population of P. aeruginosa in CF lungs, as observed by the comparison between single isolate genomes and corresponding metagenomes. Furthermore, single isolates also show the same mutational patterns as metagenomes sampled from the same patient, and is different from metagenomes and single isolates sampled from other patients. The evolutionary dynamics of the population represented by single isolates is supported by the dominant SNP alleles from metagenomes. According to these results, the genomic information from longitudinally collected single isolates is sufficient for investigations of the dynamics of P. aeruginosa populations in the CF airways.
WS19.8 Comparative proteomic profiling of two Swedish CF Pseudomonas aeruginosa isolates reveals possible factors associated with the transmissible cystic fibrosis strain J-2 E.A. Johansson1,2 , C. Welinder-Olsson1,2 , S. Folkesson Hansson3 . 1 Sahlgrenska University Hospital, Gothenburg, Sweden; 2 University of Gothenburg/Inst of Biomedicine, Sahlgrenska Academy, Gothenburg, Sweden; 3 University of Gothenburg, Sambio Core Facility, Gothenburg, Sweden Objectives: Two strains of Pseudomonas aeruginosa, B and J-2, have been isolated from sputa of Swedish cystic fibrosis patients and were identified as the most frequent genotypes. Their pathogenic potential differs distinctly: while strain J-2 is highly transmissible and multi-resistant, strain B is not. The aim of the present work is to investigate the proteome to reveal differences between these two strains. Methods: We employed a comparative proteomic approach involving twodimensional gel electrophoresis coupled to matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry with isobaric tags for relative and absolute quantitation (iTRAQ) to map the proteomes of B and J-2 and to identify differentially expressed proteins. Results: We identified 1873 proteins and quantified 1769 proteins of which about 21% were differentially expressed at statistically significant altered abundance between the two analysed strains. Functional analysis of the different abundant proteins showed they could be clustered into five major groups i. secreted factors; ii. cell wall/LPS/capsule; iii. chemotaxis; iv. motility & attachment; v. adaption/protection. Within the functional clusters, upregulated proteins (q < 0.05) involved in virulence were more common in strain J-2 than strain B, 17% respectively 6% of the total number of upregulated proteins. Conclusion: This work provides the first overview of P. aeruginosa strains J-2 and B on proteome changes occurring during adaptation to the CF lung and/or antibiotic treatments. The identified altered virulence profile of strain J-2 might contribute to its pathogenicity and transmission ability.
Workshop 19. Influence of host environment on bacterial diversity
Oral Presentations
WS19.5 Alginate phenotypes of Pseudomonas aeruginosa from a Brazilian CF center compared to Scandinavian CF centers
WS19.7 Pseudomonas aeruginosa genotyping: Predicting transition to chronic colonization in cystic fibrosis patients
N.C. Ca¸cador1,2 , A.L.C. Darini1 , L.A.G.M.M. Torres3 , O. Ciofu2 , N. Høiby2,4 . 1 School of Pharmaceutical Sciences of Ribeir˜ ao Preto, University of S˜ao Paulo, Ribeir˜ao Preto, Brazil; 2 Institute of International Health, Immunology and Microbiology, University of Copenhagen, Rigshospitalet, Copenhagen, Denmark; 3 Department of Pediatrics, Ribeir˜ ao Preto Medical School, University of S˜ao Paulo, Hospital das Cl´ınicas, Ribeir˜ao Preto, Brazil; 4 Department of Clinical Microbiology, University Hospital, Rigshospitalet, Copenhagen, Denmark
L. Jonckheere1 , M. Vaneechoutte2 , F. De Baets3 , S. Van Daele3 , F. Haerynck3 , L. Van Simaey2 , P. Schelstraete3 . 1 Ghent University, Ghent, Belgium; 2 Ghent University, Laboratory for Microbiology Research, Ghent, Belgium; 3 Ghent University Hospital, Pediatric Pulmonology, Ghent, Belgium
Objectives: Alginate production (mucoid phenotype) of P. aeruginosa is a marker of chronic biofilm infection in the lungs of CF patients. Little is known about the occurrence of alginate-producing P. aeruginosa in CF patients outside Europe and North America. We therefore decided to study the occurrence of the alginate phenotype in P. aeruginosa from chronically infected CF patients in a CF center in Brazil and CF centers in Scandinavia (Ciofu et al., 2008). Methods: The phenotype was classified according to Schurr et al. (1994): Type I: mucoid (M) on PIA (Pseudomonas isolation agar) and LB agar, type II: mucoid on PIA but non-mucoid (NM) on LB, type III: non-mucoid on both, type IV: very slight but detectable mucoidy on both media after 4 days incubation. Results: See the table. Table: Distribution of different alginate phenotypes Type I Type II Type III Type IV Both Both Only Only
M & NM isolates from 20 Brazilian patients 95% M & NM isolates from 83 Scandinavian patients 89% NM isolates from 12 Brazilian patients NM isolates from 8 Scandinavian patients
17%
40% 87% 92% 75%
65% 6% 8% 25%
Objectives: Chronic respiratory infection with Pseudomonas aeruginosa (Pa) is known to increase morbidity in cystic fibrosis (CF) patients. Following a first ever Pa isolation, patients may go through subsequent episodes of intermittent colonization, preceding chronic colonization by a variable time span, before chronic infection eventually is established. Currently, definitions of chronic colonization are based on the frequency of Pa isolation. This study evaluates whether genotyping of subsequent Pa isolates can be predictive towards persistence of Pa, and therefore towards transition from intermittent to chronic colonization. Methods: Between January 2002 and December 2014 respiratory samples were prospectively collected and cultured from 80 CF patients with a first ever Pa isolation and all available isolates were compared using McRAPD genotyping (Deschaght et al.). Results: Sixty-eight patients, 22 of which became chronically colonized − according to the European consensus criteria − during the study period, had at least one subsequent Pa isolate. Isolates were genotyped for a sample group of 25 patients. For 10 out of the 11 patients becoming chronically colonized during the study period. the first and second isolate were identical, as compared to for only 7 out of the 14 patients remaining non-chronically colonized (p < 0.0001). Conclusion: Our results indicate that the presence of a genotypically identical subsequent Pa isolate can predict impending chronic colonization. As such, genotyping and comparison of the subsequent Pa isolates could − for many patients − speed up the diagnosis of chronic colonization, compared to the currently used criteria.
Conclusion: Generally, the results show rather similar distributions of the Brazilian and Scandinavian isolates on the different phenotypes (Table). This probably reflects common selective environment in the lungs of CF patients in Brazil and Scandinavia, since the countries are widely separated and have completely different climate and geography, whereas the public medical systems are tax-based in all 4 countries. We are now sequencing the mucA and algT genes of the isolates which are responsible for the alginate phenotypes. WS19.6 Diversity of Pseudomonas aeruginosa in cystic fibrosis airways L.M. Sommer1 , R.L. Marvig2 , A. Luj´an3 , A. Koza1 , T. Pressler4 , S. Molin1,5 , H.K. Johansen2 . 1 Technical University of Denmark, Novo Nordic Foundation, Center for Biosustainability, Hørsholm, Denmark; 2 Rigshospitalet, Department of Clinical Microbiology, Copenhagen, Denmark; 3 Exeter University, Cornwall, United Kingdom; 4 Rigshospitalet, CF Centre, Copenhagen, Denmark; 5 Technical University of Denmark, Systems Biology, Lyngby, Denmark Objectives: Previous investigations of the population dynamics of P. aeruginosa in the CF airways have been based on longitudinally collected single isolates, but recently this approach has been questioned. The aim of this study is to determine whether genotypic characterization of single isolates can represent the adaptation of P. aeruginosa populations in CF airways. Methods: Five metagenomes from sputum samples from four CF patients were sequenced without pre-culturing. The metagenomes were analysed and compared with single isolates from the same patients. Results: For all the investigated patients the most recent clone type identified among the single isolates was compared with the metagenome of the specific patient. In all cases the metagenomes were linked to a significantly higher degree of the mutations found in the single isolates sampled from the same patient, than to single isolates sampled from other patients(p < 0.05). Conclusion: Our results document that single isolates represent the dominant clone type in the population of P. aeruginosa in CF lungs, as observed by the comparison between single isolate genomes and corresponding metagenomes. Furthermore, single isolates also show the same mutational patterns as metagenomes sampled from the same patient, and is different from metagenomes and single isolates sampled from other patients. The evolutionary dynamics of the population represented by single isolates is supported by the dominant SNP alleles from metagenomes. According to these results, the genomic information from longitudinally collected single isolates is sufficient for investigations of the dynamics of P. aeruginosa populations in the CF airways.
WS19.8 Comparative proteomic profiling of two Swedish CF Pseudomonas aeruginosa isolates reveals possible factors associated with the transmissible cystic fibrosis strain J-2 E.A. Johansson1,2 , C. Welinder-Olsson1,2 , S. Folkesson Hansson3 . 1 Sahlgrenska University Hospital, Gothenburg, Sweden; 2 University of Gothenburg/Inst of Biomedicine, Sahlgrenska Academy, Gothenburg, Sweden; 3 University of Gothenburg, Sambio Core Facility, Gothenburg, Sweden Objectives: Two strains of Pseudomonas aeruginosa, B and J-2, have been isolated from sputa of Swedish cystic fibrosis patients and were identified as the most frequent genotypes. Their pathogenic potential differs distinctly: while strain J-2 is highly transmissible and multi-resistant, strain B is not. The aim of the present work is to investigate the proteome to reveal differences between these two strains. Methods: We employed a comparative proteomic approach involving twodimensional gel electrophoresis coupled to matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry with isobaric tags for relative and absolute quantitation (iTRAQ) to map the proteomes of B and J-2 and to identify differentially expressed proteins. Results: We identified 1873 proteins and quantified 1769 proteins of which about 21% were differentially expressed at statistically significant altered abundance between the two analysed strains. Functional analysis of the different abundant proteins showed they could be clustered into five major groups i. secreted factors; ii. cell wall/LPS/capsule; iii. chemotaxis; iv. motility & attachment; v. adaption/protection. Within the functional clusters, upregulated proteins (q < 0.05) involved in virulence were more common in strain J-2 than strain B, 17% respectively 6% of the total number of upregulated proteins. Conclusion: This work provides the first overview of P. aeruginosa strains J-2 and B on proteome changes occurring during adaptation to the CF lung and/or antibiotic treatments. The identified altered virulence profile of strain J-2 might contribute to its pathogenicity and transmission ability.