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Berner Kolloquien für Labormedizin UNIBE, 10.12.2018

Berner Kolloquien für Labormedizin UNIBE, 10.12.2018 Microbiota analyses of the upper airways: Clinical relevance Markus Hilty Institut für Infektionskrankheiten, Universität Bern Universitätsklinik für Infektiologie , Inselspital Bern. Overview.

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Berner Kolloquien für Labormedizin UNIBE, 10.12.2018

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  1. Berner Kolloquien für Labormedizin UNIBE, 10.12.2018 Microbiota analyses of the upper airways: Clinical relevance Markus Hilty Institut für Infektionskrankheiten, Universität Bern Universitätsklinik für Infektiologie, Inselspital Bern

  2. Overview • Why is the characterization of the human microbiota important?; Disordered microbial communities in asthmatic, COPD and ILD airways (PLoSOne, 2010) • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Healthy cohort; Cystic fibrosis cohort - Importance of personalized microbiota - Necessity to do Longitudinal analyses • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Example pig farming - Importance of personalized microbiota - S. aureus is not so important after all?

  3. V7 V4 V6 V5 V3 V8 V1 V9 V2

  4. Taxonomy • Advantages of Sanger sequencing method: - Good sequence information (1000 bp) • Disadvantages: - redundant sequencing - Laborious and expensive

  5. Site specific distributions of bacterial phyla in healthy humans Size of circles is proportionate to average number of species-level phylotypes per individual Dethlefsen et al. Nature 2007

  6. Overview • Why is the characterization of the human microbiota important?; Disordered microbial communities in asthmatic, COPD and ILD airways (PLoSOne, 2010) • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Healthy cohort; Cystic fibrosis cohort - Importance of personalized microbiota - Necessity to do Longitudinal analyses • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Example pig farming - Importance of personalized microbiota - S. aureus is not so important after all?

  7. Disordered microbial communities in asthmatic airways • Asthma: Most prevalent in children and young adults • North-South Gradient • Familial • Rich microbial environment protective • Asthma exacerbation is triggered by infections

  8. Hygiene hypothesis N Engl J Med. 2011 Feb 24;364(8):701-9.

  9. To develop a molecular, culture independent tool based on small ribosomal sequencing of the 16S rRNA gene to characterize clinical samples from the respiratory tract. To identify the microbial flora of the left upper lobe in healthy compared to asthmatic and COPD subjects Study aims

  10. Patient characteristics Hilty et al. PLoS One, 2010;

  11. D C E Swabs and brushings A B A: Nasal swab B: Throat swab C: Left upper lobe prox. D: Left upper lobe distal E: Right lower lobe

  12. V7 V4 V6 V5 V3 V8 V1 V9 V2

  13. Extract DNA, PCR Native microbial populations Mixed 16S rRNA Separation Sequencing Phylogenetic identification Separate by cloning into E. coli Cloning and sequencing

  14. ‘Denaturing Gradient Gel Electrophoresis’ (DGGE) within the respiratory tract

  15. UniFrac bacterial community comparisons of PSB-derived PCR libraries

  16. Quantification

  17. Disordered microbial communities in asthmatic airways Hilty et al. PLoS One, 2010;

  18. Ecribal: Relative abundances of the top 20 bacterial families Garzoni C. et al., Thorax

  19. Comparison of the microbiota between upper and lower airways Garzoni C. et al., Thorax

  20. Conclusions (I) • Development of molecular tools to define the respiratory tract microbiota in health and disease • High percentage of Bacteroidetes (>40 %) and Proteobacteria in the microbiota of ‘normal’ and asthmatic subjects, respectively. • Function? Infectious pathogens? BUT: Disease phenotype does not always correlate with distinct disease phenotype (e.g. interstitial lung disease). Cave: antibiotic treatment, vaccination, smoking etc. BUT: No replacement for bacterial culture!

  21. Overview • Why is the characterization of the human microbiota important?; Disordered microbial communities in asthmatic, COPD and ILD airways (PLoSOne, 2010) • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Healthy cohort; Cystic fibrosis cohort - Importance of personalized microbiota - Necessity to do Longitudinal analyses • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Example pig farming - Importance of personalized microbiota - S. aureus is not so important after all?

  22. Charlson et al., Am J Respir Crit Care Med, 2011 Picture adapted from: clipartpanda.com Dynamics of the healthy nasal microbiota in infancy Microaspiration colonisation • Factors influencing the colonisation within the first year of life: • Type of birth; Environment • Season; Age; Vaccines? • Prerequisite for investigating the potential role of the microbiota in various respiratory diseases

  23. Dynamics of the healthy nasal microbiota in infancy http://www.bild-cohort.ch/; Mika et al. JACI 2015

  24. Dynamics of the healthy nasal microbiota in infancy): DOB February March April May June July October November December August September 382 374 395 401 403 405 409 410 414 380 381 387 396 391 392 393 397 399 400 dates of birth: January 421 425 456 407 408 415 450 384 388 483 484 417 419 Exclusion criteria: respiratory tract infection and antibiotic therapy during sampling http://www.bild-cohort.ch/; Mika et al. JACI 2015

  25. 16S rRNA PCRV3-V5454 Seq V7 V4 V6 V5 V3 V8 V1 V9 V2 primers → SequencesGenBank; 97% OTUs

  26. Relative abundance of bacterial families Cough 381 391

  27. Results: dynamics by season Relative bacterial abundances by season Mika et al. JACI 2015, Teo et al. CHM 2015

  28. Personalized or not? between- and within-subject dissimilarity Important: Microbiota is personalized and therefore relevant

  29. Study Design CF infants n=30 healthy infants n=47 nasal swab every second week DNA extraction, 16S rRNA PCR, 454 sequencing quality control sequence reads of n=462 samples sequence reads of n=872 samples before first AB therapy samples, n=229 (infants, n=24) at/after AB therapy samples, n=233 (infants, n=23) after 1x AB therapy samples, n=41 (infants, n=7) at AB therapy samples, n=30 (infants, n=15) after >1x AB therapy samples, n=162 (infants, n=17) www.scild.ch

  30. Bern: 13 Zurich: 7 St. Gallen: 1 Aarau: 1 Basel: 3 Lausanne: 4 Geneva: 1 www.scild.ch

  31. Results – significant differences • Microbiota composition differs between CF and healthy • The difference is mainly caused by • - Staphylococcaceae - Pasteurellaceae • - Corynebacteriaceae www.scild.ch

  32. Antibiotic-naïve CF Samples H: healthy infants NMDS: non-metric multidimensional scaling • microbiota composition differs between healthy and CF, also without the effect of antibiotic therapy • CF environment of the airways select for specific bacteria Mika et al., Lancet Respir Med, 2016

  33. Results – Effect of antibiotics on the microbiota in CF • Staphylococcaceae remains increased after antibiotic therapy • S. aureus decreased but coagulase-negative staphylococci show a trend of increased abundance

  34. Overview • Why is the characterization of the human microbiota important?; Disordered microbial communities in asthmatic, COPD and ILD airways (PLoSOne, 2010) • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Healthy cohort; Cystic fibrosis cohort - Importance of personalized microbiota - Necessity to do Longitudinal analyses • Influence of the environment/interventions on the respiratory tract/nasal microbiota? Example pig farming - Importance of personalized microbiota - S. aureus is not so important after all?

  35. Study design pig study 4x 4x n=26 n=16 cow-farmers nasal swab non-exposed nasal swab n=28 pig farms controls 4x n=26 n=56 n=43 pigs nasal swab pig-farmers nasal swab air impingement Krämer et al. Science of the Total Environment; Krämer et al., in preparation

  36. 16S rRNA PCRMiSeqDada2 V7 V4 V6 V5 V3 V8 V1 V9 V2 primers → SequencesGenBank; 97% OTUs

  37. Alpha diversity measurements Krämer et al., AEM; 2018

  38. Betadiversity measurements (I) Abundance based Binary based clustering of pigs, air, pig-farmers, cow-farmers and non-exposed. pig, air and pig-farmer closer clustering in the plot with abundance based values →share more SVs →clustering mainly abundance-based Krämer et al., AEM; 2018

  39. Corynebacteriaceae and Staphylococcaceae Higher abundance in controls

  40. Conclusions (II) • Healthy microbiota in infants is personalized though not so much in summer • Pneumococcal vaccines influence the microbiota; Replacement by many different bacteria? Microbiota pattern more stable over time in the PCV13 era? • Longitudinal analyses important for adjusting natural fluctuation • Pig farm environment influences the nasal microbiota – stability? Longitudinal study needed • => Personalized AND influenced by the environment

  41. Acknowledgments P. Latzin, I. Korten, C. Casaulta, N. Regamey, Linda Béul-Beguin, Sandra Lüscher, U. Frey, I. Mack, R. Neumann(BILD Cohort) Moana Mika, Aurélie Allemann, Suzanne Aebi, Marianne Küffer, Julia Krämer, Josua Maurer, Lukas Troxler and Stephen Leib Anne Oppliger

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