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BRN Symposium 03/06/16 Microbiome in COPD: Pitfalls and Progress
- 1. The Microbiome in COPD Progress and Pitfalls Sanjay Sethi MD Professor of Medicine Division Chief, Pulmonary, Critical Care and Sleep Medicine Assistant Vice President for Health Sciences University at Buffalo, SUNY ssethi@buffalo.edu
- 2. British Hypothesis Mucus Hypersecretion Infective exacerbations Fletcher and Peto BMJ 1977,1:1645-8 Ciba Guest Symposium 1959, Thorax;14:286-99
- 4. Learning Objectives Does the lung microbiome change in Stable COPD? Does the lung microbiome impact disease progression in COPD? Does the lung microbiome change at Exacerbation and with its treatment? Does conventional microbiology have a role in the microbiome era?
- 5. Learning Objectives Does the lung microbiome change in Stable COPD? Does the lung microbiome impact disease progression in COPD? Does the lung microbiome change at Exacerbation and with its treatment? Does conventional microbiology have a role in the microbiome era?
- 6. Microbiome Diversity in COPD Cabrera-Rubio J Clin Micro 2012; 50 (11):3562 Six former smokers with moderate COPD No controls Sputum, BAL, Bronchial biopsy and Bronchial aspirates obtained
- 7. Microbiome diversity in COPD Erb-Downward et al PLoS ONE 6(2): e16384.
- 8. Tissue microbiome in COPD Sze et al AJRCCM 2012,185:1073-80
- 9. Are these Upper or Lower Airway Samples? Hilty et al, Plos ONE 5 (1): e58578 Erb-Downward et al PLoS ONE 6(2): e16384.
- 10. Bacterial Colonization in Ex-smokers with COPD Group % positive for PPM >/=102/ml Pathogens isolated Titer 1 (ex-smokers with COPD) 34.6 % (9/26) 1) NTHI and HP 2) NTHI and SP 3) HP 4) PA 5) SA 6) NTHI 7) NTHI 8) HP 9) HP 3 x 102 2 x 102 7 x 104 2 x 104 1 x 102 6 x 102 1 x 102 1.5 x 104 5 x 105 6.5 x 105 3 x 105 2 (ex-smokers without COPD) 0% (0/20) 3 (healthy non- smokers) 6.7% (1/15) 1) HP 2 x 102 Sethi et al AJRCCM 2006, 173:991-8 • A sequential two-scope procedure was performed • The right middle lobe was lavaged with 50 x 3 ml of saline.
- 11. Endotoxin levels in BAL in COPD Sethi et al AJRCCM 2006, 173:991-8
- 12. Contamination of Lower Airway Specimens Sethi et al AJRCCM 2006, Charlson et al AJRCCM 2011 Unless measures are taken - to avoid scope contamination and - control for environmental contamination - the results of Bronchoscopic microbiome studies are uninterpretable
- 13. Does the Lung Microbiome change in Stable COPD? Yes, by conventional microbiology Current microbiome studies are inadequate Increase in Proteobacteria Decrease in Diversity Similar Community structure changes are seen in Bronchiectasis and Cystic Fibrosis
- 14. Learning Objectives Does the lung microbiome change in Stable COPD? Does the lung microbiome impact disease progression in COPD? Does the lung microbiome change at Exacerbation and with its treatment? Does conventional microbiology have a role in the microbiome era?
- 15. Bacterial Colonization in Ex-smokers with COPD Group % positive for PPM >/=102/ml Pathogens isolated Titer 1 (ex-smokers with COPD) 34.6 % (9/26) 1) NTHI and HP 2) NTHI and SP 3) HP 4) PA 5) SA 6) NTHI 7) NTHI 8) HP 9) HP 3 x 102 2 x 102 7 x 104 2 x 104 1 x 102 6 x 102 1 x 102 1.5 x 104 5 x 105 6.5 x 105 3 x 105 2 (ex-smokers without COPD) 0% (0/20) 3 (healthy non- smokers) 6.7% (1/15) 1) HP 2 x 102 Sethi et al AJRCCM 2006, 173:991-8 • A sequential two-scope procedure was performed • The right middle lobe was lavaged with 50 x 3 ml of saline.
- 16. 0 10 20 30 40 50 60 70 80 90 COPD PPB+ COPD PPB- Ex-smokers Non-smokers %PMN Groups p<0.001 p<0.001 p=0.03 p=0.02 p=0.004 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 PMN/ml p=0.02 p=0.007 p<0.001 COPD PPB+ COPD PPB- Ex-smokers Non-smokers Groups Sethi S et al Am J Resp Crit Care Med 2006;173:991-8. PMN, polymorphonuclear neutrophil; PBB, potentially pathogenic bacteria, including Haemophilus spp, Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Gram-negative enteric bacteria Bacterial Colonization and Airway Inflammation in Ex-smokers with COPD
- 17. Groups Groups 1 10 100 1000 IL-8pg/ml p=0.006 p<0.001 p<0.001 p=0.02 1 10 100 1000 10000 100000 1000000 ActiveMMP-9units/ml p=0.04 p<0.001 p=0.01 p=0.002 p=0.007 IL-8, interleukin 8; MMP-9, matrix metalloproteinase-9 Bacterial Colonization and Airway Inflammation in Ex-smokers with COPD COPD PPB+ COPD PPB- Ex-smokers Non-smokers COPD PPB+ COPD PPB- Ex-smokers Non-smokers Sethi S et al Am J Resp Crit Care Med 2006;173:991-8.
- 18. Greater Induction of Cytokines by Proteobacteria from Dendritic Cells Larsen et al, PLoS ONE 2012, 7(2): e31976
- 19. Potential Consequences of Altered Airway Microbiome in COPD Hogg et al, NEJM 2004, Martínez-García et al. Chest 2011
- 20. Vicious Circle Hypothesis Initiating factors e.g. smoking, childhood respiratory disease Impaired innate lung defense Microbial Colonization Airway epithelial injury Inflammatory response Increased proteolytic activity Altered proteinase anti-proteinase balance Progression of COPD Bacterial products Acute Exacerbation Sethi and Murphy NEJM 2008
- 21. Learning Objectives Does the lung microbiome change in Stable COPD? Does the lung microbiome impact disease progression in COPD? Does the lung microbiome change at Exacerbation and with its treatment? Does conventional microbiology have a role in the microbiome era?
- 22. COPD Study Clinic Aims Dynamics of bacterial infection in COPD Hypothesis Acquisition of new strains of bacterial pathogens is associated with an increased risk of exacerbation Clinic visits: Monthly Suspected exacerbation At each visit: Clinical evaluation Serum sample Sputum sample for quantitative bacteriology
- 23. Naturally Occurring Exacerbations: Changes in the Microbiome -150 -100 -50 0 50 100 150 Days Relative to Exacerbation Exacerbationonset Antibiotics only Corticosteroids only Antibiotics and corticosteroids Exacerbation treatment group Huang et al, JCM, J Clin Microbiol. 2014 ;52(8):2813-23
- 24. Changes in Microbiome at Exacerbation Huang et al, JCM, J Clin Microbiol. 2014 ;52(8):2813-23
- 25. 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co cc ac ea e En teroco cc ac ea e En teroco cc ac ea e En teroco ccac ea e En teroco ccac ea e En teroco ccac ea e En ter ococcaceae En teroco cc ac ea e En teroco cc ac ea e En teroco cc ac ea e Lacto bacillac eae Lacto bacillaceae Lacto bacillaceae Lacto bacillaceae Lacto bacillaceae Lacto bacillace ae Lactobacillace ae Lac tob acillac eae Lac tobacillac eae Lac tobacillaceae Lac tobacillaceae Lac tobacillace ae Leuconostoc aceae Le ucon ostoc aceae Stre ptoc occaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococcaceae Streptococca ceae Streptococca ceae Streptococcaceae Strep tococcaceae Streptococcaceae Streptococcacea e Bac illi Stre ptococcaceae Pa eniba cillac eae Sp oro lac tob acilla ceae Ca ryo phanaceae Ba cillac ea e Ba cillac ea e Ba cillac ea e Ba cillac ea e Ba cillace ae Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Bac illac ea e Baci lla ce ae Baci lla ce ae Sta ph yloc oc ca ce ae Sta ph yloc oc ca ce ae Sta ph yloc oc ca ce ae Sta ph yloc oc ca ce ae Sta ph yloc oc ca ce ae Sta ph yloc oc ca ce ae Sta ph yloc oc ca ce aeSta ph yloc oc ca ce ae Sta ph yloc oc ca ce ae Baci lla ce ae Ha loba cillace ae Ha loba cillace aeBa cilla ce ae Ba cilla ce ae Bac illac ea e Ba cilla ce ae Ha loba cilla ce ae Fus oba cteriac eae Mycop lasmataceae Acholeplasmataceae Ery sipelotric haceae Ery sipelo tric haceae Erysipe lotrichaceae Erysipe lotrichaceae Clostridiaceae Clostridiaceae Clostridiaceae Clostridiaceae Clostridiaceae ClostridiaceaePeptostreptococcaceaePeptostreptococcaceaePeptostreptococcaceae ClostridiaceaeClostridiaceaeClostridiaceaePeptostreptococcaceae ClostridiaceaeClostridiaceaePeptostreptococcaceae Peptostreptoco ccaceae Peptostreptoco ccaceae Pep tostrep tococcaceae Pep tostrep tococcaceae Peptostre ptococ cac eae Pe ptoc oc c/Ac idam inoc oc c Pe ptoc c/A cid am ino co cc Pe ptococ c/A cid am ino co cc Pe ptoc oc c/A cid am ino co cc Pe pto cc/Acid am ino cocc Pe pto cc/Acid am ino cocc Firm icutes Natrono an ae robium Sy mbiob ac teria Ba cillace ae Firm icu tes Pol ya ng ia ce ae Pol ya ng ia ce ae Pol ya ng ia ce ae Pol ya ng ia ce ae Pol ya ng ia ce ae Delta pr ot eo ba cter ia Delta pr ot eo ba cter ia Delta pr ot eo ba cter ia Del ta pr ot eo ba cter ia Delta pr ot eo ba cter ia Des ulfoba cter ac ea e Syn tro ph ac ea e Deltaproteo ba cteria Des ulfobu lbac ea e De su lfo bu lbac ea e De su lfo bu lbac ea e Des ulfoba cter ac ea e Deltaproteo ba cteria D esu lfu ro m onace ae G eob ac te ra ce ae Syn trop ho ba ct er ac ea e Del ta pr ot eo ba ct er ia Syn trop ho ba ct er ac ea e Del ta pr ot eo ba ct er iaD elta pro te obact eria De su lfo ha lobiac ea e De su lfo vib rio na ce ae De su lfovib rio na ce ae Rhodocyclaceae Nitro somonadaceae Betaproteobacteria Methylophilaceae Rhodocyclaceae Rhodocyclaceae Neisseriaceae Procabacteriaceae Burkholderiaceae Ralstoniaceae Ralstoniaceae Ralstoniaceae Ralstoniaceae Oxalobacteraceae Oxalobacteraceae Oxalobacteraceae Oxalobacteraceae Oxalobacteraceae Oxalobacteraceae Oxalobacteraceae Oxalobacteraceae Burkholderiaceae Alcaligenaceae Alcaligenaceae Alcaligenaceae Gammaproteobacteria Xanthomonadaceae Xanthomonadaceae Xanthomonadaceae Xanthomonadaceae Xanthomonadaceae Legionellaceae Moraxellace ae Moraxellaceae Moraxellaceae Moraxellaceae Pseud omona dac eae Pseud omonadace ae Pseudomo nadaceae Pseudomo nadaceae Pseudomo nadaceae Ocean osp irilla cea e Pseud omona dac eae Pis cirick ettsia ceae Pis cirick ettsia ceae Ga mm aproteobacte ria Me thy loc occaceae Ga mm ap roteob acter ia Ga mm ap roteo ba cte ria Ga mm aproteobacte ria Gammaproteobacteria Halomonadaceae Alte ro m on ad ac ea e Alte ro m on ad ac ea e Pse ud oa lte ro m on ad ac ea e Alte ro m on ad ac ea eAl tero mon ad ac ea e Ps eu do altero mon ad ac ea e Alteromon ad ac ea e Alteromon ad ac ea e Alteromon ad ac ea e Alteromon ad ac ea e Alteromon ad ac ea e Alteromon ad ac ea e Alteromon ad ac ea e Al tero mon ad ac ea e Al tero mon ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e She wan ellace ae Alte ro m on ad ac ea e Alte ro m on ad ac ea e G am m ap ro te ob ac te ria Alte ro m on ad ac ea e Alte ro m on ad ac ea e Alte ro m on ad ac ea e Shew an el la ce ae Shew anella ce ae Shew anella ce ae G am m apro te obact eria Vibriona ce ae Vi br iona ce ae Vibrio na ce ae Vibrio na ce ae Vibrio na ce ae Pa ste urellacea e Pa ste urellacea e Pa ste urellaceae Pa ste urellacea e Pa ste ure llaceae Pa ste ure llaceae Paste ure llac eae Paste ure llac eae Paste ure llac eae Pa ste ure llaceae Pa ste ure llaceae Enterobac teriace ae Enterobac teriace ae Enterobacteriaceae Enterobacteriaceae EnterobacteriaceaeEnterobacteriaceaeEnterobacteriaceaeEnterobacteriaceaeEnterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteria ceae Enterobacteriaceae Enterobacteriaceae Enterobacteriacea e Ente roba cteriaceae Enterobacteria ceae Enterobacteriaceae Enterobacteriaceae Enterobac teriace ae Enterobac teriace ae Gammaproteobacteria Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterob acte riaceae Enterob acte riaceae Ente roba cteriaceae Enterobacteriaceae Enterobacteria ceaeEnterobacteriaceaeEnterobacteriaceaeEnterobacteriaceae Enterobacteriaceae Ente robacteriaceae Enterobacteria cea e Entero bac teriace ae X6 04 12 .2 Ae romon ad ac ea e Ae romon ad ac ea e S3 92 32 .2 Ae romon ad ac ea e Ae romon ad ac ea e Ch romatiac ea e Gammaproteobacteria Coxiellaceae Coxiellaceae Coxiellaceae Thiotrichaceae Thiotrichaceae Chromatiaceae Gam map roteobacteria Ro seococcaceae Alph ap roteo ba cte ria Rickettsia ceae Rickettsia ceae Ca ediba cte rac eae Alp hapro teo bacte ria Alp hapro teo bacte ria Rhodocyclaceae Caulobac terace ae Caulobac tera cea e Cau lobacteraceae Cau loba cteraceaeCaulobactera cea e Alp hapro teo bacte ria Bra dyrhizobiaceae Bra dyrhizobiaceae Ac idob ac teria Acido ba cter iace ae Acido ba cter iace ae Acido ba cter iace ae Acido ba cter iace ae Acido ba cter ia Acido ba cter iace ae Acido ba cter ia ce ae Nitros pina ce ae Ac idob ac teria ce ae Ac idob ac teria De lta proteo ba cteria De lta proteo ba cteria NC 10 −2Un cla ss ifie d Leptospiraceae Leptospiraceae Spirochaetaceae Spirochaetaceae Spirochaetaceae Spirochaetaceae Spirochaetaceae Spirochaetaceae Chlorobi Cren otrichace ae Ba cte roide tes Ba cte roide tes Sp hin go ba cte ria Ba cte roide tes Ba cte roide tes Ba cte roidetes Ba cte roidetes Ba cte roide tes Rik enella cea e Bacte roidetes Porph yro mo nadaceae Ba cte roidaceae Ba cte roidaceae Bacteroid etes Bacteroid ete s Flavob acteria ceae Flavob acteria cea e Blattab acteria cea e SphingobacteriaFlex ibac teraceae Flam meo virgaceae Flexiba cteraceae CrenotrichaceaeCrenotrich aceaeCrenotrichaceaeCren otrichace ae Helicobacteraceae Helicobacteraceae Epsilonproteobacteria Helicobacteraceae Campylobacteraceae mgA−2 mgA−1 TM7−3 CyanobacteriaChloroplasts Chloroplasts Chloroplasts Cop ro th er m ob ac te ria Syn er gi st es Syn er gi st es Syn er gist es Syn er gi stesOP9 Deh aloc oc co idet es Deh aloc oc co idetes Deh aloc oc co idetes Ana er olinea e Chlor of lexi Cellulom on ad ac ea e M icro co cc ac ea e M icro co cc ac ea e Acido ba ct er ia Act in om yc et ac ea e Bifido ba ct er ia ce ae Bifido ba ct er ia ce ae Bifi do ba ct er ia ce ae Acido ba cter ia Acido ba cter ia Cor ioba cter iace ae Rub ro ba cter ac ea e Ac idob ac teria 0.01 Classes (tree branches): Gammaproteobacteria Verrucomicrobiae Chlamydiae Aquificae gut clone group Catabacter Bacilli Fusobacteria Mollicutes Symbiobacteria Deltaproteobacteria Betaproteobacteria Alphaproteobacteria Acidobacteria N C 10− 2 Spirochaetes Sphingobacteria Bacteroidetes Flavobacteria Epsilonproteobacteria m gA − 1 T M 7− 3 Cyanobacteria OP9 Actinobacteria Enterobacteriaceae Pasteurellaceae (Haemophilus-related) Pseudomonadaceae Moraxellaceae Oxalobacteraceae (Betaproteobacteria) Lactobacillus spp. Streptococcaceae (Lactobacillales) Helicobacter Campylobacter (Epsilonproteobacteria) Deltaproteobacteria Staphylococcus spp. Clostridiaceae/Lachnospiraceae (Clostridia) Bacillaceae Abundance Higher Lower Positive correlation Negative correlation Huang et al, JCM, J Clin Microbiol. 2014 ;52(8):2813-23
- 26. Antibiotics and Steroids have Differential Effects on the Microbiome in AECOPD Positive changes in relative abundance (log2) indicate an increase in the latter time point compared. Huang et al, JCM, J Clin Microbiol. 2014 ;52(8):2813-23
- 27. MAESTRAL Study: Clinical Failure Rates with Oral Corticosteroid use 0 5 10 15 20 25 30 35 40 During therapy EOT 4 weeks post- therapy 8 weeks post- therapy Clinicalfailure(%ofpatients) Sethi et al, Infection 2016
- 28. Changes in Airway Microbiome after Rhinovirus inoculation Molyneaux; et al, Am J Respir Crit Care Med 188, 1224-1231. *P < 0.001
- 29. Control: no significant differences Distribution of bacterial phyla at each time point after rhinovirus (RV) inoculation. Molyneaux et al, Am J Respir Crit Care Med 188, 1224-1231. COPD: significant increases in Proteobacteria (dark blue) were observed on Day 15 (P = 2.2 × 10–16).
- 30. Bacterial Load (16s) and Inflammatory Changes following Rhinovirus Infection Molyneaux et al, Am J Respir Crit Care Med 188, 1224-1231. p=0.08 p=0.0001 p=0.007 p=0.045p=0.001 p=0.07
- 31. Learning Objectives Does the lung microbiome change in Stable COPD? Does the lung microbiome impact disease progression in COPD? Does the lung microbiome change at Exacerbation and with its treatment? Does conventional microbiology have a role in the microbiome era?
- 32. TMI
- 33. Losing the Trees in the Forest
- 34. Species vs Strain level Differentiation Han et al Thorax 2013 Sethi et al NEJM 2008
- 35. NTHI Colonization vs Exacerbation strains •10 exacerbation strains •7 colonization strains •In vivo mouse model •In vitro respiratory epithelial cell line Chin et al AJRCCM 2005
- 36. Does Conventional Microbiology have a Role in the Microbiome Era? Identifying a pathogen by sequence is the first step Immune and inflammatory consequences of putative pathogens need to be determined Ability to manipulate the pathogen in culture is essential Vaccine development Pathogen virulence
- 37. Conclusions The ability to assess the lung microbiome could radically change our understanding of lung disease and lung infection COPD and COPD exacerbations are clearly associated with changes in the lung microbiome Restoring the healthy lung microbiome could make a significant difference in COPD management Maybe, the British were right all the time!
- 38. Acknowledgments Co-Investigators Tim Murphy Charles Berenson Karin Provost Ilya Berim Study coordinators Nancy Evans Karen Eschberger Jane Maloney Ellana Eberhardt Laboratory personnel Catherine Wrona Lori Grove Phyllis Lobbins Regina Clare Collaborators Yvonne Huang (UCSF) U of Iowa