- The respiratory microbiome is a new frontier in medicine, but there are still many uncertainties regarding normal flora, sampling methods, disease states, and interactions. - Studies have found similar microbiomes in healthy subjects' oropharynx and bronchial areas, but disease like COPD is associated with loss of diversity and overgrowth of Proteobacteria. - Exacerbations of COPD are linked to increases in potential pathogens like Moraxella and decreases in diversity, though the role of bacteria-virus interactions is still unclear. Interventions aimed at the microbiome may be possible but the best approaches require more research.

2. Certainities and uncertainites in
the respiratory microbiome
The respiratory microbiome: a
new frontier in medicine
Eduard Monsó
Head of the Respiratory Diseases Department
Hospital Universitari Parc Taulí
Universitat Autònoma de Barcelona

3. ~5 % of microorganisms currently culturable
Staley and Konopka 1985 Annual Review of Microbiology 39 pp 321-346
Microbiome is there

4. Normal flora
• Something grows below the larynx?.

5. Healthy Human Respiratory Tract
Charlson. Am J Respir Crit Care Med 20011
6 subjects

6. Healthy Human Respiratory Tract
Charlson. Am J Respir Crit Care Med 20011
Control BAL 1st 2nd return
Oropharynx wash
BAL other lobe PSB

7. Healthy Human Respiratory Tract
Charlson. Am J Respir Crit Care Med 2011
Similar microbioma in the
oropharynx and the bronchial
tree in the healthy subject
Nasopharinx Oropharinx and bronchi
Firmicutes
Bacteroidetes
*
*
*
* proteobacteria with clinical meaning:
Pseudomonas / Haemophilus / Moraxella

8. Aspiration as bacterial flora source in the lung
• Swallowed saliva: 1 liter per day.
• Aspiration during sleep 50% subjects.
• Temporal variability.
Hypothesis:
Infrequent aspiration
Frequent aspiration
Gleeson. Chest 1997; 111:1266
Huxley. Am J Med 1978; 64:564
Bassis. mBio 6(2):e00037-15

9. Respiratory flora
Significantly different from supraglotis…
Dickson. Ann Am Thorac Soc 2015.

10. Selective elimination Prevotella (Lung)
Bassis. mBio 6(2):e00037-15
Bacterial richness (OTU 97% identity / 700 reads)
Overrepresented in BAL

11. Severe COPD
Microbiota of the bronchi
Erb-Downward JR. PLoS ONE 2011; 6(2): e16384
Heterogeneity of bronchial mucosa microbiota in severe COPD

12. Normal flora
• Something grows below the larynx?: YES.
• Oropharyngeal secretions aspiration.
• Selective growing in the bronchial-lung ecosystem.
• Heterogeneous in advanced COPD.
• When is it acquired?.
• Is changes with age?.

13. Sample Extract DNA
Amplifiy and
sequence16S
rRNA gene
GATACAGAGATGCAT
GATACAGTGATGCAT
GATACAGTAGATGCAT
GATACAGTAGATGCAT
TACCAGATTTACATAC
TACCAGATTACATAC
TACCAGATTACATAC
OTU 1
OTU 2
Group sequences into
Operational Taxonomic
Units (OTUs)
GreenGenes
Identify OTUs
Community analysis
Which microorganisms are
present (percentage)
Evenness
Richness
PC1 20%
PC2 14%
PC3 11%
α-diversity β-diversity
What is the community
structure
METHOD

14. Taxonomy
Bacteria: Difficult at species level

15. Relative abundance
Wang. Eur Respir J 2016.

16. Relative abundance
0 50 100
0
5
10
15
r=0.7
p<0.0001
RA Pseudomonas
log10P.aeruginosaculture
Garcia-Nuñez (submitted)

17. Diversity
 Shannon index

18. Sampling approaches
Charlson. Am J Respir Crit Care Med 20011
Oropharynx aspirates
Oropharynx wash
Higher abundance in the oropharynx, when sampled through washing

19. Sampling volume
Bronchoalveolar Lavage (150 mL)
Gastric wash (50 mL)
Oral wash (20 mL)
Nasal swab
Bassis. mBio 6(2):e00037-15
28 asymptomatic subjects
71% never smokers
FEV1% range 59-120
Dililution effect

20. BAL and PSB
Dickson. Ann Am Thorac Soc 2015
Bronchoalveolar lavage
(BAL)
Protected specimen brush
(PSB)

21. Respiratory flora
equivalent according with region and method
Dickson. Ann Am Thorac Soc 2015.
BAL (first)
BAL (second)
Same channel
PSB
PSB
Equivalent results

22. 4 respiratory tract sample – healthy subject
microbial composition - sequence of the 16S rRNA gene
EI
BAL
BB
BAS
EI
BAL
BB
BAS
Sputum different from bronchial biopsy and bronchoalveolar lavage
Bronchial biopsy and bronchoalveolar lavage cluster together
sputum

23. Disease
• COPD.
• Cystic fibrosis.
• Idiopathic pulmonary fibrosis.
• Clinical status: Exacerbation.

24. Proteobacteria in advanced disease
Distal bronchi – Lung explants
Sze. Am J Respir Crit Care Med 2015; 192:438.

25. Bronchial secretions
Loss of diversity in advanced disease
Garcia-Nuñez M. Journal of Clinical Microbiology 2014; 52:4217
P<0.03
←lessdiversity

26. Loss of richness in advanced disease
Sze. Am J Respir Crit Care Dis 2015
Diversity:
Number of OTU present
≈ number of species
← increase in emphysema destruction

27. Loss of Prevotella and Streptococcus
advanced disease
Sze. Am J Respir Crit Care Dis 2015.

28. Exacerbation: relative abundance
Proteobacteria up
Firmicutes down
Wang. Eur Respir J 2016

29. Variability in relative abundance
Exacerbation
Streptococcus
Acromobacter
Moraxella
Hemophilus
Streptococcus
Moraxella
Hemophilus
Millares 2014

30. Variability in relative abundance
Stability
Millares 2014

31. Moraxella in exacerbations
Wang. Eur Respir J 2016
Increase in relative abundance
of Moraxella en 36/87 patients
during exacerbation (41%).
Significant increase in Moraxella
relative abundance (p=0.022),
identified as Moraxella
catarrhalis.
Correlated with sputum
neutrophilia (r=0.18 p<0.002)

32. COPD Exacerbation
Identification of pathogens

33. Severe COPD
Pseudomonas colonization
stability and exacerbation
Millares 2014

34. Exacerbation
Pseudomonas colonization
Standard pathogens in COPD colonized by
Pseudomonas aeruginosa when exacerbated

35. Bacterial and eosinophilic exacerbations
Wang. Eur Respir J 2016
Positive culture eosinophilia >3%

36. Healthy subject COPD
Molyneaux. Am J Respir Crit Dis 2013
Interaction
Rhinovirus infection

37. Loss of diversity
in exacerbations
Wang. Eur Respir J 2016
Increase in Proteobacteria
Loss in diversity
Decrease in Proteobacteria
Recovery of the diversity

38. Dominant patogen
George Hajishengallis 2012

39. Keystone patogen
George Hajishengallis 2012

40. Keystone patogen
George Hajishengallis 2012

41. Interaction network
Wang. Eur Respir J 2016 Overgrowth of Haemophilus or Moraxella related to loss of diversity

42. Symbiosis
close and often long-term interaction between two
different biological species
+ Mutualist
Commensal
Parasitic (PPM)
Effect on
Microbe
Type of Microbial
Symbiont
+
0
_
Effect on
Host
+
+

43. Mutualist?
I thank you
for your attention

45. Interactions
• Bacteria-Bacteria:
– Exists: +
– Has a clinical meaning: ?
• Virus-Bacteria:
– Changes in behaviour: ?
– Favour overgrowth: +
– Role of bacteriophages: ?
• Bacteria-viruses-Bronchial mucosa/lung:?

46. The respiratory microbiome: a
new frontier in medicine
• Normality: ?
• Sampling: ?
• Processing: ?
• Disease: ?
• Interactions: ?
• Functional analyses: ?
• Host response: ?
• Interventions?

48. Keystone

50. Proteobacteria and Firmicutes
in exacerbation
Wang. Eur Respir J 2016

51. COPD exacerbation
Millares. PlosOne 2015

52. COPD exacerbation
Millares.
PlosOne 2015

53. The respiratory microbiome: a
new frontier in medicine
• Host response:
• Inflammation?.
• Remodelling?.
• None?.
• Symbiotic?

54. COPD
Bronchial inflammatory response in colonized patients
Marin. Eur Respir J 2010;35:295
Statistically significant Inflammatory response associated with H. influenzae

55. Asthma and Microbes
• Asthma complex relationship to infection:
– Infections cause exacerbations.
– Rich microbial environment childhood: protective.

56. Asthma Hygiene Hypothesis

57. Asthma Hygiene Hypothesis
In the kids room

58. Diversity and inflammation

59. Diversity and inflammation

60. The respiratory microbiome: a
new frontier in medicine
• Interventions:
• Possible?.
• Useful?.
• Which approach?.

61. Ecuador and Asthma
• Cardenas. Upper Airways Microbiota in Antibiotic-Naıve
Wheezing and Healthy Infants from theTropics of Rural
Ecuador. PLoS ONE 2012; 7: e46803.
• Ecuadorian Jungle.
• No steroids / No antibiotics.
• Pyrosequencing 16S rRNA gene oropharyx.
• 24 infants with non-infectious early onset wheezing.
• 24 healthy controls.
• 76,627 high quality sequences.
• 182 operational taxonomic units (OTUs).

62. ActinobacteriaFusobacteriaFirmicutesBacteroiditesProteobacteria
Staphylococci
Haemophilus
Ecuador
Heatmap
Controls Cases

63. Ecuador
• Streptococcus most common genus (49.7%).
• Known pathogens case compared to control:
– Haemophilus spp. (OR=2.12, P=5.46x10-23).
– Staphylococcus spp. (OR=124.1, P=1.87x10-241).
• Veillonella spp.:
– More common in controls (OR=0.59, P=8.06x10-86).

64. In the natural state
• In absence of antibiotics/inhaled steroids:
• core community airways similar to West.
• similar pathogens present in asthmatics.

66. There are more bacteria out there
than we first imagined.....
Zoetendal. Gut 2008; 57:1605-15
Molecular methods like PCR amplification of 16S rRNA gene followed by
cloning and sequencing identificate many bacteria undetected by culture.

67. Respiratory flora
similarities supraglotis and RUL - intersubject
Dickson. Ann Am Thorac Soc 2015.

68. Microbiota COPD - Asthma
Hilty M. PLoS ONE 2010; 5(1): e8578
Proteobacteria
Other
Neisseria
Haemophilus
Bronchial brushing - Distal bronchi
>Proteobacteria in asthma and COPD
5 COPD
11 asthma
8 healthy

69. Exacerbation treatment
Huang Y, Sethi S. J Clin Microbiol 2014

70. COPD exacerbation treatment
Huang Y, Sethi S. J Clin Microbiol 2014

71. COPD exacerbation treatment
Huang Y, Sethi S. J Clin Microbiol 2014