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BRN Symposium 03/06/16 Certainties and uncertainties in the respiratory microbiome
1.
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
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
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
18. Sampling approaches
Charlson. Am J Respir Crit Care Med 20011
Oropharynx aspirates
Oropharynx wash
Higher abundance in the oropharynx, when sampled through washing
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
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.
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)
37. Loss of diversity
in exacerbations
Wang. Eur Respir J 2016
Increase in Proteobacteria
Loss in diversity
Decrease in Proteobacteria
Recovery of the 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
+
+
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.
65.
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.