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A study to define the microbiome
subtypes in the adult asthmatic
airway
A Sullivan, EB Hunt, S Lapthorne, C Ward, J Eustace, PM O’Byrne, BJ Plant,
DM Murphy, J MacSharry

13th September 2017

UCC, Cork, Ireland
I have no real or perceived conflicts of interest that relates to this presentation
 Introduction
Asthma is a chronic inflammatory disease of the lungs

Microorganisms are present and can add to onset and

progression of asthma

Microbes are associated with acute exacerbations [1]

Macrolide antibiotics are used and are effective in asthma [2]

Despite this we still have not characterised the microbiome

S. Wenzel. Nature Medicine 18, 716–725 (2012)

1. Papadopoulos, N.G., et al., Viruses and bacteria in acute asthma exacerbations – A GA2LEN-DARE* systematic review. Allergy, 2011.
66(4): p. 458-468.
2. Gibson, P.G., et al., Effect of azithromycin on asthma exacerbations and quality of life in adults with persistent uncontrolled asthma
(AMAZES): a randomised, double-blind, placebo-controlled trial. The Lancet. 390(10095): p. 659-668.
 Factors influencing the lung microbiome

The microbiome possibly both influences and is influenced by asthma disease severity

Sullivan, A., et al., The Microbiome and the Pathophysiology of Asthma.

Respiratory Research, 2016. 17(1): p. 163.
 Methods
80 patients recruited at our Asthma clinic at CUH, Cork, Ireland.

Measures of disease severity, FEV1, ACQ-7 and blood testing

were taken on the morning of bronchoscopy.

Bronchoscopy: 3x60ml lavage from lingula or RML

Endobronchial biopsy from RLL or LLL

 Methods
Neat BAL n=80
• Cytospin onto microscope slides (400g x 2min)
• Slides stained – Differentially (Kwik-Diff) and Gram

BAL n=80
• Centrifuged (500g x 10min) Host Cell Pellet
• Supernatant removed and aliquoted – pellet is saved for DNA extraction
• 30 ml of SN centrifuged (20,000g x 5min)
Microbial Pellet
Endobronchial Biopsies n=52
• Paraffin embedded and microtome cut (5mm) biopsy slides
• Slides stained – H&E
 Patient demographics
80 Patients
Asthma severity
FEV-1
GINA (1-5)
• Mild (GINA 1+2) 34%
• Moderate (GINA 3) 21%
• Severe (GINA 4+5) 45%
ACQ-7
• ACQ-7 <1.5 controlled 37%
• ACQ-7 ≥1.5 uncontrolled 63%
63% Female

Hunt, E.B., et al., The Potential Role of Aspiration in the Asthmatic Airway. CHEST. 151(6): p. 1272-1278.
BAL – Initial findings
A B

10X 60X
N=80 D
C

100X 100X
Asthma- BAL qPCR

N=80
 Results- Neutrophils
Increased
• Pseudomonas fluorescens P=0.0003
• Moraxella catarrhalis P=0.0006
• Neisseria bacilliformis P=0.0017
• Staphylococcus aureus P=0.0023
• Streptococcus pneumoniae P=0.0041
• Veillonella P=0.0258
P=0.0102
• Haemophilus influenzae P=0.0479

Decreased
• Bordetella P=0.0318

N=80
Results- Eosinophils
No correlation for ;
• Total Bacteria
• Firmicutes
• Pseudomonas
• Pseudomonas fluorescens
• Escherichia
• Bordetella
• Prevotella
• Moraxella catarrhalis
• Neisseria bacillliformis
• Staphylococcus aureus
• Streptococcus pneumoniae
• Veillonella
P=0.0102 • Haemophilus influenzae
• Chlamydophilia pneumoniae
• Mycoplasma pneumoniae
N=80
Biopsy – bacterial adhesion

Endobronchial biopsy N=52

Biopsy – Biopsy scoring score

N=52
Asthma- BAL qPCR-II
Microbial Pellet
BAL - Free microbes

Host Cell Pellet

BAL – Cell assoc. microbes

S. aureus
Strep. pneumoniae

N=14
 Conclusion
Conclusions
• In summary in our cohort the presence of total bacteria as measured by qPCR
correlates with airway Neutrophils.

• Airway Eosinophils did not correlate with the presence of total bacteria but did
correlate with increased presence of Neisseria meningitidis

• Initial findings suggest that measures of asthma control and severity do not
correlate with total bacteria

• We hope our findings will advance asthma characterisation but also further
understanding and rational with current therapeutic strategies such as
azithromycin
 Acknowledgements
Dr. John MacSharry
Dr. Desmond Murphy
Dr. Eoin Hunt
Dr. Susan Lapthorne
Dr. Chris Ward
Prof. Barry Plant
Prof. Joseph Eustace
Prof. Paul M O’Byrne

Funded by: the Wilton Respiratory Research Fund, UCC TRAP 2015,
Denis O’Sullivan Award and the APC Microbiome Institute
Innovation Fund with the financial support of Science Foundation
Ireland (SFI) under Grant Number SFI/12/RC/2273.